JP2004135656A - Transgenic non-human mammalian animal expressing chimeric 7 times membrane-penetrating type receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provided a non-human mammalian animal having different reactivities to a human 7 times membrane-penetrating receptor and to the ortholog of the receptor of a non-human animal with which a pharmacological evaluation of a substance acting specifically with the human 7 times membrane-penetrating type receptor can be performed. <P>SOLUTION: This transgenic non-human mammalian animal is constituted so as to have a DNA encoding the chimeric 7 times membrane-penetrating type receptor obtained by substituting an intracellular domain of the human 7 times membrane-penetrating receptor with a corresponding intracellular domain of the ortholog of the non-human mammalian animal in its genome, and express the chimeric 7 times membrane-penetrating receptor. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】図１は、本発明にて決定したマウスＡ３アデノシン受容体ｃＤＮＡ配列と、公知であるマウスＡ３アデノシン受容体ｃＤＮＡ配列を比較した結果を示す。下線は両者で異なる塩基配列を示している。
【図２】図２は、本発明にて決定したマウスＡ３アデノシン受容体ｃＤＮＡ配列より推定されるアミノ酸配列と、公知であるマウスＡ３アデノシン受容体ｃＤＮＡ配列より推定されるアミノ酸配列を比較した結果を示す。＊は両者で異なるアミノ酸配列を示している。
【図３】図３は、Ａ３アデノシン受容体遺伝子のエクソン１を含む約９ｋｂｐのマウスゲノムＤＮＡ配列に存在する、各制限酵素に対する認識配列を示す。
【図４】図４は、Ａ３アデノシン受容体遺伝子のエクソン２を含む約６．５ｋｂｐのマウスゲノムＤＮＡ配列に存在する、各制限酵素に対する認識配列を示す。
【図５】図５は、本発明で決定したＡ３アデノシン受容体遺伝子を含む約５．２ｋｂｐのマウスゲノムＤＮＡ配列、および公知であるＡ３アデノシン受容体遺伝子を含む約６ｋｂｐのマウスゲノムＤＮＡ配列の領域を示す。
【図６】図６は、ヒトＡ３アデノシン受容体ｃＤＮＡを含むプラスミドｐＢＳ−ｈＡ３Ｒの構築を示す。
【図７】図７は、マウスＡ３アデノシン受容体遺伝子をヒトＡ３アデノシン受容体をコードするＤＮＡで置換するためのターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１工程、プラスミドｃｈｉｍｅｒａ−１の構築を示す。
【図８】図８は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２工程、プラスミドｃｈｉｍｅｒａ−２の構築を示す。
【図９】図９は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第３工程、プラスミドｃｈｉｍｅｒａ−３の構築を示す。
【図１０】図１０は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第４工程、プラスミドｃｈｉｍｅｒａ−４の構築を示す。
【図１１】図１１は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第５工程、プラスミドｃｈｉｍｅｒａ−１＋２の構築を示す。
【図１２】図１２は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第６工程、プラスミドｃｈｉｍｅｒａ−３＋４の構築を示す。
【図１３】図１３は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第７工程、プラスミドｃｈｉｍｅｒａ−１＋２＋３＋４の構築を示す。
【図１４】図１４は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第８工程、プラスミドＡ３Ｒ−Ｎ１の構築を示す。
【図１５】図１５は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第９工程、プラスミドＡ３Ｒ−Ｎ２の構築を示す。
【図１６】図１６は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１０工程、プラスミドＡ３Ｒ−Ｎ３の構築を示す。
【図１７】図１７は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１１工程、プラスミドｃＡ３Ｒ−Ｎの構築を示す。
【図１８】図１８は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１２工程、プラスミドＡ３Ｒ−Ｎ５の構築を示す。
【図１９】図１９は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１３工程、プラスミドａｒｍ１＋ｃＡ３Ｒ−Ｎの構築を示す。
【図２０】図２０は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１４工程、プラスミドｃｈｉｍｅｒａ−５の構築を示す。
【図２１】図２１は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１５工程、プラスミドｃｈｉｍｅｒａ−６の構築を示す。
【図２２】図２２は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１６工程、プラスミドｃｈｉｍｅｒａ−５＋６の構築を示す。
【図２３】図２３は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１７工程、プラスミドＡ３Ｒ−Ｃ１の構築を示す。
【図２４】図２４は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１８工程、プラスミドＡ３Ｒ−Ｃ２の構築を示す。
【図２５】図２５は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第１９工程、プラスミドｃＡ３Ｒ−Ｃの構築を示す。
【図２６】図２６は、マウスＡ３アデノシン受容体遺伝子をヒトＡ３アデノシン受容体をコードするＤＮＡで置換するためのターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２０工程、プラスミドｐｌｏｘＰ−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築を示す。
【図２７】図２７は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２１工程、プラスミドｐｌｏｘＰ−ＲＥ−ＨＰＲＴ−ｈＡ３Ｒ−ａｒｍ２の構築を示す。
【図２８】図２８は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２２工程、プラスミドｐｌｏｘＰ−ＲＥ−Ｃ−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築を示す。
【図２９】図２９は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２３工程、プラスミドｐｌｏｘＰ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築を示す。
【図３０】図３０は、ターゲットベクターｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築（全２４工程）の第２４工程、プラスミドｐｌｏｘＰ−ＤＴ−ａｒｍ１−ＨＰＲＴ−ｃｈｉＡ３Ｒ−ａｒｍ２の構築を示す。
【図３１】図３１は、上段にキメラＡ３アデノシン受容体の模式図を、下段にヒトとマウスのＡ３アデノシン受容体のアミノ酸配列を示す。上段の実線はキメラＡ３アデノシン受容体中のヒトＡ３アデノシン受容体の領域を、点線はマウスＡ３アデノシン受容体の領域を示す。下段の下線はＡ３アデノシン受容体の細胞内領域を、アステリスクはヒトとマウスで共通するアミノ酸配列を、数字はそれぞれの段の右端のアミノ酸残基の番号を示す。
【図３２】図３２は、ヒトＡ３アデノシン受容体の発現ベクターｈＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全４工程）の第１工程、プラスミドｐｏｌｙＡ　ｉｎ　ｐＢＳの構築を示す。
【図３３】図３３は、ヒトＡ３アデノシン受容体の発現ベクターｈＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全４工程）の第２工程、プラスミドｈＡ３Ｒ　ｉｎ　ｐＢＳの構築を示す。
【図３４】図３４は、ヒトＡ３アデノシン受容体の発現ベクターｈＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全４工程）の第３工程、プラスミドｈＡ３Ｒ−ｐｏｌｙＡ　ｉｎ　ｐＢＳの構築を示す。
【図３５】図３５は、ヒトＡ３アデノシン受容体の発現ベクターｈＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全４工程）の第３工程、プラスミドｈＡ３Ｒ　ｉｎ　ｐＣＡＧの構築を示す。
【図３６】図３６は、キメラＡ３アデノシン受容体の発現ベクターｃＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全３工程）の第１工程、プラスミドｃＡ３Ｒ（Ｎ）　ｉｎ　ｐＢＳの構築を示す。
【図３７】図３７は、キメラＡ３アデノシン受容体の発現ベクターｃＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全３工程）の第２工程、プラスミドｃＡ３Ｒ（Ｃ）　ｉｎ　ｐＣＡＧの構築を示す。
【図３８】図３８は、キメラＡ３アデノシン受容体の発現ベクターｃＡ３Ｒ　ｉｎ　ｐＣＡＧの構築（全３工程）の第３工程、プラスミドｃＡ３Ｒ　ｉｎ　ｐＣＡＧの構築を示す。
【図３９】図３９は、ヒトＡ３アデノシン受容体またはキメラＡ３アデノシン受容体を一過的に発現させたマウスミエローマ細胞Ｐ３Ｕ１の、Ａ３アデノシン受容体に特異的なアゴニストＣｌ−ＩＢ−ＭＥＣＡに対する反応性（細胞内Ｃａ^２＋の上昇量）を測定した結果を示す。横軸は時間（秒）で、矢印の時点でＣｌ−ＩＢ−ＭＥＣＡを細胞に添加した。縦軸はエクオリンの発光量で、細胞内Ｃａ^２＋の上昇量を表す。●はヒトＡ３アデノシン受容体を発現させたＰ３Ｕ１細胞、▲はキメラＡ３アデノシン受容体を発現させたＰ３Ｕ１細胞、△はコントロールのＰ３Ｕ１細胞、□はＣｌ−ＩＢ−ＭＥＣＡを添加しなかったコントロールのＰ３Ｕ１細胞の測定結果である。
【図４０】図４０は、ヒトＡ３アデノシン受容体を安定発現させたマウスミエローマ細胞株（計４株）のＡ３アデノシン受容体に特異的なアゴニストＣｌ−ＩＢ−ＭＥＣＡに対する反応性（細胞内Ｃａ^２＋の上昇量）を測定した結果を示す。横軸は時間（秒）で、矢印の時点でＣｌ−ＩＢ−ＭＥＣＡを細胞に添加した。縦軸はエクオリンの発光量で、細胞内Ｃａ^２＋の上昇量を表す。●、▲、黒い四角および◆はヒトＡ３アデノシン受容体を安定発現させたＰ３Ｕ１細胞、□はコントロールのＰ３Ｕ１細胞の測定結果である。
【図４１】図４１は、キメラＡ３アデノシン受容体を安定発現させたマウスミエローマ細胞株（計９株）のＡ３アデノシン受容体に特異的なアゴニストＣｌ−ＩＢ−ＭＥＣＡに対する反応性（細胞内Ｃａ^２＋の上昇量）を測定した結果を示す。横軸は時間（秒）で、矢印の時点でＣｌ−ＩＢ−ＭＥＣＡを細胞に添加した。縦軸はエクオリンの発光量で、細胞内Ｃａ^２＋の上昇量を表す。●、○、▲、黒い四角、囲んだ×、△、◆、×および◇はヒトＡ３アデノシン受容体を安定発現させたＰ３Ｕ１細胞、□はコントロールのＰ３Ｕ１細胞の測定結果である。
【図４２】図４２は、プラスミドｐＢＳ−ｐｒｏｂｅ５’が含むマウスゲノムＤＮＡのＳａｃＩ−ＳｍａＩ断片の位置を模式的に示す。
【図４３】図４３は、プラスミドｐＢＳ−ｐｒｏｂｅ３’が含むマウスゲノムＤＮＡのＮｈｅＩ断片の位置を模式的に示す。
【図４４】図４４は、Ａ３アデノシン受容体遺伝子周辺のマウスゲノム構造および、相同組換えによりマウスＡ３アデノシン受容体遺伝子がキメラＡ３アデノシン受容体をコードするＤＮＡに置換された時の同マウスゲノム構造を示す。
【図４５】図４５は、相同組換えによりマウスＡ３アデノシン受容体遺伝子がキメラＡ３アデノシン受容体をコードするＤＮＡに置換された時のマウスゲノム構造および、ＣｒｅリコンビナーゼによりｌｏｘＰ配列とｌｏｘＰ配列の間に存在する薬剤耐性マーカー（ＰＧＫプロモーター及びｈｐｒｔ遺伝子）が除去された時の同マウスゲノム構造を示す。
【図４６】図４６は、Ｃ５７ＢＬ／６Ｊマウス、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したヘテロ接合体マウスおよびホモ接合体マウスのゲノムＤＮＡに対する５’側プローブを使用したサザンハイブリダイゼーションの解析結果を示す。レーン１はＣ５７ＢＬ／６Ｊマウス、レーン２はヘテロ接合体マウス、レーン３はホモ接合体マウスの結果を示す。
【図４７】図４７は、Ｃ５７ＢＬ／６Ｊマウス、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したヘテロ接合体マウスおよびホモ接合体マウスのゲノムＤＮＡに対する３’側プローブを使用したサザンハイブリダイゼーションの解析結果を示す。レーン１はＣ５７ＢＬ／６Ｊマウス、レーン２はヘテロ接合体マウス、レーン３はホモ接合体マウスの結果を示す。
【図４８】図４８は、ＴＮＰ特異的ＩｇＥおよび抗ｃ−ｋｉｔ抗体を用いたＣ５７ＢＬ／６Ｊマウス由来ＢＭＭＣのＦＡＣＳによる解析を示す。ａ）は未染色のＢＭＭＣ、ｂ）は抗体によって染色したＢＭＭＣのＦＡＣＳ解析結果を示す。縦軸はＰＥ標識した抗ｃ−ｋｉｔ抗体により染色された細胞の蛍光強度、横軸はＦＩＴＣ標識したＴＮＰ特異的ＩｇＥにより染色された細胞の蛍光強度を示す。
【図４９】図４９は、ＴＮＰ特異的ＩｇＥおよび抗ｃ−ｋｉｔ抗体を用いた、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したホモ接合体マウス由来ＢＭＭＣのＦＡＣＳによる解析を示す。ａ）は未染色のＢＭＭＣ、ｂ）は抗体によって染色したＢＭＭＣのＦＡＣＳ解析結果を示す。縦軸はＰＥ標識した抗ｃ−ｋｉｔ抗体により染色された細胞の蛍光強度、横軸はＦＩＴＣ標識したＴＮＰ特異的ＩｇＥにより染色された細胞の蛍光強度を示す。
【図５０】図５０は、Ｃ５７ＢＬ／６Ｊマウス由来ＢＭＭＣの細胞内Ｃａ^２＋量の変動を示す。ａ）はカルシウム測定緩衝液のみを添加した結果、ｂ）は最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを添加した結果、ｃ）は最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを加える１０分前にヒトＡ３アデノシン受容体アンタゴニストＫＦ２６７７７を添加した結果、ｄ）は最終濃度１０ｎｇ／ｍＬのＴＮＰ−ＢＳＡを添加した結果を示す。縦軸はＣａ^２＋により励起されるＦｌｕｏ−３の波長５３０ｎｍの蛍光量を、薬物添加前の値を１．０とした相対値で示す。横軸は薬物添加後の時間を秒で示す。
【図５１】図５１は、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したマウス由来ＢＭＭＣの細胞内Ｃａ^２＋量の変動を示す。ａ）はカルシウム測定緩衝液のみを添加した結果、ｂ）は最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを添加した結果、ｃ）は最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを加える１０分前にヒトＡ３アデノシン受容体アンタゴニストＫＦ２６７７７を添加した結果、ｄ）は最終濃度１０ｎｇ／ｍＬのＴＮＰ−ＢＳＡを添加した結果を示す。縦軸はＣａ^２＋により励起されるＦｌｕｏ−３の波長５３０ｎｍの蛍光量を、薬物添加前の値を１．０とした相対値で示す。横軸は薬物添加後の時間を秒で示す。
【図５２】図５２は、Ｃ５７ＢＬ／６Ｊマウス由来ＢＭＭＣの脱顆粒を示す。縦軸は細胞外に放出されたβヘキサミニダーゼ活性（全活性を１００％とする）を示す。黒のバーは最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡ添加の場合、白のバーは非添加の場合を示す。
【図５３】図５３は、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したマウス由来ＢＭＭＣの脱顆粒を示す。縦軸は細胞外に放出されたβヘキサミニダーゼ活性（全活性を１００％とする）を示す。黒のバーは最終濃度１μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡ添加の場合、白のバーは非添加の場合を示す。
【図５４】図５４は、ＰＷＭ−ＳＣＭ非含培地での培養時における、Ｃ５７ＢＬ／６Ｊマウス由来ＢＭＭＣの細胞数変動を示す。縦軸は細胞数、横軸は培養開始からの時間（時間）を示す。▲は最終濃度１μｍｏｌ／ＬのヒトＡ３アデノシン受容体アンタゴニストＫＨ２６７７７添加の場合、●は非添加の場合を示す。
【図５５】図５５は、ＰＷＭ−ＳＣＭ非含培地での培養時における、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したマウス由来ＢＭＭＣの細胞数変動を示す。縦軸は細胞数、横軸は培養開始からの時間（時間）を示す。▲は最終濃度１μｍｏｌ／ＬのヒトＡ３アデノシン受容体アンタゴニストＫＨ２６７７７添加の場合、●は非添加の場合を示す。
【図５６】図５６は、ＴＵＮＥＬ染色により測定した、ＢＭＭＣ培地およびＰＷＭ−ＳＣＭ非含ＢＭＭＣ培地で４８時間培養したＣ５７ＢＬ／６Ｊマウス由来ＢＭＭＣにおける、細胞のアポトーシス率を示す。縦軸はアポトーシスを示す細胞の割合（％）を示す。白のバーはＢＭＭＣ培地、黒のバーはＰＷＭ−ＳＣＭ非含ＢＭＭＣ培地での結果を示す。
【図５７】図５７は、ＴＵＮＥＬ染色により測定した、ＢＭＭＣ培地およびＰＷＭ−ＳＣＭ非含ＢＭＭＣ培地で４８時間培養したＡ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したマウス由来ＢＭＭＣにおける、細胞のアポトーシス率を示す。縦軸はアポトーシスを示す細胞の割合（％）を示す。白のバーはＢＭＭＣ培地、黒のバーはＰＷＭ−ＳＣＭ非含ＢＭＭＣ培地での結果を示す。
【図５８】図５８は、Ｃ５７ＢＬ／６Ｊマウス由来膵臓細胞の細胞内Ｃａ^２＋量の変動を示す。ａ）はカルシウム測定緩衝液のみを添加した結果、ｂ）は最終濃度１００μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを添加した結果、ｃ）は最終濃度１００μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを加える１０分前に最終濃度１００μｍｏｌ／ＬのヒトＡ３アデノシン受容体アンタゴニストＫＦ２６７７７を添加した結果を示す。縦軸はＣａ^２＋により励起されるＦｌｕｏ−３の波長を薬物添加前の値を１．０とした相対値で示す。横軸は薬物添加後の時間を秒で示す。
【図５９】図５９は、Ａ３アデノシン受容体遺伝子をキメラＡ３アデノシン受容体をコードするＤＮＡに置換したホモ接合体マウス由来膵臓細胞の細胞内Ｃａ^２＋量の変動を示す。ａ）はカルシウム測定緩衝液のみを添加した結果、ｂ）は最終濃度１００μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを添加した結果、ｃ）は最終濃度１００μｍｏｌ／ＬのＡ３アデノシン受容体アゴニストＣｌ−ＩＢ−ＭＥＣＡを加える１０分前に最終濃度１００μｍｏｌ／ＬのヒトＡ３アデノシン受容体アンタゴニストＫＦ２６７７７を添加した結果を示す。縦軸はＣａ^２＋により励起されるＦｌｕｏ−３の波長を薬物添加前の値を１．０とした相対値で示す。横軸は薬物添加後の時間を秒で示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a chimera 7 in which all the intracellular regions or any part of the intracellular region of the human seven-transmembrane receptor are substituted with a part corresponding to the part of the ortholog of a non-human mammal of the receptor. The present invention relates to a transgenic non-human mammal expressing a transmembrane receptor. The present invention also relates to a method for producing the above-mentioned transgenic non-human mammal, a method for pharmacological evaluation of a test substance using the transgenic non-human mammal, and a DNA used for producing the transgenic non-human mammal.
[0002]
[Prior art]
In drug development, evaluation in human disease models using animals is indispensable, and it is required in drug development to verify the efficacy and toxicity of drug development candidate compounds using animal models. In this case, the problem of species difference that the main action and side effects are very different depending on the animal species often occurs, which is a major problem in drug development. For example, there are many known examples of drugs that show significant efficacy in mice, but have no effect on humans, and compounds that inhibit human receptors well, but do not show inhibitory effects on mouse or rat receptors. (See Non-Patent Document 1).
[0003]
As a target molecule of a drug, a receptor which is one of the molecules involved in cell signal transmission is the most common, and many drugs targeting a seven-transmembrane receptor are commercially available.
The seven-transmembrane receptor is a general term for receptors that are composed of an extracellular region, a transmembrane region, and an intracellular region, and that are present on the cell membrane surface and have seven transmembrane regions.
[0004]
It has been shown that the seven-transmembrane receptor couples to the G protein to transmit information into the cell, and the second intracellular domain loop and the third intracellular domain loop from the N-terminus are linked to the G protein. There is a report that it is important for interaction (see Non-Patent Document 2). However, a consensus sequence on the primary structure involved in the interaction between the receptor and the G protein has not been found, and the three-dimensional structure of the whole intracellular region rather than a specific amino acid sequence has been improved. Considered important. In addition, it is known that a palmitic acid binding site may be present in the C-terminal intracellular region, thereby affecting the interaction with the G protein (see Non-Patent Documents 3 and 4). Furthermore, the intracellular region of the seven-transmembrane receptor has a site that is phosphorylated by the action of a kinase, and is thought to be involved in the control of intracellular signal transmission (Non-patent document) 2, 5, and 6).
[0005]
It has been reported that a drug such as an agonist or an antagonist that selectively acts on a seven-transmembrane receptor binds to an extracellular region or a transmembrane region of a target receptor and exhibits pharmacological activity. Patent Documents 7, 8, 9, 10 and 11).
[0006]
In the seven-transmembrane receptor, different pharmacology is obtained when targeting a human seven-transmembrane receptor and when targeting a receptor (ortholog) corresponding to the human receptor of a non-human mammal. The effect may be shown. Specific examples include antagonists to the A3 adenosine receptor (see Patent Literature 1), antagonists to the 5-HT1B serotonin receptor (see Non-Patent Literatures 12, 13, and 14), and CP # 96345 and RP # 67580 for the NK1 tachykinin receptor. (See Non-Patent Documents 10, 11, 15, 16 and 17), agonists or antagonists for B2 bradykinin receptor (see Non-Patent Documents 18 and 19), agonists such as CGP-12177 for β3 adrenergic receptor (non-patent documents). Patent Document 20), agonists for calcitonin receptor (see Non-Patent Document 21), antagonies such as AF-DX116 and pirenzepine for M2 muscarinic acetylcholine receptor (See Non-Patent Documents 22 and 23), antagonists such as yohimbine and rauwolsine (see Non-Patent Documents 24 and 25) for α2A adrenergic receptor, CGP 20712A and atenolol (for atenolol) for β1 adrenergic receptor (See Non-Patent Documents 26 and 27), antagonists such as L-365260 and L-364718 for CCKB cholecystokinin receptor (see Non-Patent Documents 28 and 29), xenopsin (xenopsin) for the neurotensin receptor, and [Trp¹¹And analog compounds such as neurotensin (see Non-Patent Documents 30 and 31), and prostaglandin D2 analog compounds against DP prostanoid receptor (see Non-Patent Documents 32 and 33).
[0007]
As one means for solving such a species difference problem, humanization of a model animal, for example, a method of pharmacological evaluation of a compound by expressing a human gene in an animal has attracted attention.
Expression of human genes in animals can be performed using transgenic technology. For example, transgenic mice expressing human adrenergic β2 receptor (see Non-Patent Document 34) and transgenic mice expressing human bradykinin B2 receptor (see Non-Patent Document 35) have been reported. However, simply using the transgenic technique cannot control the expression of the orthologous gene corresponding to the introduced human gene. By introducing a human gene using a transgenic technique after disruption of the animal ortholog gene, a transgenic animal that does not express its own ortholog gene but expresses only the human gene can be produced. For example, mice that do not express mouse adrenaline β3 receptor but express only human adrenaline β3 receptor (see Non-Patent Document 20) have been reported. However, this technique cannot control the site of introduction of a human gene, and therefore cannot control the destruction of the gene existing at the site of introduction or the change in secondary phenotype such as peripheral gene activity due to the introduced gene.
[0008]
In contrast, a genetic modification called knock-in that introduces a human gene into the corresponding non-human mammal orthologous gene using a method combining positive selection and negative selection solves the above-mentioned disadvantages. . For example, a mouse in which a human apolipoprotein E gene is knocked in at the position of a mouse apolipoprotein E gene (see Non-Patent Document 36), a human cystic fibrosis transmembrane regulatory protein (Cystic fibrosis transmembrane \ conductance \ regulator, hereinafter abbreviated as Cftr). ) A mouse in which the gene is knocked in at the position of the mouse Cftr gene (see Non-Patent Document 37), a mouse in which the human leukemia gene Cbfb-MyH11 is knocked in at the position of the Cbfb gene in the mouse (see Non-Patent Document 38), Production of a mouse in which the gene is knocked in at the position of the mouse KDR gene (see Patent Document 2) has been reported.
[0009]
However, in such genetically modified animals, since the drug resistance gene used for positive selection remains near the target gene, expression of the human gene knocked-in by the promoter or enhancer of the drug resistance gene is required during phenotypic analysis. Need to be taken into account. As a method for keeping a portion other than the human gene to be replaced as little as possible, a double replacement method in which homologous recombination is performed twice (see Non-Patent Documents 39, 40 and 41), and a drug resistance gene portion used for positive selection are used. A Cre / LoxP method for deletion (see Non-Patent Document 42) has been developed, and a gene-substituted mouse in which a mouse α-lactalbumin gene is replaced with a human α-lactalbumin gene (see Non-Patent Documents 43 and 44). Production of a gene-replaced mouse in which a mouse prion gene is replaced with a human prion gene (see Non-Patent Document 45) has been reported.
[0010]
In such a transgenic animal, the human gene product is expressed in place of the non-human mammal's factor corresponding to that gene product, but the introduced human gene product is sufficient in place of the non-human mammal's gene product. The proper function depends on the individual properties of the replaced gene. For non-human mammals, the sequence of the human gene is merely a mutated sequence and may not be functionally acceptable. When the physiological function of the replaced human gene product is not sufficient for the host non-human mammal due to species differences between the human gene and the corresponding non-human mammal gene, the target gene is destroyed. The same expression system appears. In particular, replacement of a molecule that exerts a function by intracellular signal transmission through complex protein-protein interaction, such as a seven-transmembrane receptor, impairs the function of the replaced gene product as an expression system. Probably a big problem.
[0011]
[Patent Document 1]
WO 98/15555 pamphlet
[0012]
[Patent Document 2]
WO 99/18191 pamphlet
[0013]
[Non-patent document 1]
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[0014]
[Non-patent document 2]
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[0015]
[Non-Patent Document 3]
The Journal of Biological Chemistry, (USA), 1989, Vol. 264, No. 13, p. 7564-7569
[0016]
[Non-patent document 4]
FEBS Letters, (Netherlands), 1988, Vol. 230, No. 1-2, p. 1-5
[0017]
[Non-Patent Document 5]
The Faseb Journal, (USA), 1990, Vol. 4, No. 11, p. 2881-2889
[0018]
[Non-Patent Document 6]
The Faseb Journal, (USA), 1992, Vol. 6, No. 6, p. 2323-2331
[0019]
[Non-Patent Document 7]
Trends in Pharmaceutical Sciences, (UK), 1991, Vol. 12, No. 5, p. 199-203
[0020]
[Non-Patent Document 8]
Nature, (UK), 1993, 362, 6418, p. 350-353
[0021]
[Non-Patent Document 9]
The Faseb Journal, (USA), 1989, Vol. 3, No. 7, p. 1825-1832
[0022]
[Non-Patent Document 10]
The Journal of Biological Chemistry, (USA), 1992, Vol. 267, No. 36, p. 25668-25671
[0023]
[Non-Patent Document 11]
The Journal of Biological Chemistry (USA), 1993, Vol. 268, No. 4, p. 2319-2323
[0024]
[Non-Patent Document 12]
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[0025]
[Non-patent document 13]
Nature, (UK), 1992, 360, 6400, p. 161-164
[0026]
[Non-patent document 14]
Journal of Neurochemistry, (UK), 1993, Vol. 60, No. 1, p. 380-383
[0027]
[Non-Patent Document 15]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 262
[0028]
[Non-Patent Document 16]
Science, (USA), 1991, Vol. 251, No. 4992, p. 435-437
[0029]
[Non-Patent Document 17]
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[0030]
[Non-Patent Document 18]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 68-70
[0031]
[Non-Patent Document 19]
Biochemical and Biophysical Research Communications, (USA), 1992, Vol. 187, No. 3, p. 1306-1311
[0032]
[Non-Patent Document 20]
Diabetes, (USA), 1998, Vol. 47, No. 9, p. 1464-1468
[0033]
[Non-Patent Document 21]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 74-76
[0034]
[Non-Patent Document 22]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 11
[0035]
[Non-Patent Document 23]
The Journal of Biological Chemistry (USA), 1991, Vol. 266, No. 26, p. 17382-17387
[0036]
[Non-Patent Document 24]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 42-44
[0037]
[Non-Patent Document 25]
Molecular @ Pharmacology, (USA), 1992, Vol. 42, No. 1, p. 16-27
[0038]
[Non-Patent Document 26]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 47-49
[0039]
[Non-Patent Document 27]
Molecular @ Pharmacology, (USA), 1980, Vol. 17, No. 1, p. 1-7
[0040]
[Non-Patent Document 28]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 92
[0041]
[Non-Patent Document 29]
Nature, (UK), 1993, 362, 6418, p. 348-350
[0042]
[Non-Patent Document 30]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 199-201
[0043]
[Non-Patent Document 31]
British Journal of Pharmacology, (UK), 1980, Vol. 69, No. 4, p. 689-692
[0044]
[Non-patent document 32]
Edited by Steve Watson and Steve Arkinstall, "The G-Protein Linked Receptor, The Facts Book (The Facts, Book of Facts, UK), The G-Protein Linked Receptor. Press (Academic @ Press), 1994, p. 241-251
[0045]
[Non-Patent Document 33]
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[0046]
[Non-Patent Document 34]
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[0047]
[Non-Patent Document 35]
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[0048]
[Non-Patent Document 36]
The Journal of Biological Chemistry (USA), 1997, Vol. 272, No. 29, p. 17972-17980
[0049]
[Non-Patent Document 37]
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[0050]
[Non-Patent Document 38]
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[0051]
[Non-Patent Document 39]
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[0052]
[Non-Patent Document 40]
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[0053]
[Non-Patent Document 41]
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[0054]
[Non-Patent Document 42]
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[0055]
[Non-Patent Document 43]
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[0056]
[Non-Patent Document 44]
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[0057]
[Non-Patent Document 45]
Biochemical and Biophysical Research Communications, (USA), 1996, Vol. 222, No. 3, p. 742-747
[0058]
[Problems to be solved by the invention]
Non-human that can perform pharmacological evaluation of substances that have different reactivities between human seven-transmembrane receptor and orthologs of the receptor of non-human animals and that specifically act on human seven-transmembrane receptor There is a need for mammals.
[0059]
[Means for Solving the Problems]
The present invention provides the following (1) to (37).
(1) A DNA encoding a chimeric seven-transmembrane receptor in which all intracellular regions of a human seven-transmembrane receptor are replaced with corresponding portions of a non-human mammal ortholog of the receptor is contained in the genome. And a transgenic non-human mammal expressing said chimeric seven transmembrane receptor.
(2) A receptor in which any part of the intracellular region of the human seven-transmembrane receptor is replaced with a corresponding part of the ortholog of a non-human mammal of the receptor, and A transgenic non-human having in its genome a DNA encoding a chimeric seven-transmembrane receptor that exhibits a cellular response to an agonist acting on the human receptor, and expressing the chimeric seven-transmembrane receptor Mammals.
(3) The transgenic non-human mammal according to (2), wherein any part of the intracellular region is a portion containing at least one of the following (a) to (d).
(A) The second intracellular region from the N-terminus
(B) Third intracellular region from the N-terminus
(C) Palmitic acid binding site in C-terminal intracellular region
(D) Phosphorylation site by kinase in intracellular region
(4) Both homologous chromosomes in which the DNA encoding the chimeric seven-transmembrane receptor has been inserted in place of the region encoding the seven-transmembrane receptor of the gene of its own ortholog have been inserted into the genome. The transgenic non-human mammal according to any one of (1) to (3), wherein
(5) The transgenic non-transgenic cell according to any one of (1) to (4), wherein the seven-transmembrane receptor is the receptor described in any one of the following (a) to (l): Human mammal.
(A) A3 adenosine receptor
(B) 5-HT1B serotonin receptor
(C) NK1 tachykinin receptor
(D) B2 bradykinin receptor
(E) β3 adrenergic receptor
(F) Calcitonin receptor
(G) M2 muscarinic acetylcholine receptor
(H) α2A adrenergic receptor
(I) β1 adrenergic receptor
(J) CCKB cholecystokinin receptor
(K) neurotensin receptor
(L) DP prostanoid receptor
[0060]
(6) The non-human mammal is a non-human mammal selected from the group consisting of mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, goats, cows and monkeys, (1) to ( The transgenic non-human mammal according to any one of 5).
(7) A transgenic mouse which has a DNA encoding the chimeric A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 32 in its genome and expresses the chimeric A3 adenosine receptor.
(8) Both homologs in which the DNA encoding the chimeric A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 32 has been inserted in place of the region encoding the A3 adenosine receptor of the own A3 adenosine receptor gene The transgenic mouse according to (7), which has a chromosome in the genome.
(9) The method for producing a transgenic non-human mammal according to any one of (1) to (3), comprising the following steps (a) to (d).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing an expression vector containing DNA encoding the body
(B) introducing the expression vector into a fertilized egg of a non-human mammal
(C) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal
(D) a step of breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor;
(10) The method for producing a transgenic non-human mammal according to any one of (1) to (3), comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing an expression vector containing DNA encoding the body
(B) a step of introducing the expression vector into a non-human mammal embryonic stem cell
(C) a step of introducing the embryonic stem cells into a fertilized egg of a non-human mammal
(D) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal
(E) breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor
[0061]
(11) The method for producing a transgenic non-human mammal according to any one of (1) to (3), comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing an expression vector containing DNA encoding the body
(B) a step of incorporating the expression vector into sperm
(C) fertilizing an unfertilized egg of a non-human mammal with the sperm
(D) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal
(E) breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor
(12) The method for producing a transgenic non-human mammal according to any one of (1) to (3), comprising the following steps (a) to (f).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing a retroviral vector containing a body-encoding DNA
(B) a step of producing a recombinant retrovirus using the retrovirus vector
(C) a step of infecting an unfertilized egg of a non-human mammal with the recombinant retrovirus
(D) a step of in vitro fertilizing the unfertilized egg
(E) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal
(F) breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor;
(13) The method for producing a transgenic non-human mammal according to (4), comprising the following steps (a) to (h).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing a target vector containing DNA encoding the body
(B) a step of introducing the target vector into embryonic stem cells of a non-human mammal
(C) selecting embryonic stem cells having a chromosome inserted by replacing the DNA encoding the chimeric seven-transmembrane receptor with the region encoding the seven-transmembrane receptor of the gene of its own ortholog Process
(D) a step of introducing the selected embryonic stem cells into a fertilized egg
(E) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal
(F) breeding the non-human mammal and selecting a germline chimera from the offspring
(G) the germ-line chimera is crossed, and the DNA encoding the chimeric seven-transmembrane receptor is replaced with a region encoding the seven-transmembrane receptor of the gene of its own ortholog from the offspring born. For selecting an individual having a chromosome inserted by insertion
(H) The individuals obtained in (g) are bred to each other, and the DNA encoding the chimeric seven-transmembrane receptor on both chromosomes is transduced from the offspring of the offspring by the seven transmembrane times of the gene of its own ortholog. For selecting a homozygote inserted in place of the region encoding the type receptor
(14) The method for producing a transgenic non-human mammal according to any one of (1) to (3), comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing an expression vector containing DNA encoding the body
(B) Step of introducing the constructed expression vector into cells of a non-human mammal
(C) a step of introducing the nucleus of the cell obtained in (b) into a non-fertilized egg of a denucleated non-human mammal to start development
(D) transplanting the unfertilized egg that has started to develop into the oviduct or uterus of a pseudopregnant female non-human mammal
(E) breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor
(15) The method for producing a transgenic non-human mammal according to (4), comprising the following steps (a) to (g).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor For constructing a target vector containing DNA encoding the body
(B) Step of introducing the constructed target vector into a non-human mammal cell
(C) a step of selecting a cell having a chromosome inserted by replacing the DNA encoding the chimeric seven-transmembrane receptor with the region encoding the seven-transmembrane receptor of its own ortholog gene
(D) introducing the nucleus of the selected cell into an unfertilized egg of a non-human mammal that has undergone enucleation and initiating development
(E) transplanting the unfertilized egg that has started development into the oviduct or uterus of a pseudopregnant female non-human mammal
(F) breeding the non-human mammal, and replacing the DNA encoding the chimeric seven-transmembrane receptor with the region encoding the seven-transmembrane receptor of the gene of its own ortholog from the offspring born Selecting an individual having the chromosome inserted in the genome
(G) The individuals obtained in (f) are bred to each other, and the DNA encoding the chimeric seven-transmembrane receptor is expressed on both chromosomes from the offspring of the offspring. A step of selecting a homozygote inserted in place of the region encoding the penetrating receptor
[0062]
(16) The cell derived from the transgenic non-human mammal according to any one of (1) to (8).
(17) The cell according to (16), wherein the cell is an egg, sperm or embryonic stem cell.
(18) A method for producing a transgenic non-human mammal, further comprising introducing a foreign gene into the cell according to (16) or (17).
(19) A transgenic non-human mammal produced by the method according to (18).
(20) A method for producing a knockout non-human mammal, comprising introducing a foreign gene into the cell according to (16) or (17) to delete a specific gene.
[0063]
(21) A knockout non-human mammal produced by the method according to (20).
(22) A chimeric seven-transmembrane receptor, wherein the transgenic non-human mammal according to any one of (1) to (8) is crossed with a disease model animal of the same or different strain. A method for producing a disease state model non-human mammal that expresses
(23) A pathological model non-human mammal expressing the chimeric seven-transmembrane receptor produced by the method according to (22).
(24) A disease model that expresses a chimeric seven-transmembrane receptor, wherein the disease is induced in the transgenic non-human mammal according to any one of (1) to (8). A method for producing a human mammal.
(25) A pathological model non-human mammal expressing the chimeric seven transmembrane receptor produced by the method according to (24).
[0064]
(26) A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) and (b):
(A) a step of administering a test substance to the non-human mammal according to any one of (1) to (8), (19), (21), (23) and (25) and examining a pharmacological effect;
(B) examining the pharmacological effect of the test substance on the non-human mammal by comparing the pharmacological effect with the test substance not administered to the non-human mammal;
(27) A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) and (b).
(A) a step of administering a test substance to the cells according to (16) or (17) and examining the pharmacological effect;
(B) examining the pharmacological effect of the test substance on the cell by comparing the pharmacological effect with the test substance not administered to the cell;
(28) A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) to (c).
(A) a step of inducing differentiation of the embryonic stem cells according to (17) and obtaining the differentiated cells (b) a step of administering a test substance to the differentiated cells obtained in (a) and examining the pharmacological effect
(C) a step of examining the pharmacological effect of the test substance given to the cells by comparing the pharmacological effect obtained when no test substance is administered to the differentiated cells obtained in (a)
(29) The test substance is an agonist whose human agonist activity on a seven-transmembrane receptor is at least 10 times that of a non-human mammal ortholog of the receptor or a human seven-transmembrane receptor (26) The method for evaluating a pharmacological effect according to any one of (26) to (28), wherein the antagonistic activity of the receptor is at least 10 times the antagonistic activity of the receptor to an ortholog of a non-human mammal.
(30) The evaluation of the pharmacological effect according to any one of (26) to (28), wherein the test substance is an agonist or antagonist of the receptor according to any one of the following (a) to (l): Method.
(A) A3 adenosine receptor
(B) 5-HT1B serotonin receptor
(C) NK1 tachykinin receptor
(D) B2 bradykinin receptor
(E) β3 adrenergic receptor
(F) Calcitonin receptor
(G) M2 muscarinic acetylcholine receptor
(H) α2A adrenergic receptor
(I) β1 adrenergic receptor
(J) CCKB cholecystokinin receptor
(K) neurotensin receptor
(L) DP prostanoid receptor
[0065]
(31) a DNA encoding a chimeric A3 adenosine receptor having the amino acid sequence represented by SEQ ID NO: 33;
(32) A DNA having the base sequence of SEQ ID NO: 32.
(33) a DNA encoding a mouse A3 adenosine receptor having the amino acid sequence of SEQ ID NO: 4;
(34) a DNA having the nucleotide sequence of SEQ ID NO: 3;
(35) a DNA having the base sequence of SEQ ID NO: 11;
(36) A target vector comprising the DNA according to (31) or (32).
(37) DNA consisting of a sequence of 500 or more consecutive bases of the 1st to 637th sequence of the base sequence shown in SEQ ID NO: 11 and 500 consecutive bases of the 3735th to 5270th base of the base sequence shown in SEQ ID NO: 11 The target vector according to (36), which comprises a DNA having the above sequence.
[0066]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the seven-transmembrane receptor includes any receptor that has seven transmembrane regions and can transmit information into cells in combination with G protein. An agonist activity or an antagonist activity for a human seven-transmembrane receptor is at least 10 times, preferably 20 times or more, more preferably 50 times or more, and still more preferably, an activity of the receptor for orthologs of non-human mammals. Is preferably 100-fold or more, particularly preferably 1000-fold or more, and is preferably a seven-transmembrane receptor having a human selective agonist or antagonist. Examples of the seven-transmembrane receptor in which a human selective agonist or antagonist is present include A3 adenosine receptor, 5-HT1B serotonin receptor, NK1 tachykinin receptor, B2 bradykinin receptor, β3 adrenergic receptor, calcitonin receptor And seven-transmembrane receptors such as M2 muscarinic acetylcholine receptor, α2A adrenergic receptor, β1 adrenergic receptor, CCKB cholecystokinin receptor, neurotensin receptor and DP prostanoid receptor.
[0067]
The seven-transmembrane receptor expressed by the transgenic non-human mammal of the present invention can be obtained by converting all of the intracellular region of the human seven-transmembrane receptor or any part of the intracellular region to a non-human receptor of the receptor. A receptor (hereinafter, also referred to as a chimeric seven-transmembrane receptor) substituted with a corresponding portion of a mammalian ortholog, which is a non-human mammal cell capable of reacting with an agonist acting on the human receptor. Chimeric seven transmembrane receptors that respond are preferred.
[0068]
In a non-human mammal cell, chimeric seven transmembrane receptors that show a cellular response to an agonist acting on the human receptor are all intracellular regions or intracellular regions of human seven transmembrane receptors. A DNA encoding a chimeric seven-transmembrane receptor in which an arbitrary portion of the chimera is replaced with a corresponding portion of a non-human mammal ortholog is prepared, and an expression vector containing the DNA is introduced into cells. Expressing the transmembrane receptor in a cell of a non-human mammal, measuring a cell response when an agonist acting on the human receptor is acted on, and selecting a cell showing a cell response to obtain the transmembrane receptor. Can be. In the seven-transmembrane receptor, an extracellular domain or a transmembrane domain binds to an agonist, and an intracellular domain couples to a G protein to transmit intracellular information. Therefore, a chimeric seven-transmembrane receptor obtained by substituting all intracellular regions of a human seven-transmembrane receptor with an intracellular region of a non-human mammal, It is thought to show a cellular response to agonists acting on. In the seven-transmembrane receptor, a part involved in intracellular signal transmission includes a second or third N-terminal intracellular region, a palmitic acid binding site in a C-terminal intracellular region, or an intracellular region. And phosphorylation sites by kinases in the medium. Therefore, any part of the intracellular region to be substituted in the chimeric seven-transmembrane receptor includes (1) the second intracellular region from the N-terminal, (2) the third intracellular region from the N-terminal, 3) a palmitic acid binding site in the intracellular region at the C-terminus; and (4) a portion containing at least one of phosphorylation sites by a kinase in the intracellular region. In a non-human mammal cell, a chimeric seven-transmembrane receptor showing a cellular response to an agonist acting on a human seven-transmembrane receptor is a human A3 adenosine having an amino acid sequence represented by SEQ ID NO: 33. Chimeric A3 adenosine receptors in which all the intracellular regions of the receptor have been replaced by the corresponding intracellular regions of the mouse A3 adenosine receptor.
[0069]
The transgenic non-human mammal of the present invention, which expresses a chimeric seven-transmembrane receptor showing a cellular response to an agonist acting on a human seven-transmembrane receptor, It can be used to evaluate drugs that work. Non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, sheep, pigs, goats, cows or monkeys, and preferably mice, rats, rabbits, monkeys and the like.
[0070]
Hereinafter, the method for producing and using the transgenic non-human mammal of the present invention will be described in detail.
The transgenic non-human mammal expressing the chimeric seven-transmembrane receptor according to the present invention includes 1) a method for producing a transgenic animal for introducing a gene of interest into an animal individual, and 2) a target gene and a target gene. It can be produced by using a method for producing a gene-substituted animal in which a transgene is replaced, and 3) a method for producing a cloned individual using a nucleus of a cell subjected to a desired genetic modification. Each of these will be specifically described below.
[0071]
1. Production of the transgenic non-human mammal of the present invention using the method for producing a transgenic animal for introducing a gene of interest into an animal individual
(1) Obtaining DNA encoding human seven-transmembrane receptor
Examples of the DNA encoding the human seven-transmembrane receptor include cDNA and genomic DNA encoding the human seven-transmembrane receptor. Genomic DNA may contain introns. The codons of the coding region are not limited to codons in cDNA and genomic DNA, but may be any combination of codons as long as they code for the amino acid sequence of the human seven-transmembrane receptor. An example of a DNA encoding a human seven-transmembrane receptor is a human A3 adenosine receptor cDNA consisting of the nucleotide sequence shown in SEQ ID NO: 14.
[0072]
The base sequence database is searched for the sequence of the desired human seven-transmembrane receptor cDNA or the genomic sequence of the receptor gene, and the base sequence information is obtained. Examples of the human seven-transmembrane receptor cDNA or the genomic sequence of the receptor gene that can be obtained from the nucleotide sequence database include, for example, the genomic sequence of the human A3 adenosine receptor gene (GenBank accession numbers: L77729 and L77730). Can be.
[0073]
Based on the obtained base sequence of the DNA encoding the human seven-transmembrane receptor, a cDNA sequence or a genomic sequence region containing a region encoding the receptor is appropriately selected, and 5 ′ of this region is selected. A DNA containing a sequence of 20 to 40 bp at the 3 ′ end and a DNA containing a sequence complementary to the sequence of 20 to 40 bp of the 3 ′ end of this region at the 3 ′ end are prepared. Using these DNAs as primers, a cDNA encoding the human receptor is isolated by PCR using human cDNA as a template, and a genomic DNA encoding the human receptor is isolated by PCR using human genomic DNA as a template. can do.
[0074]
Human cDNA can be made from RNA prepared from human tissues or cells. As a method for preparing total RNA from tissues or cells, guanidine thiocyanate-cesium trifluoroacetate method (Methods @ in @ Enzymology,154, 3,） 1987), guanidine acid thiocyanate-phenol-chloroform method (Analytical Biochemistry,)162, {156, 1987; {Experimental Medicine,}9, $ 1937, $ 1991). Alternatively, kits such as Absolutely RNA RT-PCR miniprep kit (Absolutely RNA RT-PCR Miniprep Kit, manufactured by Stratagene) and RN Easy Mini Kit (RNeasy Minii Kit, manufactured by Qiagen) may be used. Can be prepared. Poly (A) from total RNA⁺As a method for preparing mRNA as RNA, oligo (dT) -immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001] (hereinafter referred to as Molecular Cloning) And the like. Furthermore, human tissues can be prepared by using kits such as FastTrack 2.0 mRNA isolation kit (FastTrack 2.0 mRNA Isolation Kit, manufactured by Invitrogen) and QuickPrep mRNA purification kit (QuickPrep mRNA Purification Kit, manufactured by Amersham Bioscience). Alternatively, mRNA can be prepared from cells. Methods for synthesizing cDNA from total RNA or mRNA include Molecular Cloning, Third Edition, Current Protocols in Molecular Biology, John Wiley & Sons, 1987-2001 (hereinafter abbreviated as Current Protocols in Molecular Biology). Or a commercially available kit, for example, a superscript first-strand synthesis system for RT-PCR (SUPERSCRIPT @ First-StrandSynthesis @ System @ for-RT-PCR, manufactured by Invitrogen), a Prostar first-strand RT-PCR kit (ProSTAR @ First) Strand RT-PCR Kit, manufactured by Stratagene), PCR-Select c NA subtraction Kit (PCR-Select cDNA Subtraction Kit, Clontech) a method using thereof. CDNAs of various human tissues commercially available from Clontech and the like can also be used. An example of the DNA encoding the human seven-transmembrane receptor thus obtained is a DNA having the nucleotide sequence shown in SEQ ID NO: 14.
[0075]
Human genomic DNA can be prepared from human tissues or cells by the method described in Molecular Cloning, Third Edition. Alternatively, it can be prepared by using a kit such as an easy DNA kit (Easy-DNA @ Kit, manufactured by Invitrogen) or a DNA extraction kit (DNA @ Extraction @ Kit, manufactured by Stratagene). Human genomic DNA commercially available from Clontech, etc. can also be used.
[0076]
(2) Obtaining DNA encoding orthologs of non-human mammals
Examples of the DNA encoding the ortholog of the human seven-transmembrane receptor of the non-human mammal producing the transgenic animal include cDNA and genomic DNA encoding the ortholog of the non-human mammal. Genomic DNA may contain introns. In addition, the codons of the coding region are not limited to codons in cDNA and genomic DNA, but may be any combination of codons as long as they code for the amino acid sequence of the ortholog. Examples of the DNA encoding the ortholog of a non-human mammal include a mouse A3 adenosine receptor cDNA consisting of the nucleotide sequence shown in SEQ ID NO: 3. In the same manner as in (1), cDNA or genomic DNA encoding the ortholog of the non-human mammal can be isolated from the information on the nucleotide sequence of the cDNA or genomic DNA encoding the ortholog of the non-human mammal. If the sequence information cannot be obtained from the base sequence database, a cDNA library or a genomic DNA library of the non-human mammal is prepared, and a genomic DNA clone containing the gene is isolated by plaque hybridization or colony hybridization. According to the method, cDNA or genomic DNA encoding an ortholog of a non-human mammal can be isolated. When information on a partial sequence can be obtained from a base sequence database, a probe obtained by labeling an isolated partial fragment by PCR using primers based on the sequence can be used as the probe. When partial sequence information cannot be obtained from the database, a DNA obtained by labeling the DNA encoding the human seven-transmembrane receptor obtained in (1) can be used as a probe.
[0077]
Preparation of a library, plaque hybridization, colony hybridization, and labeling and preparation of a probe can be performed by methods described in Molecular Cloning, 3rd edition, Current Protocols in Molecular Biology, and the like.
[0078]
(3) Preparation of DNA encoding chimeric seven-transmembrane receptor
The amino acid sequence of human seven-transmembrane receptor and the domain structure of orthologs of non-human mammals and the domain structure of intracellular regions of the receptor are compared, and the amino acid sequence of chimeric seven-transmembrane receptor to be designed is designed. I do. The DNA fragment encoding the chimeric receptor is isolated as follows. First, as the nucleotide sequence encoding the designed chimeric receptor, a region obtained by substituting the amino acid sequence of the human receptor ortholog with the human receptor in the human nucleotide sequence of the cDNA of the receptor is used. The base sequence is designed by substituting the portion to be replaced with the base sequence of the cDNA of the ortholog of a non-human mammal. In the nucleotide sequence encoding this chimeric receptor, a sequence containing a restriction enzyme site present in or near a region where the nucleotide sequence of the human receptor cDNA is replaced by the nucleotide sequence of the non-human mammal ortholog cDNA. Select If there is no appropriate restriction enzyme site, the base sequence is redesigned so that an appropriate restriction enzyme site can be formed by selecting codons without changing the amino acid sequence of the chimeric receptor. A primer having the selected base sequence or a base sequence complementary to the selected base sequence is designed and prepared based on the base sequence of DNA encoding a human receptor or DNA encoding a non-human mammal ortholog as a template. I do. Using the primers thus prepared, a fragment of the DNA encoding the chimeric receptor is amplified by PCR using DNA encoding a human receptor or DNA encoding a non-human mammalian ortholog as a template. By connecting the obtained DNA fragments using a restriction enzyme site, a DNA encoding the chimeric receptor can be obtained.
[0079]
The DNA encoding the chimeric seven-transmembrane receptor obtained as described above encodes a chimeric A3 adenosine receptor in which the intracellular region of human A3 adenosine receptor is replaced by the intracellular region of mouse A3 adenosine receptor. And a DNA having the base sequence shown in SEQ ID NO: 32.
An expression vector containing the DNA is prepared according to the method described in (4) below, and the chimeric seven-transmembrane receptor is expressed in the cells by introducing the vector into a non-human mammal cell. An agonist acting on the human seven-transmembrane receptor is added to the cells, and the cell response according to the human seven-transmembrane receptor is measured. It can be confirmed that human mammalian cells show a cellular response to an agonist acting on a human seven-transmembrane receptor. The cellular response includes intracellular Ca²⁺, A decrease in intracellular cAMP level, an increase in intracellular cAMP level, and the like.
[0080]
(4) Preparation of transgene containing DNA encoding chimeric seven-transmembrane receptor
When introducing the DNA encoding the chimeric seven-transmembrane receptor obtained in (3) into a non-human mammal, an expression vector obtained by ligating the DNA downstream of an appropriate promoter is used as a transgene. Is preferred.
[0081]
As the promoter used for the expression vector, any promoter can be used as long as it can initiate transcription in cells of a non-human mammal into which a DNA encoding a chimeric seven-transmembrane receptor is introduced. For example, promoters of genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, simian virus, retrovirus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) And genes derived from birds (such as chickens) [eg, albumin, insulin II, erythropoietin, endothelin, osteocalcin, muscle creatine kinase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, atrial natriuretic Factor, dopamine β-hydroxylase, endothelial receptor tyrosine kinase (Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallo Thionein I and IIA, metalloproteinase 1 tissue inhibitor (TIMP1), MHC class I antigen (H-2L), smooth muscle α-actin, polypeptide chain elongation factor 1α (EF-1α), β-actin, α and β myosin heavy chain , Myosin light chains 1 and 2, myelin basic protein, serum amyloid P component, glycolytic enzymes such as myoglobin, renin, phosphoglycerate kinase (PGK), and genes such as heat shock proteins], enhancers of the above genes A promoter fused with a sequence or a promoter sequence [for example, an SRα promoter consisting of a promoter of an early gene of simian virus 40 (SV40) and a part of a long terminal repeat of human T-cell leukemia virus 1; CAG promoter comprising an early (IE) gene enhancer and a chicken β-actin promoter], and the like. It is preferable to use the promoter of the objective seven-transmembrane receptor gene, It is particularly preferred to use a promoter. By using a promoter of a gene showing tissue-specific expression, it can be expressed in a specific tissue of a transgenic non-human mammal into which DNA encoding the receptor has been introduced. In addition, it is also possible to control the expression timing, expression amount, etc. of the introduced foreign gene in the following manner in combination with the Cre-loxP system.
[0082]
A DNA encoding a chimeric seven transmembrane receptor in an expression vector is inserted between two loxP sequences, and a transgenic non-human mammal is prepared using the expression vector by the method described later in (5). . By expressing Cre recombinase in the transgenic non-human mammal using an adenovirus vector, a chimeric seven-transmembrane receptor specifically at the site or stage of infection with the adenovirus for Cre recombinase expression And the expression of the receptor can be suppressed. In addition, in addition to the expression vector of the chimeric seven-transmembrane receptor by the method described later in (5), for example, a Cre recombinase expression vector using a promoter showing tissue-specific expression is introduced to transgenic non-human mammals. By preparing an animal, a transgenic non-human mammal in which the expression of the chimeric seven-transmembrane receptor is suppressed can be prepared only in a tissue in which Cre recombinase is expressed.
[0083]
The expression vector preferably has a polyadenylation signal required for polyadenylation at the 3 'end of mRNA, downstream of the DNA encoding the chimeric seven-transmembrane receptor. Examples of the polyadenylation signal include polyadenylation signals contained in the above viruses, various mammals and birds, such as SV40 late or early genes, rabbit β globin gene, bovine growth hormone gene, and the like. Examples include polyadenylation signals of genes such as a seven-transmembrane receptor gene. In order to further express the target gene at a higher level, a part of the splicing signal, enhancer region, and intron of each gene may be linked 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region. Good. Hereinafter, a DNA construct comprising a promoter, DNA encoding a chimeric seven-transmembrane receptor and a polyadenylation signal is referred to as a chimeric seven-transmembrane receptor expression unit.
[0084]
Furthermore, Escherichia coli (Escherichia coli,Less thanE.coliAbbreviated as) to prepare the vector,E.coliReplication start point andE.coliRequires a drug resistance gene containing an endogenous promoter to be a selectable marker for the transformant. As drug resistance genes,E.coliDerived genes such as ampicillin resistance gene (β-lactamase gene), tetracycline resistance gene and kanamycin resistance gene.E.coliExamples of the replication start point that can be replicated in the above include a replication start point of pBR322 and a replication start point of colE1.
[0085]
When a retrovirus is used for gene transfer, a recombinant retrovirus vector is prepared by inserting a chimeric seven-transmembrane receptor expression unit into the retrovirus vector. By introducing the recombinant retrovirus vector into an appropriate packaging cell, a recombinant retrovirus can be produced. By infecting a fertilized egg or the like with the obtained recombinant retrovirus, a recombinant retrovirus vector can be introduced. For the introduction of the recombinant retrovirus vector into the packaging cells, known gene transfer methods (for example, calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc.) ) Can be used.
[0086]
(5) Introduction of Expression Vector into Fertilized Eggs and Selection of Transgenic Non-Human Animal The transgenic non-human mammal of the present invention contains the DNA encoding the chimeric seven-transmembrane receptor prepared in (4). The gene can be prepared by introducing a gene containing the gene into a target non-human mammal. Specifically, an individual obtained by introducing the gene into a fertilized egg, embryonic stem cell (hereinafter abbreviated as ES cell), sperm or unfertilized egg of a target non-human mammal, and generating using these cells From the above, the transgenic non-human mammal of the present invention is produced by selecting an individual in which the gene containing the DNA encoding the chimeric seven-transmembrane receptor has been integrated on the chromosome of all cells including germ cells. it can. The presence of the transgene in the created transgenic non-human mammal germ cell can be confirmed by the fact that the offspring of the produced animal have the transgene in all of its germ cells and somatic cells. The selection of the individual is performed by introducing the genomic DNA on a genomic DNA prepared from a tissue constituting the individual, for example, a blood tissue, an epithelial tissue, a connective tissue, a cartilage tissue, a bone tissue, a muscular tissue, an oral tissue or a part of a skeletal tissue. This is done by confirming the presence of the gene at the DNA level. Since the individual selected in this way is usually a heterozygote having the transgene on one of the homologous chromosomes, by mating the heterozygous individuals, the transgene has the transgene on both homologous chromosomes from among the progeny A homozygous animal can be obtained. By mating male and female animals of this homozygote, all offspring become homozygotes stably retaining the gene, so that the transgenic non-human mammal of the present invention can be propagated in a normal breeding environment. Can be replaced.
[0087]
(I) Production of a transgenic non-human mammal by introducing a gene into a fertilized egg
Production of transgenic non-human mammals by gene transfer into fertilized eggs can be performed by the following procedures: Manipulating the Mouse Mouse Embryo A Laboratory Laboratory Manual, Second Edition, Cold Spring Laboratories, and the following publications are available. ), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press, (1993), Biomanual Series 8 Gene Targeting, Generating Mutant Mice Using ES Cells, Yodosha, Trans. Ni Mouse how to make, can be carried out by the method described in Kodansha (1987), or the like.
[0088]
A fertilized egg into which a gene containing a DNA encoding a chimeric seven-transmembrane receptor is introduced can be obtained by mating a male non-human mammal with a female non-human mammal of the same species. For example, by mating a male mouse with a female mouse, preferably by mating an inbred male mouse with a female mouse, the fertilized egg of the mouse can be mated with a male rat and a female rat, preferably by Wistar. By mating a male rat of the strain and a female rat of the Wistar strain, a fertilized egg of the rat is obtained. Although a fertilized egg can be obtained by natural mating, a method of artificially regulating the estrous cycle of a female non-human mammal and then mating it with a male non-human mammal is preferred. As a method for artificially regulating the estrous cycle of a female non-human mammal, for example, a method in which follicle-stimulating hormone and then luteinizing hormone are first administered by, for example, intraperitoneal injection is preferable. Varies depending on the type of non-human mammal. When the non-human mammal is a mouse, a method in which luteinizing hormone is administered to the female mouse about 48 hours after administration of the follicle stimulating hormone to the female mouse, and a fertilized egg is obtained by mating with the male mouse, is preferred. The amount is 2-20 IU / individual, preferably about 5 IU / individual, and the dose of luteinizing hormone is 0-10 IU / individual, preferably about 5 IU / individual. When the non-human mammal is a rat, a method of administering luteinizing hormone to female rats about 48 hours after administration of follicle stimulating hormone to female rats and obtaining fertilized eggs by mating with male rats is preferable. The amount is 20-50 IU / individual, preferably about 30 IU / individual, and the dose of luteinizing hormone is 0-10 IU / individual, preferably about 5 IU / individual. When using inbred mice or rats of the Wister strain, it is preferable to use those that are reared for about 1 week under light conditions (for example, 7:00 to 19:00) for about 12 hours, and are 8 weeks old or older. .
[0089]
The obtained fertilized eggs were microinjected (W. J. Gordon et al .; Proc. Natl. Acad. Sci. USA, USA).77J., 7380, 1980; {J. {Mullins et al .; {Nature,}344, $ 541, $ 1990; E. {Hammer et al .; Nature,}315, $ 680, $ 1985; G. {Pursel et al .;} Immunol. {Immunopathol. ,17, 303, 1987) or a method using a retrovirus (manipulating mouse Embrio 2nd edition) or the like, and then introducing a gene containing DNA encoding a chimeric seven-transmembrane receptor, and transferring the fertilized egg to a female By artificially transplanting and implanting into a non-human mammal, a non-human mammal having a chromosomal DNA into which a gene containing a DNA encoding the introduced chimeric seven-transmembrane receptor is obtained is obtained.
[0090]
When a gene is introduced by the microinjection method, it is preferable to use a fertilized egg in which a male pronucleus has appeared for about 12 to about 24 hours after fertilization. The gene containing the DNA encoding the chimeric seven-transmembrane receptor introduced by the microinjection method can be used in any of the circularized state and the linearized state, and encodes the chimeric seven-transmembrane receptor. It is preferable to introduce and linearize the region and an expression control region such as a promoter without destroying the region. When a gene is introduced by a method using a retrovirus, it is preferable to use a fertilized egg at a developmental stage before the morula embryo (generally, before the 8-cell stage).
[0091]
When a fertilized egg is transplanted into a female non-human mammal, the fertilized egg obtained from the pseudopregnant female non-human mammal in which fertilization has been induced is artificially bred by crossing with a vasectomy male non-human mammal. The method of implanting and implanting is preferred. Pseudopregnant female non-human mammals can also be obtained by natural breeding, but fertilized by administering luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) or an analog thereof and mating with male non-human mammals. In addition, a pseudopregnant female non-human mammal in which has been induced can be obtained. As analogs of LHRH, for example, [3,5-diiodo-Tyr]⁵] -LHRH, [Gln⁸] -LHRH, [D-Ala⁶] -LHRH, des-Gly¹⁰-[D-His (Bzl)⁶] -LHRH ethylamide and the like. The dose of LHRH or its analog and the timing of mating with a male non-human mammal after its administration differ depending on the type of non-human mammal. When the non-human mammal is a female rat, it is usually preferable to mate with a male rat about 4 days after administration of LHRH or an analog thereof, and the dose of LHRH or an analog thereof is usually 10 to 10 days. It is 60 μg / individual, preferably about 40 μg / individual.
[0092]
(Ii) Production of transgenic non-human mammal by introducing a gene into ES cells
Production of transgenic non-human mammals by gene transfer into ES cells is described in Manipulating Mouse Embryo 2nd Edition, Gene Targeting, A Practical Approach, IRL Press At Oxford University Press, (1993), Biomanagement Series. Production of mutant mice using ES cells, Yodosha Co., Ltd. (1995), Manual for Developmental Engineering, {How to Make Transgenic Mice, Kodansha Co., Ltd.} (1987), and the like.
[0093]
After introducing a gene containing a DNA encoding a chimeric seven-transmembrane receptor into ES cells of a non-human mammal, the obtained ES cells are fertilized using a collection chimera method or an injection chimera method. A cell having a chromosomal DNA incorporating a gene containing a DNA encoding a chimeric seven-transmembrane receptor introduced by incorporation into an egg and artificially implanting and implanting the fertilized egg into a female non-human mammal Is obtained partially as a non-human mammal chimeric animal.
[0094]
Non-human mammal ES cells are mouse ES cells (M. J. Evans et al .; Nature,292, {154, {1981}; R. {Martin;} Proc. {Natl. {Acad. {Sci. USA,78, 7634, 1981), rat ES cells (P. M. Iannaccone et al .; Dev. Biol.,).163, 288, ブ タ 1994) Porcine ES cells (M. B. Wheeler; Reprod. Fertil. Dev.,)6Monkey ES cells (J. A. Thomson et al .; Proc. Natl. Acad. Sci. USA, USA).92, 7844, 1996), and the method of establishing ES cells described in US Pat. No. 5,453,357 or US Pat. No. 5,670,372. AB2.2 [manufactured by Lexicon Genetics, Inc.] can be used as mouse ES cells.
[0095]
For introducing an exogenous gene into ES cells, known gene transfer methods (for example, calcium phosphate method, electric pulse method, lipofection method, agglutination method, microinjection method, particle gun method, DEAE-dextran method, virus vector method) Etc.) can be used. The gene containing the DNA encoding the chimeric seven-transmembrane receptor can be used in any of a circularized state and a linearized state, and the region encoding the chimeric seven-transmembrane receptor and a promoter can be used. It is preferable to introduce the expression control region by linearizing it without destroying it.
[0096]
A fertilized egg can be obtained by the method described in (i).
When the ES cells are incorporated into a fertilized egg of a non-human mammal by using the chimeric assembly method, it is generally preferable to use a fertilized egg in a developmental stage before the 8-cell stage. When ES cells are incorporated into a fertilized egg of a non-human mammal using the injection chimera method, it is generally preferable to use a fertilized egg in the stage of blastocyst development from the 8-cell stage.
[0097]
When a fertilized egg is transplanted into a female non-human mammal, the fertilized egg obtained from the pseudopregnant female non-human mammal in which fertilization has been induced is artificially bred by crossing with a vasectomy male non-human mammal. A method of transplantation and implantation is preferred, and a pseudopregnant female non-human mammal can be obtained by using the method described in (i).
If the non-human mammal of the ES cell and the non-human mammal of the fertilized egg are of the same species but different in appearance phenotype such as the color of the hair, the individual is a chimeric non-human mammal, and An individual having a high chimera rate (a ratio of cells derived from ES cells, that is, cells having chromosomal DNA into which the introduced exogenous gene has been incorporated) can be easily selected.
[0098]
Among the non-human mammal chimeric animals partially having cells having chromosomal DNA into which the gene containing the DNA encoding the chimeric seven-transmembrane receptor expression is incorporated, an individual having a high chimeric rate (preferably male) and a non-human mammal Mammalian individuals (preferably females) are crossed. Chimeric seven transmembrane transfections introduced into genomic DNA prepared from a tissue of a born pup such as blood tissue, epithelial tissue, connective tissue, cartilage tissue, bone tissue, muscular tissue, oral tissue or skeletal tissue tissue Is examined by Southern hybridization, PCR, etc., for the presence of a type receptor expression unit, and a gene containing a DNA encoding a chimeric seven-transmembrane receptor introduced into the genomic DNA of all the offspring is present. In some cases, the parent non-human mammal chimeric animal is selected as the germline chimera. By mating a germline chimera with an individual of a non-human mammal, an individual having a gene containing a DNA encoding the chimeric seven-transmembrane receptor introduced on the chromosome of cells of the whole body can be obtained as a pup. . This individual is usually a heterozygote having the introduced chimeric seven transmembrane receptor expression unit in only one of the homologous chromosomes. And obtaining a transgenic non-human mammal homozygous containing a gene containing a DNA encoding a chimeric seven-transmembrane receptor introduced into both homologous chromosomes by mating the male and female of the individual. Can be.
[0099]
(Iii) Preparation of transgenic non-human mammal by gene transfer into sperm
Production of transgenic non-human mammals by gene transfer into sperm has been described by Perry et al. (Science,284, {1180, {1999)} and Wakayama et al. (Nature {Biotechnology,}).16, $ 639, $ 1998).
[0100]
After incorporating the gene containing the DNA encoding the chimeric seven-transmembrane receptor prepared in (4) into a non-human mammal sperm, the resulting sperm is transformed into an unfertilized egg using a microinsemination method. By fertilizing the egg that has begun to develop and artificially transplanting and implanting it into a female non-human mammal, a gene containing DNA encoding a chimeric seven-transmembrane receptor incorporated into sperm is placed on the chromosome. An integrated non-human mammal is obtained.
[0101]
In the case of incorporating a foreign gene into sperm, a sperm whose cell membrane has been disrupted is transformed into a medium containing a foreign gene at a concentration of about 0.5 to about 100 ng / mL (preferably 5 to 10 ng / mL) (for example, It is preferable to use a method of incubating for several minutes (preferably about 1 minute) in a CZB medium or NIM medium. As a method for destroying the cell membrane of sperm, a method of treating with a low-concentration (for example, 0.05%) surfactant (for example, Triton X-100 or the like) and a 10% fetal calf serum (hereinafter abbreviated as FCS) are used. ), A method of freeze-drying spermatozoa suspended in a medium (eg, a CZB medium or the like) containing no EDTA and a method of freeze-thawing the sperm suspension are preferred. Sperm whose cell membrane has been destroyed by such a treatment no longer has the ability to spontaneously fertilize, but it promotes embryo development by fertilizing using a microinsemination method using a micromanipulator etc. Can be. In the case of performing microinsemination, a method of injecting the head of a sperm into which an exogenous gene has been incorporated into the cytoplasm of a non-fertilized egg of a female non-human mammal of the same species in metaphase II is preferred.
[0102]
(Iv) Gene transfer into unfertilized eggs and production of transgenic non-human mammals by in vitro fertilization
Production of transgenic non-human mammals by gene transfer into unfertilized eggs and in vitro fertilization was carried out by the method reported by Chan et al. (Proc. Natl. Acad. Sci. USA, USA).95, $ 14028, $ 1998).
[0103]
Non-fertile eggs of non-human mammals (preferably mice, rats, cows, etc.) are transfected by infecting a retrovirus incorporating a gene containing a DNA encoding a chimeric seven-transmembrane receptor into a non-fertilized egg. Then, the resulting eggs are started to develop using an in vitro fertilization method, and the fertilized eggs that have started to develop are artificially transplanted and implanted in a female non-human mammal, thereby introducing the introduced chimeric seven transmembrane membranes. A non-human mammal having a chromosome into which a gene containing DNA encoding the type receptor has been obtained.
[0104]
A retrovirus into which a gene containing a DNA encoding a chimeric seven-transmembrane receptor has been incorporated can be prepared by the method described in Manipulating Mouse Embrio 2nd Edition and the like. When infecting unfertilized eggs with a recombinant retrovirus, it is preferable to use unfertilized eggs from which the zona pellucida has been removed. Methods for removing the zona pellucida of unfertilized eggs include physically removing the membrane using a micromanipulator or the like, or chemically dissolving and removing the membrane using a chemical substance (for example, acidic Tyrode). Is preferred.
[0105]
2. Production of the transgenic non-human mammal of the present invention using a method for producing a gene-replaced animal in which a target gene and a transgene of interest are exchanged.
(1) Obtaining DNA encoding human seven-transmembrane receptor
1. According to the method described in (1), a DNA encoding a human seven-transmembrane receptor is obtained. As described below, in order to add the non-coding region of the non-human mammalian seven-transmembrane receptor gene to the 5 'and 3' of the region encoding the human seven-transmembrane receptor, an initiation codon and Since an appropriate restriction enzyme site is required in the coding region near the stop codon, if such a restriction enzyme site does not exist, the sequence of the PCR primer is changed without changing the amino acid sequence to be encoded. The codons are selected and designed so that they can be formed, so that appropriate restriction enzyme sites can be formed near the start codon and stop codon in the coding region of the human seven-transmembrane receptor.
[0106]
(2) Obtaining genomic DNA containing a non-human mammal ortholog gene of the receptor
A genomic DNA containing a non-human mammal ortholog gene of the receptor is obtained. The genomic DNA must include, in addition to the region encoding the ortholog and the intron, regions 5 'and 3' to the region encoding the ortholog. The region on the 5 'side and 3' side of the region encoding the ortholog preferably has 500 bp or more, more preferably 1 kbp or more, and still more preferably 3 kbp or more.
[0107]
Examples of a method for preparing a genomic DNA containing an ortholog gene include a method of preparing a genomic DNA library of the non-human mammal and isolating a genomic DNA clone containing the gene by plaque hybridization or colony hybridization. .
A genomic DNA library is obtained by extracting genomic DNA from the cells or tissues of the non-human mammal, partially cutting with a restriction enzyme, inserting the fragment into an appropriate vector,E.coliAnd the like, and can be prepared by introducing into an appropriate host such as Preparation of genomic DNA and genomic DNA library can be prepared by methods described in Molecular Cloning Third Edition, Current Protocols in Molecular Biology, and the like. Examples of the vector include a λ phage vector such as λΔEMBL3 (manufactured by Stratagene), λ DASH II (manufactured by Stratagene), λ FIX II (manufactured by Stratagene), a bacterial artificial chromosome (BAC) vector such as BeroBACII, and pAd10sacBII. And P1 artificial chromosome (PAC) vectors such as pCYPAC1, cosmid vectors such as SuperCosII (manufactured by Stratagene) and the like.
[0108]
Probes used for plaque hybridization or colony hybridization may be those labeled with a partial fragment of the genomic gene of a seven-transmembrane receptor to be replaced by the non-human mammal, or a partial fragment of the cDNA of the receptor. it can. These partial fragments can be obtained by searching a genomic sequence or a cDNA sequence of the receptor gene of the non-human mammal from a base sequence database and performing PCR using primers prepared based on the base sequence.
[0109]
Plaque hybridization, colony hybridization, and labeling and preparation of the probe can be performed by methods described in Molecular Cloning Third Edition, Current Protocols in Molecular Biology, and the like.
When the genomic sequence of the receptor gene of the non-human mammal can be obtained by searching a base sequence database, a region of the genomic sequence to be amplified is appropriately selected, and a sequence of 20 to 40 bp at the 5 'end of this region is determined. A DNA containing a 3 'end and a sequence complementary to a sequence of 20 to 40 bp of the 3' end of this region at the 3 'end are prepared, and this DNA is used as a primer, and the genomic DNA of the non-human mammal is used as a template. Genomic DNA containing the receptor gene of the non-human mammal can be isolated.
[0110]
In addition, a genomic DNA library screening system (manufactured by Genome @ Systems) and a universal genome walker kit (Universal @ GenomeWalker)^TMBy using a kit such as Kit (manufactured by Clontech), genomic DNA containing the seven-transmembrane receptor gene of the non-human mammal can also be isolated.
As a genomic DNA containing a seven-transmembrane receptor gene of a non-human mammal obtained by the above method, a mouse genome containing a base sequence represented by SEQ ID NO: 10, which is a genomic DNA containing a mouse A3 adenosine receptor gene DNA can be mentioned.
[0111]
(3) A DNA comprising a 5 'non-coding region and a 3' non-coding region of the genomic sequence of the non-human mammalian ortholog gene of the receptor added to both ends of the DNA encoding the chimeric seven-transmembrane receptor. Production
At the 5 'end of the receptor-encoding region (region from initiation codon to stop codon) of the DNA encoding the chimeric seven-transmembrane receptor required for homologous recombination, a non-human mammalian ortholog of the receptor is provided. A non-coding region of an appropriate length 5 'to the start codon of the genomic sequence of the gene was added, and a non-coding region of an appropriate length 3' to the stop codon of the genomic sequence was added to the 3 'end. DNA can be obtained as follows. The length of the non-coding region to be added is 500 bp or more on both the 3 'side and 5' side, preferably 1 kbp or more, more preferably 3 kbp or more.
[0112]
1. 1. According to the method described in (3). 1. DNA encoding the human seven-transmembrane receptor obtained in (1) and A DNA encoding a chimeric seven-transmembrane receptor is prepared from the non-human mammal ortholog gene obtained in (2). At this time, when a DNA fragment encoding a region containing the N-terminus or C-terminus is obtained using a genomic DNA containing a non-human mammal ortholog gene as a template, one of the primers is used for the non-coding region of the genomic DNA. By making the sequence based on the base sequence, the 5 'non-coding region or 3' of the genomic sequence of the non-human mammalian ortholog gene is added to the end of the DNA fragment encoding the region containing the N-terminus or C-terminus of the chimeric receptor. Non-coding regions can be added.
[0113]
1. When preparing a DNA encoding a chimeric seven-transmembrane receptor according to the method described in (3), a DNA fragment encoding a region containing an N-terminus or a C-terminus is encoded by encoding the human receptor. (A) in the genomic sequence of the non-human mammalian ortholog gene, the non-coding region 20 further 5 'to the appropriate restriction enzyme site X 5' to the start codon. DNA having a sequence of 40 bp, (b) at the 3 ′ end of the sequence complementary to the sequence from the initiation codon to the restriction enzyme site A near the initiation codon of the DNA encoding the human seven-transmembrane receptor, DNA to which a sequence complementary to the sequence of the non-coding region 20 to 40 bp up to just before the start codon of the genomic sequence of the human mammalian ortholog gene is added, (c) encoding a human seven-transmembrane receptor DNA obtained by adding a sequence of 20 to 40 bp of a non-coding region immediately after a stop codon of a genomic sequence of a non-human mammal ortholog gene to the 3 ′ end of a sequence from a restriction enzyme site B to a stop codon near a stop codon of NA, ( d) DNA having a sequence complementary to the sequence of the non-coding region 20 to 40 bp further 3 'to the appropriate restriction enzyme site Y 3' to the stop codon in the genomic sequence of the non-human mammalian ortholog gene Is prepared. (A) and (b) as one set of primers, and (c) and (d) as one set of primers. PCR is performed using the genomic DNA containing the non-human mammal ortholog gene obtained in (1) as a template. The DNA obtained by PCR using the primers (a) and (b) was combined with the restriction enzyme site A and the 5 'non-coding region near the initiation codon of the DNA encoding the human seven-transmembrane receptor. DNA obtained by PCR using the primers (c) and (d) was cut at the restriction enzyme site X in the vicinity of the stop codon of the DNA encoding the human seven-transmembrane receptor. Cleavage at enzyme site B and restriction enzyme site Y in the 3 'non-coding region. These fragments and the genomic sequence of the seven-transmembrane receptor gene of the non-human mammal obtained in (2) are located further 5 'from the restriction enzyme sites X and X in the 5' non-coding region. The fragment cut at the appropriate restriction enzyme site W and the genomic sequence of the seven-transmembrane receptor gene of the non-human mammal are further 3 'from the restriction enzyme sites Y and Y in the 3' non-coding region. The fragment cut at the appropriate restriction enzyme site Z is ligated.
[0114]
A DNA fragment encoding the region containing the N-terminus or C-terminus of the chimeric receptor, obtained by adding the 5 'non-coding region or 3' non-coding region of the genomic sequence of the non-human mammal ortholog gene to the terminal obtained above And 1. Each partial fragment of the DNA encoding the chimeric receptor prepared according to the method described in (3) is ligated using a restriction enzyme site.
[0115]
(6) Preparation of target vector and introduction into ES cells
The target vector for homologous recombination of a non-human mammal ortholog gene was Gene Targeting, A Practical Approach, IRL Press At at Oxford University Press, (1993), Biomanual Series 8, Gene Targeting, Mutant Mouse Using Genetic Mouse (ES). (Yodosha) (1995) and the like. As the target vector, any of a replacement type and an insertion type can be used. The target vector is located at both ends of the DNA encoding the human seven-transmembrane receptor or the chimeric seven-transmembrane receptor prepared in (4) or (5), and a non-human mammalian ortholog of the receptor. DNA to which a 5 ′ non-coding region and a 3 ′ non-coding region of a genomic sequence containing a gene have been added (hereinafter also referred to as DNA for homologous recombination), genes required for selecting homologous recombinants, andE.coliRequired for the preparation of vectors usingE.coliHas a replication origin and a drug resistance gene capable of autonomous replication. Genes required for selection of homologous recombinants include drugs such as hypoxanthine phosphoribosyltransferase (hprt) gene and neomycin resistance gene used for positive selection (a method for selecting a recombinant containing the selected gene). Examples of the gene include a resistance gene, a diphtheria toxin (DT) gene used for negative selection (a method for selecting a recombinant not containing the gene used for selection), and the like. The gene used for positive selection is inserted into the non-coding region of the DNA for homologous recombination, and the gene used for negative selection is inserted at a position different from the DNA for homologous recombination.
[0116]
The gene used for positive selection may be linked to a promoter required for expression of the gene, or not linked to the promoter, and when homologous recombination occurs, the ortholog of the receptor on the chromosome of the ES cell may be deleted. You may make it express with a promoter. When two loxP sequences are added to both ends of the gene used for positive selection and inserted, homologous recombination is selected, and then the Cre recombinase expression vector is introduced into the homologous recombination. The gene used for positive selection can be removed from the chromosome.
[0117]
As a specific target vector, a vector obtained by inserting a DNA for homologous recombination and an expression unit of the DT gene into ploxpHPRT2 (WO $ 01/33957), for example, a non-coding region of genomic DNA containing a mouse A3 receptor gene in ploxpHPRT2 And a non-coding region of genomic DNA containing mouse A3 receptor gene in plaxP-DT-arm1-HPRT-hA3R-arm2 and plaxpHPRT2 in which DNA for homologous recombination consisting of human A3 receptor cDNA and DT gene expression unit are inserted. Examples include DNA for homologous recombination consisting of a DNA encoding a human and mouse chimeric A3 receptor and ploxP-DT-arm1-HPRT-cA3R-arm2 into which an expression unit of the DT gene has been inserted.
[0118]
For introducing a target vector into ES cells, known gene transfer methods such as a calcium phosphate method, an electric pulse method, a lipofection method, an agglutination method, a microinjection method, a particle gun method, a DEAE-dextran method, and a virus vector method are used. Can be used. The target vector to be introduced can be used in any of a circular and linear form, but it is preferable to introduce the homologous recombination DNA and the homologous recombinant in a form that does not destroy the gene required for selection. .
[0119]
Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press, (1993), Biomanual Series 8, Gene targeting, and sheep mutant mice using ES cells ( (Tsuchiya) (1995) and the like, such as positive selection, promoter selection, negative selection, and poly A selection. For example, in the case of a target vector containing the hprt gene, after introduction into ES cells lacking the hprt gene, the ES cells are cultured in a medium containing aminopterin, hypoxanthine, and thymidine, and aminopterin-resistant strains are selected. , A positive selection for selecting homologous recombinants containing the hprt gene can be performed. In the case of a target vector containing a neomycin resistance gene, ES cells into which the vector has been introduced are cultured in a medium containing G418, and G418-resistant strains are selected to select homologous recombinants containing the neomycin resistance gene. Can be performed. In the case of a target vector containing the DT gene, the ES cell into which the vector has been introduced is cultured, and the growing strain is selected (for a recombinant inserted randomly into a chromosome other than homologous recombination, the DT gene is Since it is integrated and expressed, it cannot grow due to the toxicity of DT), so that a negative selection for selecting a homologous recombinant containing no DT gene can be performed. Examples of a method for selecting a target homologous recombinant from the selected cell lines include a Southern hybridization method (molecular cloning, 3rd edition) for genomic DNA, PCR, and the like.
[0120]
(7) Incorporation of ES cells into fertilized eggs and production of non-human transgenic animals
Incorporation of ES cells into a fertilized egg and transplantation of the fertilized egg into a non-human mammal include: (5) It can be performed by the method described in (ii).
[0121]
From the obtained pups: (5) By the method described in (ii), a heterozygote having a gene replacement by homologous recombination on one of homologous chromosomes of whole body cells can be obtained. Further, by crossing the male and female of the individual, a transgenic non-human mammal having a homozygote having a gene replacement by homologous recombination on both homologous chromosomes can be obtained.
[0122]
The thus obtained homozygous transgenic non-human mammal expresses the human or chimeric seven-transmembrane receptor encoded by the introduced gene and does not express the ortholog possessed by itself. Non-human mammal. The transgenic non-human mammal of this homozygote is more than the transgenic non-human mammal obtained by the method (1). And 5. As a non-human mammal used for pharmacological evaluation of test substances described below, (a) the non-human mammal does not express the ortholog of the seven-transmembrane receptor originally expressed, and is used for pharmacological evaluation. It is not necessary to consider the pharmacological effects mediated by the ortholog, and (b) the secondary phenotype such as the destruction of the gene existing at the introduced site of the foreign gene or the activation of the gene around the introduced site by the introduced gene. This is preferable in that there is almost no possibility of a change.
[0123]
3. Production of the transgenic non-human mammal of the present invention using the method for producing a cloned individual using the nucleus of a cell subjected to the intended genetic modification
The transgenic non-human mammals of the present invention may be cloned sheep (I. Wilmut et al .; Nature,385, 810, 1997), cloned bovine (J. B. Cibellli et al .; Science,280, {1256, 1998, Akira Iriya; {protein nucleic acid enzyme,}44, 892, 1999), cloned goats (A. Baguisi et al .; Nature Nature Biotechnology, Inc.).17, 456, 1999), cloned mouse (T. Wakayama et al .; Nature,).394369, 1998; {Wakayama et al .; {Nature} Genetics,}22, # 127, # 1999), for example, can be manufactured as follows.
[0124]
The above 1. Or 2. A gene comprising a DNA encoding a chimeric or human seven-transmembrane receptor on the chromosome of any cell of a non-human mammal (preferably, mouse, sheep, goat, cow, etc.) Is introduced. The nucleus of the obtained cells is initialized (operation for returning the nucleus to a state in which development can be repeated again).
[0125]
Development is initiated by injecting the nuclei of the reprogrammed cells into unfertilized eggs of an enucleated non-human mammal.
The transgenic non-human mammal of the present invention that expresses the introduced gene by artificially transplanting and implanting an egg that has started to develop into a female non-human mammal is obtained. Also, 2. The transgenic non-human mammal obtained by using the nucleus of a cell subjected to gene replacement by the method described in (1), usually contains DNA encoding a chimeric or human seven-transmembrane receptor on one of homologous chromosomes. Since the heterozygote has the gene introduced therein, by crossing the obtained heterozygotes, the ortholog of the receptor possessed by itself is not expressed, and the transgene expressing the chimeric or human seven-transmembrane receptor is not expressed. A transgenic non-human mammal can be obtained. This homozygous transgenic non-human mammal also The non-human mammal used for the pharmacological evaluation is more preferable than the transgenic non-human mammal obtained by the method (1) in the same manner as the homozygous transgenic non-human mammal obtained by the method described in (1). .
[0126]
The method of initializing the nucleus of a cell differs depending on the type of non-human mammal. When the non-human mammal is a sheep, a goat, a cow, or the like, the cells into which the exogenous gene has been introduced are transformed from a medium containing 5 to 30%, preferably 10% FCS (eg, M2 medium) for 3 to 10 days, preferably 3 to 10 days. Is preferably induced in a resting state (G0 phase or G1 phase) by cell culture in an oligotrophic medium containing 0 to 1%, preferably 0.5% FCS for 5 days to initialize. When the non-human mammal is a mouse or the like, a nucleus of a cell into which an exogenous gene has been introduced is injected into an enucleated unfertilized egg of the same type of non-human mammal, and cultured for several hours, preferably for about 1 to 6 hours. It is preferable to perform initialization.
[0127]
The method of starting the reprogrammed nucleus in an enucleated unfertilized egg differs depending on the type of non-human mammal. When the non-human mammal is a sheep, a goat, a cow, or the like, the nucleus that has induced the cell cycle to a quiescent state (G0 phase or G1 phase) and initialized is removed from the same type of non-human mammal by electrofusion or the like. It is preferred that the ovum be activated to start its development by transplantation into a nucleated unfertilized egg. When the non-human mammal is a mouse or the like, an unfertilized egg into which the nucleus of the cell into which the exogenous gene has been introduced is injected is stimulated with an egg activating substance (eg, strontium) to inhibit cell division (eg, cytotoxicity). It is preferable that the generation be started by treating with calasin B) to suppress the release of the second polar body.
[0128]
4. Pharmacological evaluation method of test substance using transgenic non-human mammal of the present invention
A test substance, for example, an agonist or antagonist that selectively acts on a seven-transmembrane receptor is administered to the transgenic non-human mammal of the present invention, and the animal is compared with an animal not administered with the test substance. The pharmacological evaluation of the test substance can be performed by measuring various physical parameters such as blood pressure, respiratory rate and body weight, observing appearance and behavior, and examining pharmacological effects such as histopathological examination.
[0129]
In addition, a pathological model animal in which various diseases are induced in the transgenic non-human mammal of the present invention is prepared, and a test substance, for example, an agonist that selectively acts on a seven-transmembrane receptor on the pathological model animal Or administering an antagonist or the like, and measuring the various physical parameters such as blood pressure, respiratory rate, body weight, etc. of the disease state model animal, observing the disease state, appearance, behavior, histopathological examination, etc., and administering no test substance. By performing the test in comparison with the disease state model animal, pharmacological evaluation of the efficacy and side effects of the test substance for the disease can be performed. Further, based on this evaluation, a substance that is preferable as a therapeutic drug for the disease can be selected. In particular, a drug specific to a human receptor whose binding amount to a human seven-transmembrane receptor and an activity as an agonist or an antagonist are higher than that of a non-mammalian ortholog of the receptor, may cause a general disease state. Although pharmacological evaluation using a model animal has been difficult, pharmacological evaluation can be performed by using the transgenic animal of the present invention. The disease induced in the transgenic non-human mammal of the present invention includes heart disease (eg, acute heart failure, chronic heart failure, myocarditis, etc.), respiratory disease, joint disease (eg, rheumatoid arthritis, osteoarthritis, etc.) ), Renal diseases (eg, renal failure, glomerulonephritis, IgA nephropathy, etc.), arteriosclerosis, psoriasis, hyperlipidemia, allergic diseases (eg, asthma, allergic rhinitis, atopic dermatitis, etc.), Bone disease (eg, osteoporosis, rickets, osteomalacia, hypocalcemia, etc.), blood disease, cerebrovascular injury, traumatic brain injury, infection, dementia, cancer, diabetes, liver disease, skin disease, Examples include neurodegenerative diseases and chronic inflammatory diseases. The preparation of a disease model animal is performed using a “manual disease model mouse” [Molecular Medicine, Inc.31Extra edition, {Nakayama Shoten Co., Ltd. (1994)], "Pathological animal model for illustration and pharmacology" [Nishimura Shoten Co., Ltd. (1984)], "Arthritis model animal", "Dental Medicine Publishing Co., Ltd. (1985)", "Nerve and muscle. Disease model animals "[Ishin-Denshi Publishing Co., Ltd. (1982)]," Active oxygen and pathology {From disease models to bedside "", and so on.
[0130]
4. The present pharmacological evaluation method and And 8. As test substances to be subjected to the pharmacological evaluation method described below, antagonists to A3 adenosine receptor (WO98 / 15555; WO00 / 64894; WO00 / 02861; WO00 / 10391; WO00 / 15231; WO99 / 65512; WO99 / 64418; WO99 / WO97 / 33879; WO97 / 27177; JP-A-11-193281; JP-A-9-291089; US Pat. No. 5,444,883; US Pat. No. 5,646,156; US Pat. No. 5,573,772; Academic Press, 164, 1994; ature,360, $ 161, $ 1992; @Neurochem. ,60, 380, 1993), antagonists such as CP 96345 and RP 67580 for NK1 tachykinin receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 262, 1994; Science,).251435, 1991; Proc. {Natl. {Acad. {Sci. USA,88, $ 10208, $ 1991; {Biol. {Chem. ,267, 25668, {1992; {Biol. {Chem. ,268, 2319, 1993), agonists or antagonists to the B2 bradykinin receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 68, 1994; Biochem. Biophys. Res.187, {1306, {1992), agonists such as CGP-12177 for β3 adrenergic receptor (Diabetes.,).47, 1464, 1998), agonists for the calcitonin receptor (The G-Protein Linked Receptor, Facts ｋBook, Academic Press, 74, 1994), and antagonists such as AF-DX116 and pyrenthroin e-Gin for the M2 muscarinic acetylcholine receptor. Linked Receptor, Facts Book, Academic Press, 11, 1994; J. Biol. Chem.,266, 17382, 1991), and antagonists such as yohimbine and lauworusin against the α2A adrenergic receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 42, 1994, Mol. Pharmacol.42, 16, 1992), antagonists such as CGP 20712A and atenolol against β1 adrenergic receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 47, 1994; Mol. Pharmacol.17, 1, ８０1980), antagonists such as L-365260 and L-364718 against CCKB cholecystokinin receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 92, 1994; Nature,).362, 348, 1993), Xenopsin and [Trp for the neurotensin receptor.¹¹] Analog compounds such as neurotensin (The G-Protein Linked Receptor, Facts Book, Academic Press, 199, 1994; Br. J. Pharmacol.,69, 689, 、 1980), prostaglandin D2 analog compounds for the DP prostanoid receptor (The G-Protein Linked Receptor, Facts Book, Academic Press, 242, 1994; Can be given.
[0131]
5. Pharmacological evaluation method of test substance using cells obtained from transgenic non-human mammal of the present invention
Various cells obtained from the transgenic non-human mammal of the present invention are brought into contact with a test substance and compared with cells in the absence of the test substance.²⁺By examining pharmacological effects such as various cell responses such as an increase in concentration and changes in cell morphology, pharmacological evaluation of a test substance on the cells can be performed.
[0132]
Various types of cells can be obtained by inducing differentiation of ES cells obtained from the transgenic non-human mammal of the present invention. As a method of inducing differentiation, a method of inducing a teratoma (teratoma) mixed with various tissues by implanting ES cells subcutaneously into animals of the same type and same strain (manipulating mouse Embrio 2nd edition) By culturing in vitro under appropriate conditions, endoderm cells, ectoderm cells, mesoderm cells, blood cells, endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, nerve cells, glial cells, epithelium Method for inducing differentiation into cells, melanocytes, and keratinocytes (Reprod. {Fertil.} Dev.,}10, $ 31, $ 1998). The differentiated cells are brought into contact with a test substance, and compared with cells in the absence of the test substance, the intracellular Ca²⁺By examining pharmacological effects such as various cell responses such as an increase in concentration and changes in cell morphology, pharmacological evaluation of a test substance on the cells can be performed. By these methods, pharmacological evaluation of a test substance can be performed on cells that are difficult to remove from a human body or cells that exist only in a small number.
[0133]
6. Preparation of Genetically Modified Animal Using ES Cell, Egg, Sperm or Nucleus of Transgenic Non-Human Mammal of the Present Invention
Using ES cells, eggs, sperm or nuclei obtained from the transgenic non-human mammal of the present invention, ~ 3. And a gene-modified non-human mammal expressing a chimeric seven-transmembrane receptor and further modifying the gene on the chromosome can be obtained by the method described in (1). In particular, a gene that is known to cause a disease state that disrupts the function of the gene, Knockout animals deficient using the homologous recombination method described in 1) and dominant negative genes that inhibit the function of the gene. Or 3. The transgenic animal introduced and expressed by the method described in (1) is useful as a disease model animal.
[0134]
7. Production of a genetically modified animal by crossing the transgenic non-human mammal of the present invention with an animal of the same species or other strain, and use of the produced genetically modified animal
By crossing the transgenic non-human mammal of the present invention with an animal of the same or different strain (eg, a human disease model animal), the chimeric seven-transmembrane receptor is expressed, and a certain expression system (eg, human Genetically modified mammals exhibiting symptoms similar to pathological conditions) can be obtained. If the animal to be bred is a human disease model animal, a pathological model animal that expresses a chimeric seven-transmembrane receptor and exhibits a symptom similar to a human pathological condition as an expression system can be obtained. Methods of mating include in vitro fertilization in addition to natural mating. As the disease model animal, any disease model animal can be used, regardless of whether the disease is congenital or acquired. For example, an acquired pathological model animal is a "manual disease model mouse" [Molecular Medicine, Inc.]31Extra edition, {Nakayama Shoten Co., Ltd. (1994)], "Pathological animal model for illustration and pharmacology" [Nishimura Shoten Co., Ltd. (1984)], "Arthritis model animal", "Dental Medicine Publishing Co., Ltd. (1985)", "Nerve and muscle. Disease model animals "[Ishin-Denshi Publishing Co., Ltd. (1982)]," Active oxygen and pathology {From disease models to bedside "", Gakkai Shuppan Center (1992), and the like.
[0135]
8. Pharmacological evaluation of test substances using genetically modified animals
A test substance, for example, an agonist or antagonist acting on a seven-transmembrane receptor is administered to the genetically modified disease model animal obtained by the method described in 6 or 7, and the blood pressure, respiratory rate, Measurement of various physical parameters such as body weight, disease state, appearance, behavioral observation, histopathological examination, etc. are performed in comparison with the disease state model animal to which the test substance is not administered, whereby the test substance Pharmacological evaluation such as efficacy and side effects for diseases can be performed. Further, based on this evaluation, a substance that is preferable as a therapeutic drug for the disease state can be selected. In particular, a drug selective for a human receptor whose binding amount to a human seven-transmembrane receptor and activity as an agonist or an antagonist are higher than that of a non-mammalian ortholog of the receptor, usually causes a pathological condition of normal. Although pharmacological evaluation using a model animal was difficult, pharmacological evaluation can be performed by using the above-described genetically modified disease state model animal.
Hereinafter, examples of the present invention will be described.
[0136]
【Example】
Example 1 Isolation of mouse A3 adenosine receptor cDNA
(1) Synthesis of cDNA derived from mouse brain
Brains were excised from three males of 7-week-old C3H / HeJ @ Jcl mice (purchased from CLEA Japan), and attached using FastTrack 2.0 mRNA Isolation Kit (FastTrack 2.0 mRNA Isolation Kit, manufactured by Invitrogen). Poly A according to the manual⁺RNA was obtained.
Poly A obtained from mouse brain⁺CDNA was synthesized from 2 μg of the RNA using a PCR-Select cDNA subtraction kit (PCR-Select cDNA Subtraction Kit, manufactured by Clontech) according to the attached manual.
[0137]
(2) Isolation of mouse A3 adenosine receptor cDNA by PCR
PCR was performed using the mouse brain-derived cDNA synthesized in (1) as a template. That is, Z.報告 A report by Zhao et al. (Genomics,57, 1, 1999), a 26-mer primer having a nucleotide sequence represented by SEQ ID NO: 1 from a mouse genomic nucleotide sequence containing mouse A3 adenosine receptor (GenBank accession number: AF0697778), and represented by SEQ ID NO: 2 A 26-mer primer having a base sequence to be prepared was prepared. Using these primers and a mouse brain-derived cDNA as a template, a PCR reaction solution was prepared according to the method described in Molecular Cloning Third Edition, and reacted at 94 ° C. for 5 minutes, followed by 94 ° C. for 1 minute and 60 ° C. After repeating 10 cycles of 30 seconds and 1 minute at 72 ° C., further 15 cycles of 94 ° C. for 1 minute, 54 ° C. for 30 seconds and 72 ° C. for 1 minute, and storing overnight at 4 ° C. PCR was performed under the following conditions. This PCR reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 1 kbp containing mouse A3 adenosine receptor cDNA was recovered using a Geneclean II kit (GENECLEAN II Kit, manufactured by BIO101) according to the attached manual.
[0138]
(3) Construction of plasmid pBS-mA3R having mouse A3 adenosine receptor cDNA
A DNA fragment of about 1 kbp containing the collected mouse A3 adenosine receptor cDNA was dissolved in 100 μL of Universal Buffer H (Universal Buffer H, manufactured by Takara Shuzo), and 60 units ofEcoThe digestion reaction was performed at 37 ° C. for 2 hours with addition of RI. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1 kbp containing mouse A3 adenosine receptor cDNA was recovered using a GeneClean II kit according to the attached manual.
[0139]
Next, 2 μg of plasmid pBluescriptII @ SK (-) (manufactured by Stratagene) was dissolved in 40 μL of Universal Buffer H, and 24 units ofEcoAfter adding RI and performing a digestion reaction at 37 ° C. for 2 hours, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual. The recovered DNA fragment was dissolved in 50 μL of a buffer for alkaline phosphatase (Takara Shuzo), 40 units of bovine small intestine-derived alkaline phosphatase (Takara Shuzo) was added, and the mixture was reacted at 50 ° C. for 30 minutes, and extracted with phenol / chloroform. Thereafter, a DNA fragment of about 3 kbp whose both ends were dephosphorylated by ethanol precipitation was recovered.
[0140]
Contains the mouse A3 adenosine receptor cDNA obtained aboveEco50 ng of RI fragment (about 1 kbp) and plasmid pBluescriptII @ SK (-)Eco70 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of 2 I solution (manufactured by Takara Shuzo) was added, and the binding reaction was performed at 16 ° C. for 2 hours.
Using the reaction solutionE.coliDH5α (manufactured by Toyobo Co., Ltd.) was prepared according to the method of Cohen et al. (Proc. Natl. Acad.69, 2110, 1972), and transformed with ampicillin resistance (Amp^r) Strain was obtained. From this transformant, the method of Birnboim et al. (Nuc. {Acids Res.,})7, 1513, 1979). The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Hereinafter, this plasmid is referred to as pBS-mA3R.
[0141]
Using a DNA sequencer 377 (manufactured by Applied Biosystems), the entire nucleotide sequence of the mouse A3 adenosine receptor cDNA contained in the plasmid pBS-mA3R was determined. The sequence is shown as SEQ ID NO: 3. The stop codon sequence of the mouse A3 adenosine receptor cDNA contained in pBS-mA3R is TAG instead of TAA because it is derived from the primer sequence. Therefore, the stop codon sequence in the base sequence shown in SEQ ID NO: 3 Has been corrected based on the genomic sequence of the mouse A3 adenosine receptor gene determined in Example 2. As a result of comparing the nucleotide sequence represented by SEQ ID NO: 3 with the nucleotide sequence of a known mouse A3 adenosine receptor cDNA (GenBank accession number: AF069778) using the software GENETYX-MAC $ 7.3 (manufactured by Software Development Corporation), The nucleotide sequence differs by 15 bases (FIG. 1), and the amino acid sequence of the mouse A3 adenosine receptor deduced from the nucleotide sequence (SEQ ID NO: 4) differs by 7 amino acids (FIG. 2).
[0142]
Example 2 Isolation and Analysis of Mouse Genomic DNA Containing Mouse A3 Adenosine Receptor Gene
(1) Screening of mouse genomic DNA containing mouse A3 adenosine receptor gene
PCR primers for determining a clone having the sequence of the mouse A3 adenosine receptor gene by PCR were prepared. That is, Z.報告 A report by Zhao et al. (Genomics,57, 1, 1999), which has a base sequence represented by SEQ ID NO: 5, which amplifies a region including a part of exon 1 from the base sequence of the mouse A3 adenosine receptor gene described in GenBank accession number: AF069778. Combination of a 23-mer primer and a 23-mer primer having the nucleotide sequence of SEQ ID NO: 6 (using mouse genomic DNA as a template, an amplified band of about 360 bp corresponding to the 635 to 993 positions of the sequence of SEQ ID NO: 11 is detected. And a combination of a 20-mer primer having the nucleotide sequence of SEQ ID NO: 7 and a 20-mer primer having the nucleotide sequence of SEQ ID NO: 8 (using mouse genomic DNA as a template, 527 of the sequence of SEQ ID NO: 11) Approximately 560 bp amplification buffer corresponding to the 1088th De was prepared by selecting to) detected. Each primer was sent to a genomic DNA library PCR screening service of Kurashiki Spinning Co., Ltd., and a request for screening of a mouse A3 adenosine receptor gene from a mouse genomic DNA library (vector pBeloBACII) by PCR using these primers was made. As a result, it contains the pBeloBACII plasmid into which the mouse genomic DNA containing the sequence of the A3 adenosine receptor gene has been introduced.E.coliク ロ ー ン A clone of a transformant of the DH10B strain was obtained.
[0143]
(2) Isolation of mouse genomic DNA containing mouse A3 adenosine receptor gene
aboveE.coliプ ラ ス ミ ド A plasmid was isolated from the DH10B strain clone according to the method of Burnboim et al. The plasmid was named pmA3R / BeloBacII. Next, the obtained plasmid was treated with each restriction enzyme as follows, and Southern hybridization was performed using a radioisotope-labeled DNA probe containing the mouse A3 adenosine receptor gene sequence.
[0144]
First, 100 ng of the pmA3R / BeloBacII plasmid isolated above was used.SacI,SacII,NotI,XbaI,SpeI,BamHI,SmaI,PstI,EcoRI,EcoRV,HindIII,ClaI,HincII,AccI,SalI,XhoI,ApaI,KpnAfter complete digestion by performing a digestion reaction at 37 ° C. for 6 hours using 12 units of each restriction enzyme I (manufactured by Takara Shuzo) according to the attached manual, a pulse field device [manufactured by Bio-Rad (BIO-RAD)] Agarose gel electrophoresis (separation fragment size: using a program for 5-150 kbp) and further using the method of Southern (J. {Mol.98, $ 503, $ 1975) with a nylon filter [Hybond^TM) -N +; manufactured by Amersham Bioscience). Next, in order to prepare a probe used for Southern hybridization, PCR was performed using mouse genomic DNA as a template. That is, a 20-mer primer having the nucleotide sequence represented by SEQ ID NO: 7 and a 20-mer primer having the nucleotide sequence represented by SEQ ID NO: 8 for amplifying DNA containing a region encoding mouse A3 adenosine receptor in exon 1, exon A 23-mer primer having the nucleotide sequence shown in SEQ ID NO: 9 and a 23-mer primer having the nucleotide sequence shown in SEQ ID NO: 10 were prepared to amplify the DNA containing the region encoding the mouse A3 adenosine receptor in 2. Using these primers and the above-mentioned pmA3R / BeloBacII plasmid as a template, a PCR reaction solution was prepared and reacted at 94 ° C. for 5 minutes, followed by 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 second. After repeating the cycle for 10 minutes for 10 minutes, the cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute was further repeated 20 times, and PCR was performed under the condition of storing at 4 ° C. overnight. This PCR reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 560 bp containing the region encoding mouse A3 adenosine receptor in exon 1 and approximately 600 bp containing the region encoding mouse A3 adenosine receptor in exon 2 Was recovered using a GeneClean II kit according to the attached manual. These DNA fragments were obtained using a commercially available T7 Quick Prime Kit (T7 Quick Prime Kit, manufactured by Amersham Biosciences) [α-³²P] dCTP (manufactured by NEN).
[0145]
Each of the labeled probes was hybridized with the filter prepared above overnight, and 2 × SSPE (300 mmol / L NaCl, 20 mmol / L NaH) containing 0.1% sodium dodecyl sulfate (SDS) was used.₂PO₄, 2 mmol / L EDTA, pH 7.4) at 65 ° C. for 3 times, 0.2 × SSPE containing 0.1% SDS (30 mmol / L NaCl, 2 mmol / L NaH 2).₂PO₄, 0.2 mmol / L (EDTA, pH 7.4) twice at 65 ° C. for 15 minutes, and X-ray film [Kodak Scientific Imaging Film] X-OMAT.^TM{AR, manufactured by Kodak Company]], exposed at -80 ° C overnight, and developed the next day.
[0146]
As a result of this hybridization, the pmA3R / BeloBacII plasmid wasEcoIt was shown that cleavage with RI yields a DNA fragment of about 9 kbp including exon 1 and a DNA fragment of about 6.5 kbp including exon 2.
Subsequently, 5 μg of the above-mentioned pmA3R / BeloBacII plasmid was dissolved in 100 μL of universal buffer H, and 60 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 9 kbp containing exon 1 and a DNA fragment of about 6.5 kbp containing exon 2 were cut out, and the DNA fragments were separated using Gene Clean II kit according to the attached manual. Collected.
[0147]
Further, 1 μg of plasmid pBluescriptII @ SK (−) was dissolved in 50 μL of Universal Buffer H, and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment of about 3 kbp was recovered by an ethanol precipitation method. The recovered DNA was dissolved in 50 μL of a buffer for alkaline phosphatase, and 40 units of alkaline phosphatase derived from bovine small intestine was added, followed by a reaction at 50 ° C. for 3 hours. After phenol / chloroform extraction, a DNA fragment of about 3 kbp dephosphorylated by the ethanol precipitation method was recovered.
[0148]
from the pmA3R / BeloBacII plasmidEco10 ng of a mouse genomic DNA fragment (about 9 kbp) containing exon 1 of the mouse A3 adenosine receptor gene and 10 ng of a mouse genomic DNA fragment (about 6.5 kbp) containing exon 2 of the mouse A3 adenosine receptor were pBluescriptII. Derived from SK (-)EcoIt was dissolved in 10 μL of distilled water together with 40 ng of the RI fragment (about 3 kbp), and the ligation kit Ver. 10 µL of 2 I solution was added, and a binding reaction was performed at 16 ° C for 3 hours.
[0149]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Hereinafter, these plasmids are referred to as pBS-ex1 and pBS-ex2.
[0150]
(3) Analysis of mouse genomic DNA containing mouse A3 adenosine receptor gene
1 μg each of pBS-ex1 and pBS-ex2SacI,SacII,NotI,XbaI,SpeI,BamHI,SmaI,PstI,EcoRI,EcoRV,HindIII,ClaI,HincII,AccI,SalI,XhoI,ApaI,KpnUsing 12 units of each restriction enzyme I (manufactured by Takara Shuzo Co., Ltd.), digestion was carried out at 37 ° C. for 16 hours in accordance with the attached manual, followed by complete digestion, followed by agarose gel electrophoresis. From the results, the position of the recognition sequence for each restriction enzyme present in the mouse genomic DNA of about 15.5 kbp including exon 1 and exon 2 of the A3 adenosine receptor gene was determined. FIG. 3 shows the restriction enzyme maps of exon 1 and FIG. 4 shows the restriction enzyme maps of exon 2.
Next, using a DNA sequencer 377, the nucleotide sequence from a region of about 300 bp upstream of exon 1 to a region of about 1.1 kbp downstream of exon 2 was determined (FIG. 5, SEQ ID NO: 11).
[0151]
Example 3 Isolation of cDNA for Human A3 Adenosine Receptor
(1) Synthesis of cDNA derived from human liver
Human liver poly A purchased from Clontech⁺ＣＤＮＡ Using 4 μg of RNA as a template, cDNA was obtained according to the attached manual using a superscript preamplification system for synthesizing first-strand cDNA (SuperScript \ Preamplification \ System \ for \ first \ strand \ Synthesis, manufactured by Invitrogen).
[0152]
(2) Isolation of human A3 adenosine receptor cDNA by PCR
Subsequently, PCR was performed using the obtained human liver-derived cDNA as a template. That is, M. R. {A report by Atkinson et al. (Neurosci. {Res.,}29A 24-mer primer having the nucleotide sequence of SEQ ID NO: 12, based on the nucleotide sequence of human genomic DNA containing the human A3 adenosine receptor gene (GenBank accession numbers: L77729 and L77730) described in US Pat. And a 25-mer primer having the nucleotide sequence of SEQ ID NO: 13 was prepared. Using these primers, a PCR reaction solution was prepared using human liver-derived cDNA as a template, reacted at 94 ° C. for 5 minutes, and then repeated 30 times at 94 ° C. for 1 minute and at 68 ° C. for 2 minutes. . After performing PCR again in the same manner as described above using 2 μL of this PCR reaction solution, the PCR reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 1 kbp containing human A3 adenosine receptor cDNA was transcribed into GeneClean II. Using the kit, the cells were collected according to the attached manual.
[0153]
(3) Construction of plasmid pBS-hA3R having human A3 adenosine receptor cDNA
Next, 250 ng of the recovered DNA fragment containing the human A3 adenosine receptor cDNA (about 1 kbp) and 50 ng of the plasmid pT7Blue @ T-Vector (Novagen) were dissolved in 10 μL of distilled water, and the DNA ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
[0154]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. In addition, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence of the human A3 adenosine receptor cDNA contained in the plasmid was confirmed. As a result, the sequence was the same as that of the known human A3 adenosine receptor cDNA. SEQ ID NO: 14 shows the nucleotide sequence of the obtained human A3 adenosine receptor cDNA, and SEQ ID NO: 15 shows the amino acid sequence of the human A3 adenosine receptor encoded by the cDNA. Hereinafter, this plasmid is referred to as pBS-hA3R. FIG. 6 shows the construction steps of pBS-hA3R.
[0155]
Example 4 Construction of Target Vector for Replacing Mouse A3 Adenosine Receptor Gene with DNA Encoding Chimeric A3 Adenosine Receptor
About 5 kbp upstream of exon 1 of mouse A3 adenosine receptor geneSmaDownstream from exon 2 from I siteHincIn the region up to the site II, the DNA in which the region encoding the mouse A3 adenosine receptor and the intron have been substituted with the region encoding the chimeric A3 adenosine receptor cDNA, and the exon 2 of the mouse A3 adenosine receptor geneBamDownstream from exon 2 from HI siteApaA target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 containing the DNA in the region up to the I site was constructed by the following steps (1) to (24). The chimeric A3 adenosine receptor in Examples 4 to 9 refers to the amino acid sequence of the intracellular region of the human A3 adenosine receptor (38 to 47, 107 to 126, 199 to 230 and 285 to 285 of SEQ ID NO: 15). 318) was substituted with the amino acid sequence of the intracellular region of mouse A3 adenosine receptor (corresponding to positions 39-48, 108-127, 200-231 and 286-319 of SEQ ID NO: 4). A3 adenosine receptor consisting of an amino acid sequence. SEQ ID NO: 33 shows the amino acid sequence of the chimeric A3 adenosine receptor, and SEQ ID NO: 32 shows the nucleotide sequence of the DNA encoding the chimeric A3 adenosine receptor.
[0156]
(1) Construction of plasmid chimera-1
A plasmid chimera-1 containing the nucleotide sequence encoding the amino acid sequence at positions 190 to 225 of the chimeric A3 adenosine receptor (sequences from 568 to 678 of SEQ ID NO: 32) was constructed as follows (see FIG. 7). . In the amino acid sequence at positions 190 to 225 of the chimeric A3 adenosine receptor, Val at position 196 of the amino acid sequence at positions 191 to 226 of the mouse A3 adenosine receptor is Ala (corresponding to position 195 of the human A3 adenosine receptor). Has been replaced by
[0157]
Based on the sequence of the mouse A3 adenosine receptor gene, a 25-mer primer having the nucleotide sequence of SEQ ID NO: 16 and a 27-mer primer having the nucleotide sequence of SEQ ID NO: 17 were synthesized. The nucleotide sequence represented by SEQ ID NO: 17 was designed so as to encode a 195th Ala residue specific to the human A3 adenosine receptor by partially substituting the sequence based on the mouse A3 adenosine receptor gene. . Using these primers, a reaction solution for PCR was prepared using pBS-ex2 as a template, and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer M and 25 units ofSpeI was added and a digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 110 bp was recovered using a GeneClean II kit according to the attached manual.
[0158]
2.6 μg of plasmid pBluescriptII @ SK (−) was dissolved in 20 μL of Universal Buffer M and 5 units ofSpeI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a Geneclean II kit according to the attached manual.
[0159]
About 10 ng of the about 110 bp DNA fragment obtained above and plasmid pBluescriptII @ SK (-)-derivedSpeI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-1.
[0160]
(2) Construction of plasmid chimera-2
A plasmid chimera-2 containing the nucleotide sequence encoding the amino acid sequence at positions 127 to 190 of the chimeric A3 adenosine receptor (sequences from 379 to 570 in SEQ ID NO: 32) was constructed as follows (see FIG. 8). . The amino acids 127-190 of the chimeric A3 adenosine receptor are identical to the amino acids 127-190 of the human A3 adenosine receptor.
[0161]
Based on the cDNA sequence of the human A3 adenosine receptor gene, a 26-mer primer having the nucleotide sequence of SEQ ID NO: 18 and a 29-mer primer having the nucleotide sequence of SEQ ID NO: 19 were prepared. Using these primers and using pBS-hA3R as a template, a PCR reaction solution was prepared and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer M and mixed with 0.01% BSA and 37.5 units.XbaI was added and a digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. Dissolve in 20 μL of Universal Buffer K and add 15 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 200 bp was recovered using a GeneClean II kit according to the attached manual.
[0162]
2.6 μg of plasmid pBluescriptII @ SK (−) is dissolved in 20 μL of Universal Buffer M, and 0.01% BSA and 7.5 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer K, and 7.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0163]
About 10 ng of the about 200 bp DNA fragment obtained above and plasmid pBluescriptII @ SK (-)-derivedXbaI-Bam50 ng of the HI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-2.
[0164]
(3) Construction of plasmid chimera-3
A plasmid chimera-3 containing the nucleotide sequence encoding the amino acid sequence at positions 44 to 127 of the chimeric A3 adenosine receptor (sequences from 130 to 381 in SEQ ID NO: 32) was constructed as follows (see FIG. 9). . The amino acid sequence at positions 44 to 127 of the chimeric A3 adenosine receptor is the amino acid sequence at positions 44 to 127 of the human A3 adenosine receptor, wherein Gln at position 44 is Arg, Lys at position 119 is Arg, and Lys at position 119 are at position 120. Arg is replaced with Thr, and His at position 124 is replaced with Gln.
[0165]
Based on the sequence of the human A3 adenosine receptor cDNA, a 35-mer primer having the nucleotide sequence of SEQ ID NO: 20 and a 34-mer primer having the nucleotide sequence of SEQ ID NO: 21 were synthesized. The nucleotide sequence represented by SEQ ID NO: 20 is obtained by partially substituting a sequence based on the human A3 adenosine receptor cDNA to obtain a mouse A3 adenosine receptor specific 119th Arg, 120th Thr, and 124th Arg. Like Gln, the nucleotide sequence shown in SEQ ID NO: 21 partially substitutes a sequence based on human A3 adenosine receptor cDNA to encode Arg at position 44, which is specific for mouse A3 adenosine receptor. In addition, each was designed. Using these primers and using pBS-hA3R as a template, a PCR reaction solution was prepared and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method, and then dissolved in 50 μL of Universal Buffer K.SpeI and 37.5 unitsBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 250 bp was recovered using a GeneClean II kit according to the attached manual.
[0166]
2 μg of plasmid RpBluescript {II} SK (−) was dissolved in 50 μL of universal buffer K and 20 units ofSpeI and 30 unitsBamAfter adding HI and performing a digestion reaction at 37 ° C. for 20 hours, the reaction solution was extracted with ethanol / chloroform, and a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0167]
80 ng of the about 250 bp DNA fragment obtained above and plasmid pBS-hA3R-derivedSpeI-Bam50 ng of the HI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-3.
[0168]
(4) Construction of plasmid chimera-4
A plasmid chimera-4 containing a nucleotide sequence encoding the amino acid sequence at positions 17 to 45 of the chimeric A3 adenosine receptor (the sequence from positions 49 to 135 of SEQ ID NO: 32) was constructed as follows (see FIG. 10). . The amino acid sequence at positions 17 to 45 of the chimeric A3 adenosine receptor is such that, in the amino acid sequence at positions 17 to 45 of the human A3 adenosine receptor, Arg at position 42 is replaced with Thr and Gln at position 44 is replaced with Arg. .
[0169]
Based on the sequence of the human A3 adenosine receptor cDNA, a 34-mer primer having the nucleotide sequence of SEQ ID NO: 22 and a 28-mer primer having the nucleotide sequence of SEQ ID NO: 23 were synthesized. The nucleotide sequence represented by SEQ ID NO: 22 is obtained by partially substituting the sequence based on the human A3 adenosine receptor cDNA to encode the 42nd Thr and 44th Arg specific to the mouse A3 adenosine receptor. Designed.
[0170]
Using these primers and using pBS-hA3R as a template, a PCR reaction solution was prepared and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer M and mixed with 0.01% BSA and 37.5 units.XbaI was added and a digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoThe digestion reaction was performed at 37 ° C. for 20 hours with addition of RI. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 100 bp was recovered using a GeneClean II kit according to the attached manual.
[0171]
2.6 μg of plasmid pBluescriptII @ SK (−) is dissolved in 50 μL of Universal Buffer M and 0.01% BSA and 7.5 units ofXbaAfter addition of I and digestion reaction at 37 ° C. for 20 hours, the reaction solution was extracted with ethanol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoThe digestion reaction was performed at 37 ° C. for 20 hours with addition of RI. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0172]
About 10 ng of the about 100 bp DNA fragment obtained above and plasmid pBS-hA3R-derivedXbaI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-4.
[0173]
(5) Construction of plasmid chimera-1 + 2
A plasmid chimera-1 + 2 containing a nucleotide sequence encoding the amino acid sequence at positions 127 to 226 of the chimeric A3 adenosine receptor (sequences from 379 to 678 of SEQ ID NO: 32) was constructed as follows (see FIG. 11). .
[0174]
2.6 μg of plasmid chimera-1 was dissolved in 100 μL of universal buffer H and 30 units ofEcoRI and 25 creditsSpeI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 110 bp was recovered using a GeneClean II kit according to the attached manual.
[0175]
3 μg of plasmid chimera-2 was dissolved in 100 μL of Universal Buffer M and 37.5 units ofBamHI and 37.5 unitsXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 200 bp was recovered using a GeneClean II kit according to the attached manual.
[0176]
2.6 μg of plasmid pBluescriptII @ SK (−) is dissolved in 20 μL of Universal Buffer K and 7.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H, andEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0177]
10 ng of the DNA fragment (about 110 bp) derived from the plasmid chimera-1 and 10 ng of the DNA fragment derived from the plasmid chimera-2 (about 200 bp) and the plasmid pBluescriptII @ SK (-)BamHI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-1 + 2.
[0178]
(6) Construction of plasmid chimera-3 + 4
A plasmid chimera-3 + 4 containing the nucleotide sequence encoding the amino acid sequence at positions 17 to 127 of the chimeric A3 adenosine receptor (the sequence from positions 49 to 381 of SEQ ID NO: 32) was constructed as follows (see FIG. 12). .
[0179]
1.2 μg of plasmid chimera-3 was dissolved in 50 μL of NE buffer 3 (New England Biolabs) and 24 units ofBsiWI was added and a digestion reaction was performed at 55 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 250 bp was recovered using a Geneclean II kit according to the attached manual.
[0180]
1 μg of plasmid chimera-4 was dissolved in 50 μL of NE buffer 3 and 24 units ofBsiWI was added and a digestion reaction was performed at 55 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3.1 kbp was recovered using a GeneClean II kit according to the attached manual.
[0181]
10 ng of the DNA fragment (about 250 bp) derived from plasmid chimera-3 obtained above and plasmid chimera-4 derivedBsiWI andEco50 ng of the RI fragment (about 3.1 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-3 + 4.
[0182]
(7) Construction of plasmid chimera-1 + 2 + 3 + 4
A plasmid chimera-1 + 2 + 3 + 4 containing the nucleotide sequence encoding the amino acid sequence at positions 17 to 226 of the chimeric A3 adenosine receptor (the sequence from positions 49 to 678 of SEQ ID NO: 32) was constructed as follows (see FIG. 13). .
[0183]
3 μg of plasmid chimera-1 + 2 was dissolved in 100 μL of universal buffer H and 24 units ofEcoRI and 20 creditsBglII was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 300 bp was recovered using a GeneClean II kit according to the attached manual.
[0184]
Dissolve 2.8 μg of plasmid chimera-3 + 4 in 100 μL of Universal Buffer K and add 75 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 100 μL of Universal Buffer H, andEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a Geneclean II kit according to the attached manual.
[0185]
10 ng of the DNA fragment (about 300 bp) derived from the plasmid chimera-1 + 2 and the plasmid chimera-3 + 4BamHI andEco50 ng of the RI fragment (about 3.3 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-1 + 2 + 3 + 4.
[0186]
(8) Construction of plasmid A3R-N1
A plasmid A3R-N1 having a mouse genomic DNA sequence of about 350 bp upstream of the initiation codon of the mouse A3 adenosine receptor gene was constructed as follows (see FIG. 14).
To obtain a mouse genomic DNA fragment 350 bp upstream of the initiation codon of the mouse A3 adenosine receptor gene, PCR was performed using plasmid pBS-ex1 as a template. That is, a 27-mer primer having the nucleotide sequence of SEQ ID NO: 24 and a 28-mer primer having the nucleotide sequence of SEQ ID NO: 25 were prepared based on the nucleotide sequence of the mouse A3 adenosine receptor gene. Using these primers, a reaction solution for PCR was prepared using pBS-ex1 as a template and reacted at 94 ° C. for 5 minutes, followed by a cycle of 94 ° C. for 1 minute, 52 ° C. for 30 seconds, and 72 ° C. for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment of about 350 bp was recovered by an ethanol precipitation method. The DNA fragment is dissolved in 100 μL of Universal Buffer K and 75 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 100 μL of Universal Buffer H and 60 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 350 bp was recovered using a GeneClean II kit according to the attached manual.
[0187]
Dissolve 2.8 μg of plasmid pBluescriptII @ SK (-) in 50 μL of Universal Buffer K and add 30 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with ethanol / chloroform, a DNA fragment of about 3 kbp was recovered by an ethanol precipitation method.
[0188]
50 ng of a DNA fragment of about 350 bp upstream from the initiation codon of the mouse A3 adenosine receptor gene obtained above and plasmid pBluescriptII @ SK (-)-derivedBamHI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as A3R-N1.
[0189]
(9) Construction of plasmid A3R-N2
A plasmid A3R-N2 having a base sequence of about 200 bp downstream from the initiation codon of the human A3 adenosine receptor cDNA was constructed as follows (see FIG. 15).
[0190]
In order to obtain a DNA fragment of about 200 bp downstream from the initiation codon of the human A3 adenosine receptor cDNA sequence, PCR was performed using plasmid pBS-hA3R as a template. That is, a 28-mer primer having the nucleotide sequence of SEQ ID NO: 26 and a 26-mer primer having the nucleotide sequence of SEQ ID NO: 27 were prepared based on the sequence of the human A3 adenosine receptor cDNA. Using these primers, a reaction solution for PCR was prepared using pBS-hA3R as a template and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 52 ° C for 30 seconds, and 72 ° C for 1 minute Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight.
[0191]
After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment of about 350 bp was recovered by an ethanol precipitation method. The DNA fragment is dissolved in 100 μL of Universal Buffer K and 75 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 100 μL of Universal Buffer H and 60 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 200 bp was recovered using a GeneClean II kit according to the attached manual.
[0192]
Dissolve 2.8 μg of plasmid pBluescriptII @ SK (-) in 50 μL of Universal Buffer K and add 30 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with ethanol / chloroform, a DNA fragment of about 3 kbp was recovered by an ethanol precipitation method.
[0193]
50 ng of a DNA fragment of about 200 bp downstream from the initiation codon of human A3 adenosine receptor cDNA obtained above and plasmid pBluescriptII @ SK (-)BamHI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as A3R-N2.
[0194]
(10) Construction of plasmid A3R-N3
A DNA fragment derived from the plasmid A3R-N2 (about 200 bp downstream from the initiation codon of the human A3 adenosine receptor cDNA) downstream of the sequence 5 'to the initiation codon of the mouse A3 adenosine receptor gene of the plasmid A3R-N1 The plasmid A3R-N3 into which was introduced was constructed as follows (see FIG. 16).
[0195]
2.3 μg of plasmid A3R-N2 was dissolved in 40 μL of Universal Buffer H and 12 units ofEcoRI and 10 unitsSphI was added and a digestion reaction was performed at 37 ° C. for 20 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 200 bp was recovered using a GeneClean II kit according to the attached manual.
1.6 μg of plasmid A3R-N1 was dissolved in 40 μL of Universal Buffer H and 12 units ofEcoRI and 10 unitsSphI was added and a digestion reaction was performed at 37 ° C. for 20 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3.35 kbp was recovered using a GeneClean II kit according to the attached manual.
[0196]
From the plasmid A3R-N2 obtained aboveEcoRI-Sph10 ng of the I fragment (about 200 bp) and plasmid A3R-N1EcoRI-Sph50 ng of the I fragment (about 3.35 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as A3R-N3.
[0197]
(11) Construction of plasmid cA3R-N
A mouse genomic sequence upstream of the initiation codon of the mouse A3 adenosine receptor gene upstream of the nucleotide sequence encoding the amino acid sequence 1-226 of the chimeric A3 adenosine receptor (sequences 1-678 of SEQ ID NO: 32) A plasmid cA3R-N containing a sequence into which 350 bp was inserted was constructed as follows (see FIG. 17).
[0198]
Dissolve 2.8 μg of plasmid A3R-N3 in 100 μL of Universal Buffer K, add 0.01% BSA and 50 unitsNcoI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 100 μL of Universal Buffer L and 50 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 400 bp was recovered using a GeneClean II kit according to the attached manual.
[0199]
2.4 μg of plasmid chimera-1 + 2 + 3 + 4 is dissolved in 100 μL of universal buffer K, and 0.01% BSA and 50 units ofNcoI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 100 μL of Universal Buffer L and 50 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 3.6 kbp was recovered using a GeneClean II kit according to the attached manual.
[0200]
The plasmid A3R-N3-derivedNcoI-Sac80 ng of the I fragment (about 400 bp) and the plasmid chimera-1 + 2 + 3 + 4NcoI-Sac50 ng of the I fragment (about 3.6 kbp) was dissolved in 10 μL of distilled water, and the mixture was ligated with Ligation Kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as cA3R-N.
[0201]
(12) Construction of plasmid A3R-N5
A plasmid A3R-N5 having a mouse genomic DNA sequence 5 kbp upstream of exon 1 of the mouse A3 adenosine receptor gene was constructed as follows (see FIG. 18).
3.3 μg of plasmid pBS-ex1 was dissolved in 20 μL of Universal Buffer T containing 0.01% BSA and 4 units ofSmaI and 5 unitsSacAfter addition of I and digestion reaction at 37 ° C. for 20 hours, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 5 kbp was recovered using a GeneClean II kit according to the attached manual.
[0202]
1.1 μg of plasmid pBS-hA3R was dissolved in 50 μL of Universal Buffer H and 20 units ofNdeI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, both ends of the recovered DNA fragment were subjected to a blunting treatment using a DNA blunting kit (manufactured by Takara Shuzo) according to the attached manual. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was dissolved in 40 μL of Universal Buffer H, and 20 units ofSacAfter addition of I and digestion reaction at 37 ° C. for 16 hours, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0203]
1 μg of the DNA fragment (about 5 kbp) derived from the plasmid pBS-ex1 and the plasmid pBS-hA3RNdeI (blunted)Sac50 ng of the I fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of 2 I solution was added, and the binding reaction was performed at 16 ° C. for 16 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as A3R-N5.
[0204]
(13) Construction of plasmid arm1 + cA3R-N
A mouse genomic sequence upstream of the initiation codon of the mouse A3 adenosine receptor gene upstream of the nucleotide sequence encoding the amino acid sequence 1-226 of the chimeric A3 adenosine receptor (sequences 1-678 of SEQ ID NO: 32) Plasmid arm1 + cA3R-N containing the sequence into which 5.4 kbp was inserted was constructed as follows (see FIG. 19).
[0205]
4 μg of plasmid cA3R-N was dissolved in 50 μL of Universal Buffer L and 20 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 1 kbp was recovered using a GeneClean II kit according to the attached manual.
[0206]
Dissolve 3.4 μg of plasmid A3R-N5 in 50 μL of Universal Buffer L and add 20 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 9 kbp was recovered using a GeneClean II kit according to the attached manual.
[0207]
From the plasmid cA3R-N obtained aboveSacI-Eco10 ng of the RI fragment (about 1 kbp) and plasmid A3R-N5SacI-Eco50 ng of the RI fragment (about 9 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as arm1 + cA3R-N.
[0208]
(14) Construction of plasmid chimera-5
Plasmid chimera-5 containing the nucleotide sequence encoding the amino acid sequence at positions 225 to 284 of the chimeric A3 adenosine receptor (the sequence from positions 673 to 852 of SEQ ID NO: 32) was constructed as follows (see FIG. 20). . The amino acids 225 to 284 of the chimeric A3 adenosine receptor are identical to the amino acids 225 to 284 of the human A3 adenosine receptor.
[0209]
Based on the sequence of the human A3 adenosine receptor cDNA, a 24-mer primer having the nucleotide sequence of SEQ ID NO: 28 and a 30-mer primer having the nucleotide sequence of SEQ ID NO: 29 were synthesized. Using these primers and using pBS-hA3R as a template, a PCR reaction solution was prepared and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer K and 37.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 180 bp was recovered using a Geneclean II kit according to the attached manual.
[0210]
2.6 μg of plasmid pBluescriptII @ SK (−) is dissolved in 20 μL of Universal Buffer K and 7.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0211]
The about 180 bp DNA fragment 12 ng obtained above and the plasmid pBluescript II @ SK (-)-derivedBamHI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-5.
[0212]
(15) Construction of plasmid chimera-6
Plasmid containing a nucleotide sequence encoding the amino acid sequence at positions 282 to 318 of the chimeric A3 adenosine receptor (sequences from 844 to 957 in SEQ ID NO: 32) and a sequence of about 360 bp after the termination codon of the mouse A3 adenosine receptor gene chimera-6 was constructed as follows (see FIG. 21). In the amino acid sequence at positions 282 to 318 of the chimeric A3 adenosine receptor, Cys at position 285 of the amino acid sequence at positions 283 to 319 of the mouse A3 adenosine receptor has been substituted with Tyr.
[0213]
Based on the sequence of the mouse A3 adenosine receptor gene, a 32-mer primer having the nucleotide sequence of SEQ ID NO: 30 and a 24-mer primer having the nucleotide sequence of SEQ ID NO: 31 were synthesized. The nucleotide sequence represented by SEQ ID NO: 30 was designed to encode a 284th Tyr specific for the human A3 adenosine receptor by partially substituting a sequence based on the mouse A3 adenosine receptor gene. Using these primers, a reaction solution for PCR was prepared using pBS-ex2 as a template, and reacted at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute. Was repeated for 8 cycles, followed by a further 22 cycles of a cycle of 94 ° C. for 1 minute and 68 ° C. for 2 minutes, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer K and 37.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 20 hours. Subsequently, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 470 bp was recovered using a GeneClean II kit according to the attached manual.
[0214]
2.6 μg of plasmid pBluescriptII @ SK (−) was dissolved in 20 μL of Universal Buffer K and 7.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, this DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0215]
24 ng of the DNA fragment of about 470 bp obtained above and plasmid pBluescriptII @ SK (-)-derivedBamHI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-6.
[0216]
(16) Construction of plasmid chimera-5 + 6
Plasmid containing the nucleotide sequence encoding the amino acid sequence at positions 225 to 318 of the chimeric A3 adenosine receptor (the sequence from positions 673 to 957 of SEQ ID NO: 32) and the sequence of about 360 bp after the termination codon of the mouse A3 adenosine receptor gene chimera-5 + 6 was constructed as follows (see FIG. 22).
[0219]
2 μg of plasmid chimera-5 was dissolved in 100 μL of Universal Buffer H and 30 units ofEcoRI and 25 creditsMluI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 180 bp was recovered using a GeneClean II kit according to the attached manual.
[0218]
2 μg of plasmid chimera-6 was dissolved in 100 μL of universal buffer H and 30 units ofEcoRI and 25 creditsMluI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After the reaction solution was extracted with ethanol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 3.5 kbp was recovered using a GeneClean II kit according to the attached manual.
[0219]
Derived from plasmid chimera-5 obtained aboveEcoRI-Mlu10 ng of the I fragment (about 180 bp) and the plasmid chimera-6EcoRI-Mlu50 ng of the I fragment (about 3.5 kbp) was dissolved in 10 μL of distilled water, and the mixture was ligated with Ligation Kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as chimera-5 + 6.
[0220]
(17) Construction of plasmid A3R-C1
Plasmid A3R-C1 having a mouse genomic DNA of about 1.8 kbp containing a polyadenylation signal present in exon 2 of mouse A3 adenosine receptor gene was constructed as follows (see FIG. 23).
[0221]
5 μg of plasmid pBS-ex2 was dissolved in 100 μL of Universal Buffer K, and 30 units ofPstI and 30 unitsBamAfter adding HI and performing a digestion reaction at 37 ° C. for 16 hours, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of distilled water, and after agarose gel electrophoresis, a DNA fragment of about 1.8 kbp was recovered using a GeneClean II kit according to the attached manual.
[0222]
Dissolve 2.8 μg of pBluescriptII @ SK (-) in 100 μL of Universal Buffer K and add 30 units ofPstI and 30 unitsBamHI was added and digestion reaction was performed at 37 ° C. for 24 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method.
100 ng of the mouse genomic DNA fragment (about 1.8 kbp) containing the polyadenylation signal present in exon 2 of the A3 adenosine receptor gene and pBluescriptII @ SK (-)PstI-Bam30 ng of the HI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
[0223]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as A3R-C1.
[0224]
(18) Construction of plasmid A3R-C2
About 1.6 kbp of the sequence downstream of the polyadenylation signal was removed from the plasmid A3R-C1, andSacThe plasmid A3R-C2 to which the II recognition sequence was added was constructed as follows (see FIG. 24).
[0225]
Dissolve 2 μg of plasmid A3R-C1 in 100 μL of Universal Buffer M and add 50 units ofHinAfter cII was added and digestion reaction was performed at 37 ° C. for 16 hours, the reaction solution was extracted with phenol / chloroform, and then a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment is dissolved in 100 μL of Universal Buffer L and 50 units ofKpnI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. After dissolving the DNA fragment in 20 μL of distilled water, agarose gel electrophoresis was performed, and a DNA fragment of about 3.2 kbp was recovered using a GeneClean II kit according to the attached manual.
[0226]
Subsequently, a 17-mer synthetic oligo DNA represented by SEQ ID NO: 34 and a 13-mer synthetic oligo DNA represented by SEQ ID NO: 35 were prepared, 10 pmol of each was added to 100 μL of distilled water, heated at 90 ° C. for 10 minutes, and then room temperature. The respective synthetic oligo DNAs were annealed by returning to the above.
The plasmid A3R-C1 derived from the plasmid obtained by removing about 1.6 kbp of the mouse genomic DNA sequence obtained above.HincII andKpn30 ng of the I fragment (about 3.2 kbp) and 0.2 pmol of the annealed synthetic oligo DNA were dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 μL of 2 I solution was added, and the binding reaction was performed at 16 ° C. for 1 hour.
[0227]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Hereinafter, this plasmid is referred to as A3R-C2.
[0228]
(19) Construction of plasmid cA3R-C
A nucleotide sequence encoding the amino acid sequence at positions 225 to 318 of the chimeric A3 adenosine receptor (sequences from position 673 to position 957 of SEQ ID NO: 32) and the stop codon of the mouse A3 adenosine receptor geneHinA plasmid cA3R-C containing a sequence of about 500 bp up to the cII site was constructed as follows (see FIG. 25).
[0229]
1 μg of plasmid A3R-C2 is dissolved in 100 μL of Universal Buffer T, and 0.01% BSA and 50 units ofSacII was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer K, and 15 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 150 bp was recovered using a GeneClean II kit according to the attached manual.
[0230]
1.5 μg of plasmid chimera-5 + 6 was dissolved in 100 μL of Universal Buffer T, and 0.01% BSA and 50 units ofSacII was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 100 μL of Universal Buffer K, and 75 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 3.7 kbp was recovered using a GeneClean II kit according to the attached manual.
[0231]
From the plasmid A3R-C2 obtained aboveSacII-Bam10 ng of the HI fragment (about 150 bp) and the plasmid chimera-5 + 6-derivedSacII-Bam50 ng of the HI fragment (approximately 3.7 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as cA3R-C.
[0232]
(20) Construction of plasmid ploxP-HPRT-hA3R-arm2
Plasmid ploxpHPRT2 in which the hprt gene connected downstream of the PGK promoter is present between the two loxP sequences.SmaI /BamBetween the HI sites, in the untranslated region of exon 2 of the mouse A3 adenosine receptor geneBamDownstream of exon 2 from the HI siteApaA plasmid ploxP-HPRT-hA3R-arm2 into which a mouse genomic DNA of about 2.8 kbp up to the I site was inserted was constructed as follows (see FIG. 26).
[0233]
3 μg of the plasmid pBS-ex2 was dissolved in 100 μL of Universal Buffer M (manufactured by Takara Shuzo) containing 0.01% bovine serum albumin (hereinafter abbreviated as BSA), and 75 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 12 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 100 μL of Universal Buffer L (Takara Shuzo) and 75 units ofApaI was added and a digestion reaction was performed at 37 ° C. for 12 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 10 μL of distilled water, and after agarose gel electrophoresis, a DNA fragment of about 4 kbp was recovered using a GeneClean II kit according to the attached manual. Subsequently, both ends of the recovered DNA fragment were subjected to a blunting treatment using a DNA blunting kit (manufactured by Takara Shuzo) according to the attached manual. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was dissolved in 15 μL of Universal Buffer K (Takara Shuzo), and 7.5 units ofBamHI was added and digestion reaction was performed at 37 ° C. for 10 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 2.8 kbp was recovered using a GeneClean II kit according to the attached manual. This fragment is located in the untranslated region of exon 2 of the mouse A3 adenosine receptor gene.BamDownstream from the HI siteApaGenomic DNA corresponding to about 2.8 kbp to the I site, including the polyadenylation signal in the untranslated region of exon 2.
[0234]
2.8 μg of plasmid ploxpHPRT2 (WO # 01/33957) was dissolved in 100 μL of Universal Buffer T (manufactured by Takara Shuzo) containing 0.01% BSA, and 75 units ofSmaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment is dissolved in 100 μL of Universal Buffer K and 75 units ofBamHI was added and a digestion reaction was performed at 37 ° C. for 12 hours. After extracting the reaction solution with phenol / chloroform, DNA fragments were recovered by an ethanol precipitation method and dissolved in 20 μL of distilled water.
[0235]
90 ng of the mouse genomic DNA fragment (about 2.8 kbp) containing exon 2 of the A3 adenosine receptor obtained above and plasmid ploxpHPRT2-derivedSmaI-Bam50 ng of the HI fragment (about 5.7 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 μL of 2 I solution was added, and the binding reaction was performed at 16 ° C. for 16 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as “ploxP-HPRT-hA3R-arm2”.
[0236]
(21) Construction of plasmid ploxP-RE-HPRT-hA3R-arm2
In plaxP-HPRT-hA3R-arm2 constructed in (20)EcoWith RISacThe plasmid ploxP-RE-HPRT-hA3R-arm2 to which the recognition sequence of II was added was constructed as follows (see FIG. 27).
[0237]
2 μg of plasmid ploxP-HPRT-hA3R-arm2 was dissolved in 100 μL of Universal Buffer K and 40 units ofHpaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was dissolved in 100 μL of Universal Buffer L, and 50 units ofKpnI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. After dissolving the DNA fragment in 15 μL of distilled water, agarose gel electrophoresis was performed, and a DNA fragment of about 9 kbp was recovered using a GeneClean II kit according to the attached manual.
[0238]
Subsequently, a 38-mer synthetic oligo DNA represented by SEQ ID NO: 36 and a 34-mer synthetic oligo DNA represented by SEQ ID NO: 37 were prepared, 10 pmol of each was added to 100 μL of distilled water, heated at 90 ° C. for 10 minutes, and then heated to room temperature. To allow annealing.
Derived from plasmid ploxP-HPRT-hA3R-arm2, obtained aboveHpaI-KpnI fragment (about 9 kbp) (30 ng) and annealed synthetic oligo DNA (0.2 pmol) were dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of 2 I solution was added, and the binding reaction was performed at 16 ° C. for 1 hour.
[0239]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as ploxP-RE-HPRT-hA3R-arm2.
[0240]
(22) Construction of plasmid ploxP-RE-C-HPRT-chiA3R-arm2
A nucleotide sequence encoding the amino acid sequence at positions 225 to 318 of the chimeric A3 adenosine receptor (sequences from position 673 to position 957 of SEQ ID NO: 32) and the stop codon of the mouse A3 adenosine receptor geneHinPlasmid ploxP-RE-C-HPRT-chiA3R-arm2, in which a sequence of about 500 bp up to the cII site was introduced upstream of the hprt gene of plasmid ploxP-RE-HPRT-hA3R-arm2, was constructed as follows ( See FIG. 28).
[0241]
2 μg of plasmid cA3R-C is dissolved in 100 μL of Universal Buffer T, and 0.01% BSA and 50 units ofSacII was added and digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoThe digestion reaction was performed at 37 ° C. for 20 hours with addition of RI. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 800 bp was recovered using a GeneClean II kit according to the attached manual.
[0242]
3.2 μg of plasmid ploxP-RE-HPRT-hA3R-arm2 was dissolved in 100 μL of Universal Buffer T, and 0.01% BSA and 50 units ofSacII was added and digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoThe digestion reaction was performed at 37 ° C. for 20 hours with addition of RI. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 8.5 kbp was recovered using a GeneClean II kit according to the attached manual.
[0243]
From the plasmid cA3R-C obtained aboveEcoRI-Sac100 ng of the II fragment (about 800 bp) and the plasmid ploxP-RE-HPRT-hA3R-arm2EcoRI-Sac100 ng of the II fragment (about 8.5 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as ploxP-RE-C-HPRT-chiA3R-arm2.
[0244]
(23) Construction of ploxP-arm1-HPRT-chiA3R-arm2
A mouse genomic sequence of about 5.4 kbp upstream of the initiation codon of the mouse A3 adenosine receptor gene is added upstream of the base sequence encoding the chimeric A3 adenosine receptor (sequence of SEQ ID NO: 32), and the mouse A3 adenosine receptor downstream. After the stop codon of the geneHinA plasmid ploxP-arm1-HPRT-chiRTR-arm2 having a structure in which a sequence to which a sequence of about 500 bp was added up to the cII site was introduced upstream of the hprt gene of the plasmid ploxP-RE-HPRT-hA3R-arm2 was constructed as follows. (See FIG. 29).
[0245]
2.5 μg of plasmid arm1 + cA3R-N was dissolved in 100 μL of Universal Buffer M and 50 units ofSpeI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 7 kbp was recovered using a GeneClean II kit according to the attached manual.
[0246]
2 μg of plasmid ploxP-RE-C-HPRT-chiA3R-arm2 was dissolved in 100 μL of Universal Buffer M and 50 units ofNheI was added and a digestion reaction was performed at 37 ° C. for 20 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 9 kbp was recovered using a GeneClean II kit according to the attached manual.
[0247]
Derived from plasmid arm1 + cA3R-N obtained aboveNheI-Eco100 ng of the RI fragment (about 7 kbp) and the plasmid ploxP-RE-C-HPRT-chiA3R-arm2NheI-Eco100 ng of the RI fragment (about 9 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as “ploxP-arm1-HPRT-chiA3R-arm2”.
[0248]
(24) Construction of ploxP-DT-arm1-HPRT-chiA3R-arm2
Plasmid ploxP-DT-arm1- having a diphtheria toxin (DT) expression unit inserted upstream of a sequence of about 5.4 kbp upstream of the initiation codon of the mouse A3 adenosine receptor gene of plasmid ploxP-arm1-HPRT-chiA3R-arm2. HPRT-chiA3R-arm2 was constructed as follows (see FIG. 30).
[0249]
3.55 μg of plasmid pKO @ SelectDT (manufactured by Lexicon Genetics) was dissolved in 40 μL of NE buffer 4 and 2 units ofRsrII was added and digestion reaction was performed at 37 ° C. for 24 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 1.3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0250]
2.5 μg of plasmid ploxP-arm1-HPRT-chiA3R-arm2 was dissolved in 100 μL of NE buffer 4 and 5 units ofRsrII was added and digestion reaction was performed at 37 ° C. for 24 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Subsequently, the DNA fragment was subjected to agarose gel electrophoresis, and a DNA fragment of about 16 kbp was recovered using a GeneClean II kit according to the attached manual.
[0251]
From the plasmid pKO @ SelectDT derived aboveRsr50 ng of the II fragment (about 1.3 kbp) and the plasmid ploxP-arm1-HPRT-chiA3R-arm2RsrIIng fragment (about 16 kbp) (100 ng) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 16 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as ploxP-DT-arm1-HPRT-chiA3R-arm2.
[0252]
In addition, using a DNA sequencer 377, the nucleotide sequence (SEQ ID NO: 32) encoding the chimeric A3 adenosine receptor contained in the plasmid ploxP-DT-arm1-HPRT-chiA3R-arm2 was confirmed. FIG. 31 schematically shows the structure of the chimeric A3 adenosine receptor gene prepared in this example.
[0253]
Example 5 Construction of Expression Vector for Human A3 Adenosine Receptor
(1) Construction of plasmid polyA @ in @ pBS
A plasmid polyA in pBS containing the SV40 polyadenylation signal was constructed as follows (see FIG. 32).
Preparation of a 26-mer primer having the nucleotide sequence of SEQ ID NO: 38 containing the polyadenylation signal of SV40 of plasmid pCAG-GFP-pHPRTp (WO $ 01/33957) and a 35-mer primer having the nucleotide sequence of SEQ ID NO: 39 did. Using these primers and a plasmid pCAG-GFP-pHPRTp as a template, a PCR reaction solution was prepared and reacted at 94 ° C. for 5 minutes, followed by 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 second. The cycle for 30 minutes was repeated 30 times, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 40 μL of Universal Buffer T containing 0.01% BSA and 15 units ofXbaI and 10 unitsSacII was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 300 bp containing the SV40 polyadenylation signal was recovered using a GeneClean II kit according to the attached manual.
[0254]
3 μg of plasmid pBluescriptII @ SK (−) was dissolved in 40 μL of Universal Buffer T containing 0.01% BSA, and 15 units ofXbaI and 10 unitsSacII was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
100 ng of the DNA fragment (about 300 bp) containing the polyadenylation signal of SV40 obtained above and plasmid pBluescriptII @ SK (-)XbaI-Sac100 ng of the II fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
[0255]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. The nucleotide sequence was determined using a DNA sequencer 377, and the SV40 polyadenylation signal contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as polyA @ in @ pBS.
[0256]
(2) Construction of hA3R @ in @ pBS
A plasmid hA3R in pBS having a sequence encoding a human A3 adenosine receptor with a FLAG tag added to the N-terminus was constructed as follows (see FIG. 33).
Based on the sequence of the human A3 adenosine receptor cDNA, a 55-mer primer having the nucleotide sequence of SEQ ID NO: 40 containing a sequence encoding the FLAG tag and a 30-mer primer having the nucleotide sequence of SEQ ID NO: 41 were prepared. did. Using these primers and using the plasmid pBS-hA3R as a template, a PCR reaction solution was prepared and reacted at 94 ° C for 5 minutes, followed by 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute. The cycle was repeated 30 times, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer M containing 0.01% BSA and 37.5 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 40 μL of Universal Buffer H and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After agarose gel electrophoresis of the reaction solution, a DNA fragment of about 1 kbp containing a sequence encoding a human A3 adenosine receptor with a FLAG tag added to the N-terminus was recovered using a GeneClean II kit according to the attached manual.
[0257]
3 μg of plasmid pBluescriptII @ SK (−) is dissolved in 40 μL of Universal Buffer M containing 0.01% BSA, and 30 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. This DNA fragment was dissolved in 40 μL of Universal Buffer H, and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0258]
100 ng of the DNA fragment (about 1 kbp) containing the sequence encoding the human A3 adenosine receptor to which the FLAG tag was added at the N-terminus and the plasmid pBluescriptII @ SK (-)XbaI-Eco50 ng of the RI fragment (about 3 kbp) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 µL of 2 I solution was added, and the binding reaction was performed at 16 ° C for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. The nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid and encoding the human A3 adenosine receptor with a FLAG tag added to the N-terminus was confirmed. Hereinafter, this plasmid is referred to as hA3R in pBS.
[0259]
(3) Construction of plasmid hA3R-polyA in pBS
A plasmid hA3R-polyA in pBS having a SV40 polyadenylation signal added downstream of a sequence encoding a human A3 adenosine receptor having a FLAG tag added to the N-terminus was constructed as follows (see FIG. 34).
[0260]
Dissolve 1.25 μg of the plasmid hA3R in pBS prepared in (2) above in 40 μL of Universal Buffer M containing 0.01% BSA, and add 30 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer H and 12 units ofEcoThe digestion reaction was performed at 37 ° C. for 6 hours after adding RI. After agarose gel electrophoresis of the reaction solution, a DNA fragment of about 1 kbp containing a sequence encoding a human A3 adenosine receptor with a FLAG tag added to the N-terminus was recovered using a GeneClean II kit according to the attached manual.
[0261]
2.5 μg of the plasmid polyAｐin pBS prepared in the above (1) was dissolved in 50 μL of Universal Buffer M containing 0.01% BSA, and 30 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The recovered DNA fragment was dissolved in 40 μL of Universal Buffer H, and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 3.3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0262]
40 ng of the DNA fragment (about 1 kbp) containing the sequence encoding the human A3 adenosine receptor to which the FLAG tag was added at the N-terminus and the plasmid polyA in pBS-derivedXbaI-Eco50 ng of the RI fragment (about 3.3 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 µL of 2 I solution was added, and a binding reaction was performed at 16 ° C for 3 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as hA3R-polyA in pBS.
[0263]
(4) Construction of human A3 adenosine receptor expression vector hA3R in pCAG
A human A3 adenosine receptor expression vector hA3R in pCAG containing a FLAG tag at the N-terminus containing a CAG promoter was constructed as follows (see FIG. 35).
[0264]
3 μg of the plasmid hA3R-polyA in pBS prepared in the above (3) was dissolved in 40 μL of universal buffer H, and 12 units ofEcoRI and 15 creditsEcoRV was added and digestion reaction was performed at 37 ° C. for 16 hours. The reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of about 1.5 kbp (a DNA fragment containing a polyadenylation signal of SV40 downstream of a sequence encoding a human A3 adenosine receptor with a FLAG tag added to the N-terminus) was obtained. And using Gene Clean II kit according to the attached manual.
[0265]
4 μg of plasmid pCAG-GFP-pHPRTp was dissolved in 50 μL of Universal Buffer K and 20 units ofHpaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. Dissolve in 40 μL of Universal Buffer H and add 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 8 kbp was recovered using a GeneClean II kit according to the attached manual.
[0266]
From the plasmid hA3R-polyAｐin pBS obtained aboveEcoRI-Eco100 ng of RV fragment (about 1.5 kbp) and pCAG-GFP-pHPRTp-derivedEcoRI-Hpa50 ng of the I fragment (about 8 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 3 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as hA3R in pCAG.
[0267]
Example 6 Construction of Expression Vector for Chimeric A3 Adenosine Receptor
An expression vector for mammalian cells of a chimeric A3 adenosine receptor containing a CAG promoter, cA3R in pCAG, was constructed by the following steps (1) to (4).
(1) Preparation of DNA encoding chimeric A3 adenosine receptor with FLAG tag added to the N-terminus
Based on the sequence encoding the chimeric A3 adenosine receptor (SEQ ID NO: 32), a 55-mer primer having the nucleotide sequence represented by SEQ ID NO: 40 containing the sequence encoding the FLAG tag, and the nucleotide sequence represented by SEQ ID NO: 42 A 34-mer primer was prepared. Using these primers, a PCR reaction solution was prepared using the plasmid ploxP-DT-arm1-HPRT-chiA3R-arm2 prepared in Example 4 (24) as a template, and reacted at 94 ° C. for 5 minutes. A cycle of 30 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute was repeated 30 times, and PCR was performed under the condition of storing at 4 ° C. overnight. After extracting the PCR reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 50 μL of Universal Buffer M containing 0.01% BSA and 37.5 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 40 μL of Universal Buffer H and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The fragment amplified by PCR by this digestion reactionEcoPresent in the sequence encoding the chimeric A3 adenosine receptor as well as the RI siteEcoAlso cut at the RI site. After agarose gel electrophoresis of the reaction solution, a DNA fragment of about 700 bp encoding the N-terminus of the chimeric A3 adenosine receptor having a FLAG tag added to the N-terminus, and a DNA fragment of about 300 bp encoding the C-terminus, Was recovered using a GeneClean II kit according to the attached manual.
[0268]
(2) Construction of plasmid cA3R (N) in pBS
A plasmid cA3R (N) in pBS having a sequence encoding the N-terminus of a chimeric A3 adenosine receptor having a FLAG tag added to the N-terminus was constructed as follows (see FIG. 36).
[0269]
2 μg of plasmid pBluescriptII @ SK (−) was dissolved in 40 μL of Universal Buffer H, and 24 units ofEcoThe digestion reaction was performed at 37 ° C. for 2 hours with addition of RI. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual. The recovered DNA is dissolved in 50 μL of alkaline phosphatase buffer, 40 units of bovine small intestine-derived alkaline phosphatase is added, the reaction is carried out at 50 ° C. for 1 hour, phenol / chloroform extraction is performed, and the ends are dephosphorylated by ethanol precipitation. The obtained DNA fragment of about 3 kbp was recovered.
[0270]
Derived from the above plasmid pBluescriptII @ SK (-)Eco100 ng of the RI fragment (about 3 kbp) and 100 ng of the DNA fragment (about 700 bp) containing the sequence encoding the N-terminal of the chimeric A3 adenosine receptor having a FLAG tag added to the N-terminus prepared in (1) were distilled at 10 μL. Dissolve in water and ligate kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
[0271]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. In addition, the nucleotide sequence was determined using a DNA sequencer 377, and a 700 bp sequence encoding the N-terminal side of the chimeric A3 adenosine receptor with a FLAG tag added to the N-terminus contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as cA3R (N) in pBS.
[0272]
(3) Construction of plasmid cA3R (C) in pCAG
Plasmid cA3R (C) in pCAG having a sequence obtained by adding the SV40 polyadenylation signal downstream of the sequence encoding the C-terminal of the chimeric A3 adenosine receptor and the CAG promoter was constructed as follows (see FIG. 37). ).
[0273]
2.5 μg of the plasmid polyA in pBS prepared in Example 5 (4) was dissolved in 50 μL of Universal Buffer M containing 0.01% BSA, and 37.5 units ofXbaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 40 μL of Universal Buffer H and 24 units ofEcoRV was added and digestion reaction was performed at 37 ° C. for 16 hours.
[0274]
After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 300 bp containing the SV40 polyadenylation signal was recovered using a GeneClean II kit according to the attached manual.
4 μg of plasmid pCAG-GFP-pHPRTp was dissolved in 50 μL of Universal Buffer K and 20 units ofHpaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 40 μL of Universal Buffer H and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 8 kbp was recovered using a GeneClean II kit according to the attached manual.
[0275]
The plasmid polyA in pBS derived above obtainedXbaI-Eco50 ng of RV fragment (about 300 bp), derived from plasmid pCAG-GFP-pHPRTpHpaI-EcoDissolve 50 ng of the RI fragment (about 8 kbp) and 50 ng of the DNA fragment (about 300 bp) containing the sequence encoding the C-terminal side of the chimeric A3 adenosine receptor isolated in (1) in 10 μL of distilled water, and prepare a ligation kit. Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 16 hours.
[0276]
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as cA3R (C) in pCAG.
[0277]
(4) Construction of plasmid cA3R in pCAG
An expression vector for chimeric A3 adenosine receptor for mammalian cells, cA3R in pCAG, was constructed as follows (see FIG. 38).
[0278]
4 μg of the plasmid cA3R (N) Ｎin pBS prepared in the above (2) was dissolved in 40 μL of universal buffer H, and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 700 bp (a DNA fragment encoding the N-terminal of the chimeric A3 adenosine receptor having a FLAG tag added to the N-terminus) was attached using a GeneClean II kit. Collected according to the manual.
[0279]
2 μg of the plasmid cA3R (C) {in} pCAG} prepared in the above (3) was dissolved in 40 μL of Universal Buffer H, and 24 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 8.6 kbp was recovered using a GeneClean II kit according to the attached manual. The recovered DNA is dissolved in 50 μL of an alkaline phosphatase buffer, 40 units of bovine small intestine-derived alkaline phosphatase is added, and the reaction is carried out at 50 ° C. for 30 minutes. After phenol / chloroform extraction, the terminal is dephosphorylated by ethanol precipitation. The obtained DNA fragment of about 8.6 kbp was recovered.
[0280]
From the plasmid cA3R (N) Ｎin pBS obtained aboveEco100 ng of the RI fragment (about 700 bp) and plasmid cA3R (C) {in} pCAG-derivedEco100 ng of the RI fragment (about 8.6 kbp) was dissolved in 10 μL of distilled water, and the ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 3 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the sequence contained in the plasmid was confirmed. Hereinafter, this plasmid is referred to as cA3R in pCAG.
[0281]
Example 7 Introduction of Human A3 Adenosine Receptor Expression Vector and Chimeric A3 Adenosine Receptor Expression Vector into Mammalian Cells and Evaluation of Reactivity of Specific Drug
(1) Evaluation of drug responsiveness due to transient expression
Host cell mouse myeloma cell P3. X63 / Ag8. A stable transformant cell line in which apoaequorin expression plasmid pCAG-AEQ-pHPRTp was introduced into U1 (hereinafter abbreviated as P3U1), clone 21 ク ロ ー ン hprt strain (WO 01/33957) was constructed in Example 6 (4). The obtained expression plasmid hA3R @ in @ pCAG and the expression plasmid cA3R @ in @ pCAG constructed in Example 7 (4) were introduced, respectively, to prepare transient cell lines for human A3 adenosine receptor and chimeric A3 adenosine receptor. Apoaequorin expressed by clone 21 @ hprt forms a protein complex aequorin with the luminescent substrate coelenterazine and molecular oxygen, and aequorin is Ca²⁺Light is emitted by bonding with
[0282]
The introduction of plasmids hA3R in pCAG and cA3R in pCAG into clone 21 △ hprt cell line was carried out by electroporation as follows.
8 × 10⁶K-PBS buffer (137 mmol / L @ KCl, 2.7 mmol / L NaCl, 8.1 mmol / L @ Na₂HPO₄, 1.5 mmol / L @ KH₂PO₄, 4 mmol / L @ MgCl₂), And 200 μL of this cell suspension and 4 μg of the plasmid hA3R {in} pCAG or 4 μg of cA3R {in} pCAG} are cuvettes for electroporation [Gene \ Pulser \ Cuvette, distance between electrodes 2 mm, biorad ( BIORAD), and the gene was transfected under the conditions of a pulse voltage of 0.25 kV and an electric capacity of 125 μF, suspended in RPMIｍＬ1640 medium (manufactured by Invitrogen) containing 10 mL of FCS, and then suspended at 37 ° C. in 5% CO 2.₂The cells were cultured under the conditions for 24 hours.
[0283]
Using these transiently expressed cell lines, in order to verify whether human A3 adenosine receptor and chimeric A3 adenosine receptor can function normally in mouse cells, an agonist specific for A3 adenosine receptor was used. Some chloro-N⁶-(3-Iodobenzyl) -adenosine-5'-N-methyluronamide [chloro-N⁶-(3-iodobenzyl) -adenosine-5'-N-methyluronamide, hereinafter abbreviated as Cl-IB-MECA] (K. A. Jacobson; Trends Pharmacol. ＣSci.,)19, 5, 1998) (intracellular Ca²⁺The amount of increase) was evaluated by the amount of luminescence of aequorin as follows. Using the clone 21Δhprt strain into which each A3 adenosine receptor expression plasmid was not introduced as a control cell, the responsiveness to Cl-IB-MECA was examined.
[0284]
After culture, each cell line was⁶The suspension was suspended in RPMI 1640 medium containing 10 μmol / L of coelenterazine (Molecular Probes) at a concentration of 10 μmol / L, and 100 μL of the suspension was dispensed into a glass tube for culture (Iwaki Glass). The cells were poured and cultured for 4 hours.
After the culture, the luminescence of each cell line is measured for 5 seconds per second for 5 seconds using a luminometer Auto @ Lumat @ LB953 (manufactured by EG & G Berthold) and then a 2 μmol / L Cl-IB-MECA solution (10 mmol / L) Was added to each glass tube for culture, and the amount of luminescence immediately after the addition was measured for 60 seconds every second. The same measurement was performed using DMSO containing no Cl-IB-MECA as a control.
[0285]
FIG. 39 shows the result of this measurement. As a result of adding Cl-IB-MECA at a final concentration of 1 μmol / L, the cell line in which the human A3 adenosine receptor was transiently expressed was about twice as large as the control cell line in which each plasmid had not been introduced. In the cell line in which the chimeric A3 adenosine receptor was transiently expressed, the amount of aequorin luminescence increased about 4-fold.
From the above results, the transiently expressed human A3 adenosine receptor and chimeric A3 adenosine receptor are localized on the cell membrane of mouse myeloma cells, and have normal responsiveness to A3 adenosine receptor-specific agonist ( Ca²⁺Rise). In addition, in mouse cells, it was shown that chimeric A3 adenosine receptor had higher responsiveness to A3 adenosine receptor-specific agonists than human A3 adenosine receptor.
[0286]
(2) Evaluation of drug reactivity using a stable expression strain
The expression plasmid hA3R in pCAG constructed in Example 5 (4) and the expression plasmid cA3R in pCAG constructed in Example 6 (4) were introduced into the clone 21 △ hprt strain, and a human A3 adenosine receptor and a chimeric A3 adenosine were introduced. A cell line stably expressing the receptor was produced. Using these stably expressing cell lines, in order to verify whether the human A3 adenosine receptor and the chimeric A3 adenosine receptor can function normally in mouse cells, Cl3, an agonist specific to the A3 adenosine receptor, was used. -Response to IB-MECA (Intracellular Ca²⁺Rise) was evaluated by the amount of luminescence of aequorin.
[0287]
The introduction of plasmids hA3R in pCAG and cA3R in pCAG into clone 21 △ hprt cell line was carried out according to the electroporation method described in (1). After gene transfer and suspension in RPMIｍＬ1640 medium containing 10 mL of FCS, 37 ° C., 5% CO 2₂The cells were cultured under the conditions for 24 hours. After the culture, 200 μL of a HAT medium additive (containing HAT-Media {supplement} (50 ×), Boehringer Mannheim Biochemica, containing aminopterin, hypoxanthine and thymidine) is added for 6 days. Thus, an aminopterin-resistant strain was obtained. The number of viable cells of the obtained drug-resistant strain was counted, and a 2% HT medium supplement (HT-Media {Supplement} (50 ×), Boehringer Mannheim Biochemica Co., Ltd., hypoxanthine and thymidine was adjusted to 5 cells / mL. ) And 10% FCS in RPMI 播 1640 medium and seeded at 100 μL / well in a 96-well plate. One week later, the cells in the wells that formed a single colony were collected, thereby obtaining 4 cloned cell lines from the cell group into which the plasmid hA3R in pCAG was introduced, and 9 clone cells from the cell group from the cell group into which the plasmid cA3R in pCAG was introduced. The strain was obtained. Each clone was expanded using an RPMI # 1640 medium containing 10% FCS.
[0288]
After the culture, the responsiveness of each cell line to Cl-IB-MECA was measured according to the method described in (1). Compared with a control cell line into which each plasmid was not introduced (clone @hprt cell line), a significant increase in luminescence was observed in three out of four clone lines in the cell line expressing human A3 adenosine receptor. (See FIG. 40). In particular, clone 4 showed a 6-fold or more increase in luminescence compared to the control cell line. In addition, in the cell lines in which the chimeric A3 adenosine receptor was expressed, an increase in luminescence was observed in all nine clone lines (see FIG. 41). In particular, clone 2 showed a 6-fold or more increase in luminescence. Furthermore, by comparing the average value of the luminescence of each clone strain with the stable expression strain of the human A3 adenosine receptor and the stable expression strain of the chimeric A3 adenosine receptor, the human A3 adenosine receptor and the mouse A3 adenosine receptor were compared in the mouse cells. In comparison, it was shown that the chimeric A3 adenosine receptor had higher responsiveness to A3 adenosine receptor-specific agonists.
[0289]
From the above results, it can be seen that the stably expressed human A3 adenosine receptor and chimeric A3 adenosine receptor have normal responsiveness to an A3 adenosine receptor-specific agonist (Ca²⁺Rise). In addition, in mouse cells, it was shown that chimeric A3 adenosine receptor had higher responsiveness to agonists specific to A3 adenosine receptor than human A3 adenosine receptor.
[0290]
Example 8 Preparation of 5'-side and 3'-side probes used for Southern hybridization to detect homologous recombinants
(1) Preparation of 5'-side probe
Whether or not homologous recombination was correctly performed by the target vector prepared in Example 4 is analyzed by Southern hybridization. The mouse A3 adenosine contained in the target vector was used as a 5 ′ probe used in the Southern hybridization. A DNA containing a genomic sequence 5 ′ further from the mouse genomic sequence upstream of exon 1 of the receptor gene was prepared as follows (see FIG. 42).
[0291]
3 μg of plasmid pBS-ex1 was dissolved in 20 μL of Universal Buffer T containing 0.01% BSA, and 8 units ofSmaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment was dissolved in 20 μL of Universal Buffer L and 10 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 6 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 1.2 kbp was recovered using a GeneClean II kit according to the attached manual.
[0292]
5 μg of pBluescriptII @ SK (−) was dissolved in 50 μL of Universal Buffer T containing 0.01% BSA, and 24 units ofSmaI was added and a digestion reaction was performed at 37 ° C. for 16 hours. After extracting the reaction solution with phenol / chloroform, a DNA fragment was recovered by an ethanol precipitation method. The DNA fragment is dissolved in 40 μL of Universal Buffer L and 20 units ofSacI was added and a digestion reaction was performed at 37 ° C. for 6 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual.
[0293]
100 ng of the mouse genomic DNA fragment (about 1.2 kbp) upstream of exon 1 of the mouse A3 adenosine receptor gene obtained above and pBluescriptII @ SK (-)-derivedSmaI-SacI fragment (about 3 kbp) (100 ng) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the mouse genomic sequence contained in the plasmid was confirmed. SEQ ID NO: 43 shows its nucleotide sequence. Hereinafter, this plasmid is referred to as pBS-probe 5 '.
[0294]
The 5'-side probe used in Southern hybridization was prepared by PCR using plasmid pBS-probe 5 'as a template. That is, based on the mouse genomic sequence contained in the plasmid pBS-probe5 ', a 26-mer primer having the nucleotide sequence of SEQ ID NO: 44 and a 26-mer primer having the nucleotide sequence of SEQ ID NO: 45 were prepared. A PCR reaction solution was prepared using these primers and pBS-probe 5 ′ as a template, and reacted at 94 ° C. for 5 minutes, followed by 94 ° C. for 1 minute, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute. The cycle was repeated 30 times, and PCR was performed under the condition of storing at 4 ° C. overnight. This PCR reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 300 bp was recovered using a GeneClean II kit according to the attached manual. Hereinafter, this DNA fragment is used as a 5'-side probe.
[0295]
(2) Preparation of 3'-side probe
A DNA containing a mouse genomic sequence 3 ′ further from the mouse genomic sequence downstream of exon 2 of the mouse A3 adenosine receptor gene contained in the target vector as a 3 ′ probe used in Southern hybridization is as follows: (See FIG. 43).
[0296]
3 μg of plasmid pBS-ex2 was dissolved in 20 μL of Universal Buffer M and 10 units ofNheI was added and a digestion reaction was performed at 37 ° C. for 16 hours. The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 600 bp was recovered using a GeneClean II kit according to the attached manual.
3 μg of pBluescriptII @ SK (−) was dissolved in 40 μL of Universal Buffer M and 20 units ofSpeAfter addition of I and digestion reaction at 37 ° C. for 16 hours, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 3 kbp was recovered using a GeneClean II kit according to the attached manual. Next, the recovered DNA was dissolved in 50 μL of an alkaline phosphatase buffer, 40 units of bovine small intestine-derived alkaline phosphatase was added, the reaction was carried out at 50 ° C. for 30 minutes, and phenol / chloroform extraction was performed. A dephosphorylated DNA fragment of about 3 kbp was recovered.
[0297]
100 ng of a mouse genomic DNA fragment (about 600 bp) downstream of exon 2 of the mouse A3 adenosine receptor obtained above and pBluescript II @ SK (-)-derivedSpeI fragment (about 3 kbp) (100 ng) was dissolved in 10 μL of distilled water, and ligation kit Ver. 10 μL of Solution I was added, and a binding reaction was performed at 16 ° C. for 2 hours.
Using the reaction solutionE.coliDH5α was transformed by the method of Cohen et al.^rGot the strain. Plasmid DNA was isolated from this transformant according to the method of Burnboim et al. The structure of the obtained plasmid was confirmed by restriction enzyme digestion. Further, the nucleotide sequence was determined using a DNA sequencer 377, and the mouse genomic sequence contained in the plasmid was confirmed. SEQ ID NO: 46 shows the nucleotide sequence. Hereinafter, this plasmid is referred to as pBS-probe 3 '.
[0298]
To prepare a 3 'probe for use in Southern hybridization, PCR was performed using plasmid pBS-probe 3' as a template. That is, a 26-mer primer having the base sequence of SEQ ID NO: 47 and a 26-mer primer having the base sequence of SEQ ID NO: 48 were prepared based on the mouse genomic DNA sequence contained in the plasmid pBS-probe 3 '. A PCR reaction solution was prepared using these primers and pBS-probe 3 ′ as a template, reacted at 94 ° C. for 5 minutes, and then repeated 30 times at 94 ° C. for 1 minute and at 68 ° C. for 2 minutes. PCR was performed under the condition of storing overnight at 4 ° C. This PCR reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 240 bp was recovered using a GeneClean II kit according to the attached manual. Hereinafter, this DNA fragment is used as a 3'-side probe.
[0299]
Example 9 Preparation of Transgenic Mice Expressing Chimeric A3 Adenosine Receptor
(1) Introduction of target vector into ES cells
Culture of mouse ES cells AB2.2 (manufactured by Lexicon Genetics, catalog number C100, hereinafter referred to as AB2.2 cells) was performed using M15 medium [15% FCS (catalog number B100, manufactured by Lexicon Genetics).^-4mol / L β-mercaptoethanol (manufactured by Lexicon Genetics, Catalog No. M260), 2 mmol / L L-glutamine (manufactured by Lexicon Genetics), 50 units / mL penicillin and 50 μg / mL streptomycin (Lexicon) Using a Dulbecco's modified Eagle medium (DMEM, Lexicon Genetics, catalog number M205) containing Genetics, catalog number M250) on a feeder plate at 37 ° C, 5% CO2₂Performed under conditions.
[0300]
The feeder plate is obtained by culturing mouse embryonic fibroblasts (manufactured by Lexicon Genetics, catalog number C200, hereinafter referred to as STO cells) treated with mitomycin C in a cell culture dish that has been subjected to a gelatin coating treatment as described below. Produced. First, a cell culture dish (96-well plate, 24-well plate, 3 cm dish, 6 cm dish, 8.5 cm dish) was placed in a 0.1% gelatin solution (manufactured by Lexicon Genetics, catalog number M210) for 2 hours or more. A gelatin coat treatment was performed by covering the culture surface. Subsequently, after the purchased STO cells were thawed, 4.4 × 10 5 were added to STO medium (DMEM containing 7% FCS, 2 mmol / L L-glutamine, 50 units / mL penicillin and 50 μg / mL streptomycin).⁵Cells / mL, suspended in a gelatin-coated cell culture dish, 0.07 mL / well for a 96-well plate, 0.5 mL / well for a 24-well plate, 2 mL / sheet for a 3 cm dish 4 mL / plate for 6 cm dish, 12 mL / plate for 8.5 cm dish, 37 ° C, 5% CO₂A plate cultured for 48 hours or more under the conditions was used as a feeder plate.
[0301]
AB2.2 cells were suspended in an M15 medium and then seeded on a feeder plate to start culturing. The M15 medium was 0.1 mL / well for 96-well plates, 2 mL / plate for 3 cm dishes, 4 mL / plate for 6 cm dishes, 10 mL / plate for 8.5 cm dishes, and 0.1 mL / plate for multi-dish and 24-well plates. 5 mL / well was used. Before the cells reached confluence, they were passaged as follows. First, prior to the passage, the M15 medium was replaced two hours before. After washing the cells twice with PBS (manufactured by Lexicon Genetics), 0.025 mL / well for a 96-well plate, 0.25 mL / well for a 24-well plate, 1 mL / plate for a 3 cm dish, 6 cm dish 2 mL / plate, 8.5 mL dish with 2 mL / plate of 0.25% trypsin solution containing 0.04% EDTA (manufactured by Lexicon Genetics, catalog number M220, hereinafter abbreviated as trypsin solution) And 37 ° C, 5% CO₂It was left under the conditions for 15 minutes. An equal volume of M15 medium was added, and the cells were dispersed by vigorous pipetting 40 times using a 3 mL transfer pipette (Falcon, Catalog No. 7575). The cells were precipitated and recovered from the solution containing the cells by centrifugation. The recovered cells were resuspended in M15 medium and seeded on a new feeder plate for subculture.
[0302]
The introduction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 into AB2.2 cells was performed by the electroporation method as follows. The target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 obtained in Example 4 (24) is a restriction enzymeSalI was cut into linear form, extracted with phenol / chloroform, ethanol-precipitated, and dissolved in 1 mmol / L {Tris-HCl} (pH 8.0) containing 0.1 mmol / L {EDTA}. AB2.2 cells purchased 3.0 × 10⁶The seeds were seeded on a 6 cm dish, cultured for 48 hours, then subcultured to an 8.5 cm dish, and further cultured for 24 hours. Twenty-four hours later, the cells were washed twice with 7 mL of PBS, and the cells were collected according to the above passage method. 1.0 × 10 cells recovered⁷The cells were suspended in 0.9 mL of PBS, and 25 μg of linear plaxP-DT-arm1-HPRT-hA3R-arm2 was cuvette for electroporation (Gene Pulser Cuvette, distance between electrodes 4 mm, manufactured by Bio-Rad) , Catalog number 165-2088). Using an electroporation apparatus Gene Pulser (manufactured by Bio-Rad), the gene was introduced under the conditions of a pulse voltage of 0.23 kV and an electric capacity of 500 μF, and suspended in 70 mL of M15 medium, followed by seven 8.5 cm feeder plates. Was seeded at 10 mL each and cultured for 24 hours.
[0303]
After 24 hours, the cells were cultured for 5 days in an M15 medium containing 1 × concentration of 100 × HAT supplement (HAT {supplement} (100 ×), manufactured by Invitrogen, catalog No. 3992, including aminopterin, hypoxanthine and thymidine). An aminopterin-resistant colony is further cultured for 3 days in a M15 medium containing a 100 × HT additive (HT {Supplement} (100 ×), manufactured by Invitrogen, catalog number 11067-030, containing hypoxanthine and thymidine) at a 1 × concentration. Appeared. Each colony was isolated as follows, and the culture was continued. First, after appearing colonies were washed twice with PBS, a total of 512 colonies were directly picked up using a sterile chip made of plastic, and a round-bottomed 96-well plate (Falcon, catalog No., added with 30 μL of trypsin solution) 3077) at 1 colony / well. 37 ° C, 5% CO₂After standing for 15 minutes or more under the conditions, 70 μL / well of M15 medium was added, and the cells were dispersed by vigorous pipetting 40 times with a sterile plastic tip. The cell suspension was inoculated on a 96-well feeder plate having 100 μL of M15 medium added to each well, and cultured for 3 days.
[0304]
Three days later, a replica plate was prepared as described below, and cultured for another five days. First, the cells cultured in the above 96-well feeder plate were washed twice with 100 μL / well of PBS, and then 25 μL of a trypsin solution was added thereto.₂It was left for more than 15 minutes under the conditions. 25 μL of M15 medium was added, and the mixture was vigorously pipetted 40 times with a sterile plastic tip to disperse. Subsequently, 50 μL of 20% dimethyl sulfoxide (DMSO, manufactured by Sigma-Aldrich, catalog number D2650) and DMEM containing 40% FCS (hereinafter referred to as 2 × freezing medium) were added and mixed from the cell suspension. A replica plate was prepared by dispensing 50 μL, pre-gelatin-coated, and transferring to a 96-well plate to which 100 μL / well of M15 medium had been added. The plate containing the remaining cell suspension was stored frozen at -80 ° C as a master plate.
[0305]
(2) Selection of homologous recombinants by Southern hybridization
The replica plate of 288 colonies of the replica plate of 512 colonies prepared in (1) was cultured for 5 days, the medium was removed, and the plate was washed twice with 100 μL / well of PBS, and then a cell lysis solution [10 mmol / L Tris -HCl (pH 7.5), 10 mmol / L EDTA, 10 mmol / L NaCl, 0.5% sarkosyl, 1 mg / mL proteinase K (manufactured by Invitrogen, catalog number 25530-049)] is added in an amount of 50 μL / hole, and the container is sealed. After the paper was put together with paper sufficiently containing water, the cells were kept at 60 ° C. for 24 hours to lyse the cells. 100 μL / well of ethanol containing 75 mmol / L NaCl was added to the cell lysate, left at room temperature for 30 minutes or longer to precipitate genomic DNA, and the 96-well plate was inverted to discard the solution. Subsequently, the plate was washed three times with 150 μL / well of 70% ethanol, and left at room temperature for 20 minutes to evaporate the remaining ethanol.
[0306]
12 unitsEcoAfter adding 30 μL / well of universal buffer H containing RI and 5 μg of ribonuclease A (manufactured by Sigma; Catalog No. R5125), vigorously pipetting to thoroughly dissolve the genomic DNA, and then digestion reaction at 37 ° C. for 20 hours. Was performed. After the reaction, the reaction solution was subjected to agarose gel electrophoresis, and subjected to the method of Southern (J. {Mol.98, $ 503, $ 1975) with a nylon filter (Hybond)^TM-N +, manufactured by Amersham Bioscience). The 5'-side probe and the 3'-side probe prepared in Example 8 were subjected to [α-probe using a T7 quick prime kit (manufactured by Amersham Bioscience).³²P] ＰdCTP (manufactured by NEN). Each of the labeled probes was hybridized with the filter prepared above overnight, washed three times for 15 minutes at 65 ° C. using 2 × SSPE containing 0.1% SDS, and then washed with 0.1% SDS. Washing was performed twice at 65 ° C. for 15 minutes using 2 × SSPE, and X-ray film (Kodak Scientific Imaging Film X-OMAT) was used.^TM(AR, manufactured by Kodak Co., Ltd.), exposed at -80 ° C. overnight, and developed the next day.
[0307]
In the above Southern hybridization, wild-type mouse genomic DNAEcoWhen digested with RI, a DNA fragment of about 9 kbp is detected by using the 5 'probe, and a DNA fragment of about 6.5 kbp is detected by using the 3' probe. On the other hand, homologous recombination is normally induced by the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2, and the mouse A3 adenosine receptor gene in the mouse genomic DNA is replaced with DNA encoding the chimeric A3 adenosine receptor. In this case, a DNA fragment of about 8.5 kbp is detected by using the 5 ′ probe, and a DNA fragment of about 8.8 kbp is detected by using the 3 ′ probe (see FIG. 44).
[0308]
As a result of the above Southern hybridization, out of the 288 aminopterin resistant strains of the ES cells analyzed, 13 colonies of ES cells (1B2, 1C12, 1D7, 1D12, 2A10, 2D12, 2F10, 2H6, 3D9, 3F8, 3F11, 3H4, 3H10) was found to be a homologous recombinant in which the mouse A3 adenosine receptor gene was replaced on one chromosome by DNA encoding the chimeric A3 adenosine receptor.
[0309]
Cryopreservation stocks were prepared as described below for the 13 colony ES cells that were homologous recombinants.
Thirteen colonies of the cryopreserved master plate were thawed, and the cell suspension containing each homologous recombinant was seeded in each well of a 24-well feeder plate supplemented with 2 mL / well of M15 medium, and cultured for 24 hours. After changing the medium and culturing for 2 days, the cells were subcultured on a feeder plate of 3 cm dish and further cultured for 3 days. According to the subculturing method described in (1), the cells recovered after detachment from the dish were suspended in 0.5 mL of M15 medium. Further, a suspension obtained by adding and mixing 0.5 mL of 2 × freezing medium was dispensed in 0.1 mL portions into a cell freezing tube, cryopreserved at −80 ° C., and stored as stock.
[0310]
(3) Removal of drug resistance gene from homologous recombinant
From the homologous recombinant obtained in (2), four clones (1B2, 1D7, 3H4, 3H10) having good morphology and growth ability were selected, and a Cre expression vector was introduced to thereby introduce a drug resistance gene existing between loxP sequences. (Hprt gene) was removed. Each of the above-mentioned frozen stocks of the four clones was thawed one by one, inoculated on a 24-well feeder plate containing 2 mL / well of M15 medium, and cultured for 2 days. The medium was replaced with a 3 cm dish feeder plate, and the medium was replaced 24 hours later. Twenty-four hours later, the plate was washed twice with 1 mL of PBS, and 1 mL of a trypsin solution was added. 37 ° C, 5% CO₂After standing for 15 minutes under the conditions, 1 mL of M15 medium was added, and the cells were detached by vigorously pipetting 40 times using a 3 mL transfer pipette. The cells were precipitated and recovered by centrifugation and suspended in 0.9 mL of PBS. Using the cell suspension and 25 μg of Cre expression vector pBS185 (manufactured by Invitrogen) dissolved in 1 mmol / L Tris-HCl (pH 8.0) containing 0.1 mmol / L EDTA, described in (1). The gene was introduced into the cells according to the electroporation method described above, suspended in 10 mL of M15 medium, seeded on an 8.5 cm dish feeder plate, and cultured for 24 hours. Twenty-four hours later, the medium was replaced and the cells were further cultured for three days.
[0311]
After washing the cultured cells twice with 7 ml of PBS, the cells were detached from the dish and collected according to the subculture method described in (1), suspended in 10 mL of M15 medium, and the number of cells was measured. Subsequently, the cells were diluted to 500 cells / mL, 50 cells / mL, and 5 cells / mL using an M15 medium containing 1000X 6-thioguanine (manufactured by Sigma-Aldrich) at a concentration of 1X. Each was seeded on a feeder plate of 8.5 cm dish and cultured for 7 days, so that colonies deficient in the hprt gene appeared. Each of these colonies was isolated according to the method described in (1), inoculated on a 96-well feeder plate, and cultured for 3 days. The clones with good morphology were subcultured to 24-hole feeder plates, 12 clones for each colony, and cultured for 2 days. Further, 5 clones each having good morphology were subcultured on a 3 cm dish feeder plate and cultured for 3 days. The cells were detached from the dish according to the passaging method described in (1), collected, and suspended in 0.5 mL of M15 medium. The cell suspension was further added with 0.5 mL of 2 × freezing medium, and the mixed suspension was dispensed in 0.1 ml portions into cell freezing tubes, and a total of 20 clones (1B2-1 to -5, 1D7 -1 to -5, 3H4-1 to -5, 3H10-1 to -5) were stored at -160 ° C as stock for cryopreservation.
[0312]
(4) Confirmation of hprt gene removal by Southern hybridization
The above-mentioned cryopreservation stocks of the 20 clones were thawed one by one, seeded on a gelatin-coated 3 cm dish containing 2 mL of M15 medium, and cultured for 3 days. Three days later, the medium was removed, and the cells were washed twice with 1 mL of PBS buffer. Then, 1.75 mL of the cell lysate described in (2) was added thereto, and the resultant was placed in a sealed container together with paper sufficiently containing water. Thereafter, the cells were kept at 60 ° C. for 24 hours to lyse the cells. 3 mL of ethanol containing 75 mmol / L NaCl was added to the cell lysate, and the mixture was allowed to stand at room temperature for 30 minutes or more. Then, the genomic DNA was collected by hooking it to the tip of a sterile chip. After washing the genomic DNA three times with 70% ethanol, the ethanol was volatilized by being left at room temperature for 20 minutes. Genomic DNA was dissolved in 100 μL of 1 mmol / L Tris-HCl (pH 8.0) containing 200 μg / mL ribonuclease A and 0.1 mmol / L EDTA. 12 g of each of the obtained genomic DNAs was dissolved in 120 L of Universal Buffer H, and 12 units ofEcoRI was added and a digestion reaction was performed at 37 ° C. for 16 hours. Undigested genomic DNA was cut by pipetting the reaction solution three times with a sterile tip. Furthermore, after adding 1.2 μL of 0.5 mol / L EDTA (pH 8.0) and 300 μL of ethanol, the mixture was centrifuged at 15,000 rpm for 10 minutes to obtain a precipitate of genomic DNA.
[0313]
After dissolving the genomic DNA precipitate in 20 μL of 1 mmol / L Tris-HCl (pH 8.0) containing 0.1 mmol / L EDTA, the whole amount was subjected to agarose gel electrophoresis, and nylon was prepared according to the method described in (2). Transfer to the filter,³²Southern hybridization was performed with the 3'-side probe labeled with P. By the above Southern hybridization, mouse genomic DNA in which the mouse A3 adenosine receptor gene has been replaced with DNA encoding the chimeric A3 adenosine receptor is obtained.EcoWhen digested with RI, as shown in (2), a DNA fragment of about 8.8 kbp is detected by using the 3′-side probe. However, the PGK promoter and hprt present between the loxP sequence by the Cre protein are detected. When the gene (about 3.8 kbp) is removed, a DNA fragment as short as about 5 kbp is detected (see FIG. 45).
[0314]
From the results of Southern hybridization, in all of the 20 clones analyzed, the PGK promoter and hprt gene (about 3.8 kbp) sandwiched between the loxP sequences existing downstream of the chimeric A3 adenosine receptor gene were found on the mouse genome. It was confirmed that it was more removed.
[0315]
(5) Preparation of chimeric mouse using ES cells having DNA encoding chimeric A3 adenosine receptor
Among the ES cells in which one allele of the A3 adenosine receptor gene established in (4) was replaced with DNA encoding the chimeric A3 adenosine receptor, three clones (1D7-2, 3H4-2, To inject 3H10-2) into mouse blastocysts, the cells were cultured on feeder plates. One of the cryopreservation stocks of each clone was thawed, suspended in 10 mL of M15 medium, centrifuged and precipitated to collect cells. The cells were seeded on a 24-well feeder plate to which 0.5 mL / well of M15 medium was added, and culture was started. Two days later, the cells were passaged to a 3 cm dish feeder plate. Two days later, after washing twice with 1 mL of PBS, 0.5 mL of a trypsin solution was added, and the mixture was added at 37 ° C., 5% CO 2.₂It was left under the conditions for 15 minutes. After adding 0.5 ml of M15 medium and suspending by vigorously pipetting 40 times using a 3 mL transfer pipette, FCS was added and mixed, and the cell suspension was injected into mouse blastocysts. Using.
[0316]
Mouse blastocysts were obtained by spontaneously mating superovulated female C57B1 / 6J mice with syngeneic male mice, and after 4 days, perfusing the inside of the removed uterus with M15 medium. The obtained mouse blastocysts are subjected to 37 ° C., 5% CO 2₂After allowing the blastocyst cavity to sufficiently expand under the conditions, the blastocyst was transferred to an M15 medium containing 20 mmol / L HEPES cooled to about 4 ° C., and a microinjector (Narimo Kagaku) and a micromanipulator (Narimo) While observing under an inverted microscope (manufactured by Nikon Corporation) equipped with a scientific company, the injection needle was operated, and 10 to 15 ES cell clones (1D7-2, 3H4-2, 3H10-2) were each obtained. Microinjection into the blastocyst cavity. The blastocysts are incubated at 37 ° C., 5% CO₂After allowing the blastocyst cavity to expand under the conditions, the mouse was implanted into the oviduct-side uterus of a pseudopregnant female MCH strain mouse according to the method described in Manipulating Mouse Embryo, 2nd edition, and allowed to implant. Was. Pseudopregnant female MCH strain mice (10 weeks of age or older) were obtained by ligating female MCH strain mice in the early estrus period to the estrus stage with males from the 10-week-old vasectomy male MCH strain mice at 17:00 three days before transplantation. They were allowed to live and mate at 1: 1 and vaginal plugs were checked at 9:00 the next morning, and used 2 days later for the above purpose.
[0317]
Among the chimeric individuals in which brown hair appeared in black hair due to the contribution of the injected ES cells (1D7-2, 3H4-2, 3H10-2), male individuals having a chimera rate of more than 40% were found. 3 animals were obtained from 1D7-2 and 12 animals were obtained from 3H10-2.
[0318]
(6) Obtaining a heterozygous mouse having a DNA encoding a chimeric A3 adenosine receptor by crossing chimeric individuals
After breeding the chimeric individual obtained in (5) until the age of 8 weeks, it was mated 1: 1 with female C57B1 / 6J strain 8 weeks old or later to obtain a litter. Among the offspring, genomic DNA is prepared from the tail of an individual having a brown coat according to the method described in (4), and 12 μg of each genomic DNA is subjected to restriction enzyme.EcoAfter cleavage with RI, Southern hybridization was performed according to the method described in (4). As a result, as shown in Example 10, two bands of 6.5 kbp and 5.0 kbp were observed in offspring born from 1D7-2 and 3H10-2, and A3 on one chromosome was observed. Heterozygous mice in which the adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor were included. Therefore, a chimeric individual whose chimera rate exceeded 40% was identified as a germ-line chimeric mouse. This heterozygous mouse is a mouse that expresses the chimeric A3 adenosine receptor together with its own A3 adenosine receptor.
[0319]
(7) Obtaining mice expressing only chimeric A3 adenosine receptor
After breeding the heterozygous mouse obtained in the above (6) until the age of 8 weeks, males and females were bred to obtain offspring. Genomic DNA was prepared from the tail of the offspring individual according to the method described in (4) and subjected to Southern hybridization. As a result, as shown in Example 10, only a 5.0 kbp band was observed, and a homozygous mouse in which the A3 adenosine receptor gene was substituted on both chromosomes with DNA encoding the chimeric A3 adenosine receptor was obtained. Was included in the offspring. This homozygous mouse is a mouse that expresses a chimeric A3 adenosine receptor instead of a mouse A3 adenosine receptor.
[0320]
Example 10 Genome Structural Analysis of Mice in which A3 Adenosine Receptor Gene was Replaced with DNA Encoding Chimeric A3 Adenosine Receptor
C57BL / 6J mice (CLEA Japan), heterozygous mice in which the A3 adenosine receptor gene described in (6) of Example 9 has been replaced with DNA encoding a chimeric A3 adenosine receptor, and (7) of Example 9 A tail was collected from a homozygous mouse in which the A3 adenosine receptor gene described in (1) above was replaced with a DNA encoding a chimeric A3 adenosine receptor, and genomic DNA was prepared in the same manner as in Example 9 (4). After dissolving the genomic DNA in TE buffer (manufactured by Invitrogen), 10 μg of the genomic DNA was added for 16 hours.EcoThe cells were cut with RI (manufactured by Takara Bio Inc.), and Southern hybridization was carried out by the method described in Example 9, (2).
[0321]
As a result of Southern hybridization using the 5′-side probe described in (1) of Example 8, about 9 kbp band in the C57BL / 6J mouse, the A3 adenosine receptor gene was converted to DNA encoding the chimeric A3 adenosine receptor. About 9 kbp and about 8.5 kbp bands were detected in the substituted heterozygous mice, and about 8.5 kbp bands were detected in the homozygous mice in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor. (FIG. 46). Subsequently, Southern hybridization was performed using the 3′-side probe described in (2) of Example 8. As a result, in the C57BL / 6J mouse, a band of about 6.5 kbp, the A3 adenosine receptor gene was replaced with the chimeric A3 adenosine receptor Approximately 6.5 kbp and approximately 5.0 kbp bands in heterozygous mice substituted with DNA encoding DNA, and approximately 5.5 kbp in homozygous mice in which A3 adenosine receptor gene was substituted with DNA encoding chimeric A3 adenosine receptor. A band of 0 kbp was detected (FIG. 47). The above results are consistent with the genomic structure schematically shown in FIGS. 44 and 45. In the heterozygous mouse, the A3 adenosine receptor gene on one chromosome encodes a chimeric A3 adenosine receptor. It was confirmed that the homozygous mouse had the A3 adenosine receptor gene on both chromosomes replaced with DNA encoding the chimeric A3 adenosine receptor.
[0322]
Example 11 Preparation of bone marrow-derived mast cells from homozygous mice in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor
From bone marrow cells obtained from C57BL / 6J mice and homozygous mice obtained by substituting the DNA encoding the chimeric A3 adenosine receptor for the A3 adenosine receptor gene prepared in (9) of Example 9, Supajatura et al. Method (J. {Immunol.,}167Mast cells derived from bone marrow cells (hereinafter abbreviated as BMMC) were prepared by a method according to the method described in US Pat.
[0323]
The spleen was isolated from 20 male BALB / cA mice (CLEA Japan) and homogenized. The spleen cells were isolated from the spleen cell culture medium [10% FCS (Hyclone), 10 mmol / L @ HEPES buffer (Invitrogen). ), 0.1 mmol / L @ 2 mercaptoethanol (Invitrogen), 50 mg / L gentamicin (Nacalai Tesque), 3.4 mg / LPhytolacca americana2 × 10 6 in RPMI 1640 medium (manufactured by Invitrogen) containing lectin (manufactured by pokeweed) derived from pokeweed (Sigma-Aldrich)⁶Suspended in cells / mL, 37 ° C, 5% CO₂The cells were cultured under the conditions for 5 days. After the culture, the culture supernatant was collected. Hereinafter, the culture supernatant is referred to as PWM-SCM (abbreviation for culture supernatant of spleen cells stimulated with pokeweed mitogen).
[0324]
Subsequently, bone marrow cells were collected from femurs of C57BL / 6J mice and homozygous mice in which the A3 adenosine receptor gene prepared in (7) of Example 9 was replaced with DNA encoding a chimeric A3 adenosine receptor. BMMC medium [10% FCS, 0.1 mmol / L sodium pyruvate (Invitrogen), 0.1 mmol / L non-essential amino acid (Invitrogen), 50 μmol / L 2 mercaptoethanol, 50 mg / L gentamicin, RPMI @ 1640 medium (manufactured by Invitrogen) containing 10% PWM-SCM) at 37.degree.₂Culture was performed under the conditions. The medium was replaced with a fresh medium every four days, and the antibody staining shown below was performed on the 28th day after the start of the culture.
[0325]
2 × 10⁵After washing the cells with Dulbecco's phosphate buffer (hereinafter referred to as PBS) (Invitrogen), trinitrophenol (hereinafter abbreviated as TNP) labeled with 0.2 μg of fluorescein isothiocyanate (hereinafter abbreviated as FITC) PBS containing specific IgE (obtained from KM393 cells (ATCC No .: TIB142)) and an anti-mouse c-kit antibody (Pharmingen) labeled with 0.2 μg of phycoerythrin (hereinafter abbreviated as PE) And reacted on ice for 1 hour. After the reaction, the cells were washed twice with PBS, and the fluorescence intensity of FITC and PE was measured using FACS @ Calibur (manufactured by Becton Dickinson). It has been reported that IgE Fc receptor and c-kit exist on the cell membrane of mast cells, and both IgE and anti-mouse c-kit antibody bind (Immunity,).1695% or more of cells derived from C57BL / 6J mice (FIG. 48) and homozygous mice obtained by replacing the A3 adenosine receptor gene with DNA encoding the chimeric A3 adenosine receptor (FIG. 49). Staining with both antibodies confirmed that these cell groups had differentiated into BMMC.
[0326]
Example 12 Intracellular Ca²⁺Of Human A3 Adenosine Receptor Specific Antagonist by Assay
Intracellular Ca of BMMC by A3 adenosine receptor agonists and antagonists²⁺The variation of the amount was measured by the following method.
[0327]
In a BMMC medium supplemented with 100 ng / mL of TNP-specific IgE, a C57BL / 6J mouse prepared in Example 11 and a homozygous mouse in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor, respectively BMMC of 1 × 10⁶Cells / mL, 37 ° C, 5% CO₂For 16 hours. After the culture, the BMMC was added to a calcium measurement buffer [20 mmol / L @ HEPES (manufactured by Nacalai Tesque), 115 mmol / L NaCl (manufactured by Junsei Chemical), 5.4 mmol / L @ KCl (manufactured by Nacalai Tesque), 0.8 mmol / L MgCl₂(Manufactured by Nacalai Tesque) 1.8 mmol / L @ CaCl₂(Manufactured by Nacalai Tesque), 13.8 mmol / L glucose (manufactured by Nacalai Tesque), 0.2% 、 BSA (manufactured by Sigma-Aldrich), 2.5 mmol / L probenecid (probenecid, manufactured by Sigma-Aldrich)] After washing twice, calcium measurement was performed by adding Fluo-3 @ AM (Molecular Probes) at a final concentration of 5 μmol / L and Pluronic F-127 (Sigma-Aldrich) at a final concentration of 0.5%. Suspended in buffer, 37 ° C, 5% CO₂The cells were cultured under the conditions for 1 hour. After the culture, the cells were washed twice with a calcium measurement buffer, and⁵After suspending the cells to a cell / mL with a calcium measurement buffer, dispensing 100 μl each into a 96-well plate, 37 ° C., 5% CO 2₂The cells were cultured under the conditions for 30 minutes. After the culture, (a) only a calcium measurement buffer was added, (b) a final concentration of 1 μmol / L of an A3 adenosine receptor agonist Cl-IB-MECA was added, and (c) a final concentration of 1 μmol / L of Cl-IB-MECA was added. Ten minutes before the addition, KF26777, a human A3 adenosine receptor-specific antagonist at a final concentration of 1 μmol / L, or (d) TNP-BSA (Cosmo Bio) at a final concentration of 10 ng / mL was added. (A) to (d) Intracellular Ca for each cell²⁺Using FDSS # 6000 (manufactured by Hamamatsu Photonics KK) as an index of the amount, fluorescence having a wavelength of 530 nm generated by an excitation wavelength of 480 nm was transmitted through a blue dichroic mirror, and then measured for 1 minute with an absorption filter of 520 to 560 nm.
[0328]
As shown in FIG. 50, in BMMC derived from C57BL / 6J mice, intracellular Ca by Cl-IB-MECA addition²⁺Although an increase in the amount was observed, the increase was also observed when the human A3 adenosine receptor antagonist KF26777 at a final concentration of 1 μmol / L was added 10 minutes before the addition of Cl-IB-MECA, and inhibition by the antagonist was observed. Was not seen. On the other hand, as shown in FIG. 51, in the BMMC derived from a mouse in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor, intracellular Ca by addition of Cl-IB-MECA²⁺An increase in the amount was observed, and the increase was completely suppressed by adding a final concentration of 1 μmol / L of the human A3 adenosine receptor antagonist KF26777 10 minutes before the addition of Cl-IB-MECA. Thus, a human A3 adenosine receptor specific antagonist is capable of producing A3 adenosine receptor-mediated intracellular Ca in BMMC.²⁺Was confirmed to suppress the increase in While this could not be confirmed by normal mouse BMMC, it could be confirmed for the first time by using mouse-derived BMMC in which the A3 adenosine receptor gene was substituted with DNA encoding a chimeric A3 adenosine receptor. In both BMMCs, intracellular Ca was added by adding TNP-BSA, which is an antigen of TNP-specific IgE bound to cells.²⁺Increased amounts were observed (FIGS. 50 and 51), confirming that mast cell activation via IgE cross-linking by antigen was occurring normally.
[0329]
From the above results, pharmacological evaluation of human A3 adenosine receptor-specific antagonist (intracellular Ca²⁺Quantification) can be performed by using a mouse in which the A3 adenosine receptor gene of the present invention has been substituted with a DNA encoding a chimeric A3 adenosine receptor, and this mouse has specificity for the human A3 adenosine receptor. It was shown to be useful for pharmacological evaluation of drugs.
[0330]
Example 13 Evaluation of Human A3 Adenosine Receptor Specific Antagonist by Measurement of Degranulation
The effects of A3 adenosine receptor agonists and antagonists on the degranulation of BMMC were determined by the method of Supajatura et al. (J. {Immunol.,}167, 4, 2001), using β-hexaminidase activity released extracellularly by degranulation as an indicator. In a BMMC medium supplemented with 100 ng / mL of TNP-specific IgE, a C57BL / 6J mouse prepared in Example 11 and a homozygous mouse in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor, respectively 1 × 10⁶Cells / mL, 37 ° C, 5% CO₂For 16 hours. After culturing, the plate was washed twice with PBS, and Tyrode buffer [10 mmol / L @ HEPES (manufactured by Nacalai Tesque), 130 mmol / L @ NaCl (Pure Chemical), 5 mmol / L @ KCl (manufactured by Nacalai Tesque), 0.6 mmol / L L @ KH₂PO₄(Domestic chemical), 1 mmol / L @ CaCl₂(Manufactured by Nacalai Tesque), 0.6 mmol / L @MgCl₂(Manufactured by Nacalai Tesque), 0.1% {(w / v)} glucose (manufactured by Nacalai Tesque), 0.1% {(w / v)} BSA (Sigma), adjusted to pH 7.4]. To each BMMC, (a) No addition, (b) Addition of only A3 adenosine receptor agonist Cl-IB-MECA at a final concentration of 1 μmol / L (c) To induce degranulation in BMMC, a final concentration of 10 ng / mL of TNP-BSA was added. (d) One minute after the addition of Cl-IB-MECA at a final concentration of 1 μmol / L, TNP-BSA at a final concentration of 10 ng / mL was added. (e) Final concentration of 1 μmol / L. 1 minute after the addition of the human A3 adenosine receptor antagonist KF26777, Cl-IB-MECA at a final concentration of 1 μmol / L was added, and 1 minute later, TNP-BSA at a final concentration of 10 ng / mL was added. -Without adding IB-MECA, adding a final concentration of 1 μmol / LKF26777, and then adding a final concentration of 10 ng / mL to TNP- The drug was added under the conditions of BSA addition. (A) to (f) After reacting each cell at 37 ° C. for 30 minutes, the reaction was stopped by centrifugation at 2000 g for 5 minutes, and the supernatant was recovered. A 40 mmol / L citric acid (manufactured by Nacalai Tesque) solution containing 4 nmol / L of p-nitrophenyl N-acetyl-β-glucosaminide (manufactured by Sigma-Aldrich), which is a substrate of β hexaminidase, is used as the collected supernatant. (Adjusted to pH 4.5) and kept at 37 ° C. for 1 hour. After the incubation, an equal volume of a 0.2 mol / L glycine (manufactured by Nacalai Tesque) solution (adjusted to pH 10.7) was added, and the absorbance at 405 nm was measured using a microplate reader (Bio-Rad). Β-hexaminidase activity was measured.
[0331]
As shown in FIG. 52, as a result of degranulation induced by TNP stimulation, C57BL / 6J mouse-derived BMMC released β-hexaminidase extracellularly. Furthermore, degranulation was increased about 2-fold by adding a final concentration of 1 μmol / L @ Cl-IB-MECA before TNP stimulation. However, even when 1 μmol / L of the final concentration of human A3 adenosine receptor antagonist KF26777 was added 1 minute before the addition of Cl-IB-MECA, the degranulation was not suppressed and was similarly observed. On the other hand, as shown in FIG. 53, degranulation was promoted by the addition of Cl-IB-MECA in BMMCs derived from mice in which the A3 adenosine receptor gene was replaced by DNA encoding the chimeric A3 adenosine receptor, and the promotion was promoted by Cl-IB -Complete inhibition was achieved by adding the human A3 adenosine receptor antagonist KF26777 at a final concentration of 1 μmol / L 10 minutes before the addition of MECA. Therefore, it was confirmed that the human A3 adenosine receptor-specific antagonist inhibits degranulation of BMMC via the A3 adenosine receptor. While this could not be confirmed by normal mouse BMMC, it could be confirmed for the first time by using mouse-derived BMMC in which the A3 adenosine receptor gene was substituted with DNA encoding a chimeric A3 adenosine receptor.
[0332]
From the above results, the pharmacological evaluation of human A3 adenosine receptor-specific antagonist (measuring degranulation), which was impossible in normal mice, was confirmed by the DNA encoding the chimeric A3 adenosine receptor with the A3 adenosine receptor gene of the present invention. It was clarified that the method can be performed by using a mouse substituted with, for example, and it was shown that this mouse is useful for pharmacological evaluation of a specific drug for human A3 adenosine receptor.
[0333]
Example 14 Evaluation of Human A3 Adenosine Receptor-Specific Antagonist by Apoptosis Measurement
Hiragun et al. (Clin. {Exp. {Allergy,}30According to the method described in Example 11, the C57BL / 6J mouse-derived BMMC and the mouse-derived BMMC obtained by substituting the A3 adenosine receptor gene with DNA encoding the chimeric A3 adenosine receptor were subjected to PWM-SCM-free preparation. 4 × 10 in BMMC medium⁵Cells, 37 ° C, 5% CO₂Apoptosis was induced by culturing for 4 days under the conditions, and the number of viable cells after 48 hours and 96 hours was measured. As a result, the number of surviving cells of C57BL / 6J mouse-derived BMMC decreased to about 50% after 48 hours of culture and to about 25% after 96 hours, and a final concentration of 100 nmol / L of human A3 adenosine receptor antagonist KF26777 was added during culture. However, no change was observed in the number of surviving cells (FIG. 54). On the other hand, the number of surviving cells of mouse-derived BMMC in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor decreased to about 50% after 48 hours of culturing, to about 25% after 96 hours, and By adding the human A3 adenosine receptor antagonist KF26777 at a final concentration of 100 nmol / L, the cell viability after 48 hours of culture was further reduced to about 30% (FIG. 55).
[0334]
48 hours after the above culture, each BMMC was subjected to TUNEL staining using an in situ apoptosis detection kit (manufactured by Takara Bio Inc.) to detect apoptosis. As a result, apoptosis was observed in about 50% of C57BL / 6J mouse-derived BMMC cultured in a BMMC medium containing no PWM-SCM (FIG. 56). In addition, even when the human A3 adenosine receptor antagonist KF26777 at a final concentration of 100 nmol / L was added during the culture, the ratio of apoptosis did not change (FIG. 56). On the other hand, apoptosis was observed in about 50% of BMMCs derived from mice in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor, which had been cultured for 48 hours in a BMMC medium without PWM-SCM, and a final concentration of 100 nmol during culture. Addition of / L human A3 adenosine receptor antagonist KF26777 increased the percentage of apoptotic cells to about 70% (FIG. 57). Therefore, it was confirmed that a human A3 adenosine receptor-specific antagonist promotes apoptosis of BMMC induced by growth factor deficiency. While this could not be confirmed by normal mouse BMMC, it could be confirmed for the first time by using mouse-derived BMMC in which the A3 adenosine receptor gene was substituted with DNA encoding a chimeric A3 adenosine receptor.
[0335]
From the above results, the pharmacological evaluation of human A3 adenosine receptor-specific antagonists (apoptosis measurement), which was impossible in normal mice, was carried out using the A3 adenosine receptor gene of the present invention in the DNA encoding the chimeric A3 adenosine receptor. It became clear that this was possible by using the substituted mouse, and this mouse was shown to be useful for the pharmacological evaluation of a specific drug for the human A3 adenosine receptor.
[0336]
Example 15 Evaluation of Human A3 Adenosine Receptor-Specific Antagonist Using Pancreatic Cells
Using mouse pancreatic cells in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor, intracellular Ca by A3 adenosine receptor agonists and antagonists was used.²⁺The variation of the amount was measured by the following method.
[0337]
The pancreas was excised from C57BL / 6J mice and homozygous mice in which the A3 adenosine receptor gene was replaced with DNA encoding the chimeric A3 adenosine receptor, and then pancreatic cells were isolated by homogenization. For each of the isolated pancreatic cells, (a) a buffer alone was added, (b) a final concentration of 1 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA was added, and (c) a final concentration of 1 μmol / L of Cl- was added. When the final concentration of 1 μmol / L human A3 adenosine receptor specific antagonist KF26777 was added 10 minutes before the addition of IB-MECA, each intracellular Ca²⁺The change in concentration was measured in the same manner as in Example 12. As shown in FIG. 58, in the C57BL / 6J mouse-derived pancreatic cells, intracellular Ca by addition of Cl-IB-MECA²⁺An increase in the amount was observed, and the increase was similarly observed when the human A3 adenosine receptor antagonist KF26777 was added at a final concentration of 100 μmol / L 10 minutes before the addition of Cl-IB-MECA. I couldn't. On the other hand, as shown in FIG. 59, in mouse-derived pancreatic cells in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor, intracellular Ca by addition of Cl-IB-MECA²⁺An increase in the amount was observed, and the increase was completely suppressed by adding a final concentration of 100 (M human A3 adenosine receptor antagonist KF26777 10 minutes before the addition of Cl-IB-MECA. Adenosine receptor-specific antagonists induce intracellular Ca via A3 adenosine receptor in pancreatic cells.²⁺Was confirmed to suppress the increase in While this could not be confirmed by normal mouse BMMC, it could be confirmed for the first time by using mouse-derived BMMC in which the A3 adenosine receptor gene was substituted with DNA encoding a chimeric A3 adenosine receptor.
[0338]
From the above results, pharmacological evaluation of human A3 adenosine receptor specific antagonist (intracellular Ca²⁺Quantification) can be performed by using a mouse in which the A3 adenosine receptor gene of the present invention has been substituted with a DNA encoding a chimeric A3 adenosine receptor, and this mouse has specificity for the human A3 adenosine receptor. It was shown to be useful for pharmacological evaluation of drugs. In addition, this evaluation has shown for the first time that a human A3 adenosine receptor-specific antagonist may have a pharmacological effect on human diseases caused by abnormal intracellular signaling in human pancreatic cells.
[0339]
【The invention's effect】
According to the present invention, a pharmacological evaluation of a substance that has a different reactivity between a human seven-transmembrane receptor and an ortholog of the receptor of a non-human animal and that specifically acts on the human seven-transmembrane receptor is performed. There is provided a non-human mammal capable of:
[0340]
[Sequence List Free Text]
SEQ ID NO: 1-Inventor: Kazuya Yamano; Miho Inoue; Mitsuo Sato;
Inventor: Toro Ichimura; Shigehiro Masaki
Primers for amplifying mouse A3 adenosine receptor cDNA
SEQ ID NO: 4—Primer for amplifying mouse A3 adenosine receptor cDNA
SEQ ID NO: 5-primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 6: Primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 7—Primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 8—Primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 9—Primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 10—Primer for amplifying mouse A3 adenosine receptor gene
SEQ ID NO: 12—Primer for amplifying human A3 adenosine receptor cDNA
SEQ ID NO: 13--Primer for amplifying human A3 adenosine receptor cDNA
SEQ ID NO: 16—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 17—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 18—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 19-Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 20—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 21-Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 22—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 23—Primer for preparing DNA encoding chimeric A3 adenosine receptor
Primer for amplifying mouse genomic DNA upstream of the initiation codon of SEQ ID NO: 24-A3 receptor gene
Primer for amplifying mouse genomic DNA upstream of the start codon of SEQ ID NO: 25-A3 receptor gene
SEQ ID NO: 26—Primer for DNA amplification downstream of initiation codon of human A3 receptor cDNA
SEQ ID NO: 27—Primer for DNA amplification downstream of initiation codon of human A3 receptor cDNA
SEQ ID NO: 28-Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 29-Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 30-Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 31—Primer for preparing DNA encoding chimeric A3 adenosine receptor
SEQ ID NO: 32-Nucleotide sequence encoding chimeric A3 adenosine receptor
SEQ ID NO: 33-Amino acid sequence of chimeric A3 adenosine receptor
SEQ ID NO: 34-Oligo DNA for construction of A3R-C2
SEQ ID NO: 35-A3R-C2 oligo DNA for construction
SEQ ID NO: 36-ploxP-RE-HPRT-hA3R-arm2 construction oligo DNA
SEQ ID NO: 37-ploxP-RE-HPRT-hA3R-arm2 construction oligo DNA
Primer for amplifying DNA fragment containing polyadenylation signal of SEQ ID NO: 38-SV40
Primer for amplifying DNA fragment containing polyadenylation signal of SEQ ID NO: 39-SV40
SEQ ID NO: 40: Primer for DNA amplification encoding human A3 adenosine receptor to which FLAG tag is added
SEQ ID NO: 41: Primer for DNA amplification encoding human A3 adenosine receptor with FLAG tag
SEQ ID NO: 42: Primer for DNA amplification encoding chimeric A3 adenosine receptor with FLAG tag
SEQ ID NO: 44-Primer for amplifying probe on mouse 5 'side at mouse A3 receptor gene
SEQ ID NO: 45--Primer for amplifying probe on mouse 5 'side on mouse A3 receptor gene
SEQ ID NO: 47--Primer for amplifying probe on mouse 3 'side of A3 receptor gene
SEQ ID NO: 48-Primer for amplifying probe at 3 'side of mouse A3 receptor gene
[0341]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the results of comparison between the mouse A3 adenosine receptor cDNA sequence determined by the present invention and a known mouse A3 adenosine receptor cDNA sequence. Underlines indicate different base sequences between the two.
FIG. 2 shows the results of comparing the amino acid sequence deduced from the mouse A3 adenosine receptor cDNA sequence determined in the present invention with the amino acid sequence deduced from the known mouse A3 adenosine receptor cDNA sequence. Show. * Indicates a different amino acid sequence between the two.
FIG. 3 shows recognition sequences for each restriction enzyme present in a mouse genomic DNA sequence of about 9 kbp including exon 1 of the A3 adenosine receptor gene.
FIG. 4 shows recognition sequences for each restriction enzyme present in a mouse genomic DNA sequence of about 6.5 kbp including exon 2 of the A3 adenosine receptor gene.
FIG. 5 shows a region of about 5.2 kbp mouse genomic DNA sequence containing the A3 adenosine receptor gene determined in the present invention, and a region of about 6 kbp mouse genomic DNA sequence containing the known A3 adenosine receptor gene. Is shown.
FIG. 6 shows the construction of plasmid pBS-hA3R containing human A3 adenosine receptor cDNA.
FIG. 7 shows the construction of a target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 for replacing mouse A3 adenosine receptor gene with DNA encoding human A3 adenosine receptor (24 steps in total). The first step, the construction of plasmid chimera-1, is shown.
FIG. 8 shows the second step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-2.
FIG. 9 shows the third step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-3.
FIG. 10 shows the fourth step in the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), construction of the plasmid chimera-4.
FIG. 11 shows the fifth step in the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), construction of the plasmid chimera-1 + 2.
FIG. 12 shows the sixth step in the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-3 + 4.
FIG. 13 shows the seventh step in the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), construction of the plasmid chimera-1 + 2 + 3 + 4.
FIG. 14 shows the eighth step in the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), construction of the plasmid A3R-N1.
FIG. 15 shows the ninth step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid A3R-N2.
FIG. 16 shows the tenth step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid A3R-N3.
FIG. 17 shows the eleventh step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid cA3R-N.
FIG. 18 shows the twelfth step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid A3R-N5.
FIG. 19 shows the thirteenth step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid arm1 + cA3R-N.
FIG. 20 shows the 14th step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-5.
FIG. 21 shows the fifteenth step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-6.
FIG. 22 shows the 16th step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid chimera-5 + 6.
FIG. 23 shows the 17th step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid A3R-C1.
FIG. 24 shows the 18th step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid A3R-C2.
FIG. 25 shows the 19th step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), the construction of the plasmid cA3R-C.
FIG. 26 shows the construction of a target vector ploxP-DT-arm1-HPRT-hA3R-arm2 for replacing mouse A3 adenosine receptor gene with DNA encoding human A3 adenosine receptor (24 steps in total). Step 20 shows the construction of the plasmid ploxP-HPRT-hA3R-arm2.
FIG. 27 shows the 21st step of the construction of the target vector ploxP-DT-arm1-HPRT-hA3R-arm2 (24 steps in total), the construction of the plasmid ploxP-RE-HPRT-hA3R-arm2.
FIG. 28 shows the 22nd step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), and the construction of the plasmid ploxP-RE-C-HPRT-chiA3R-arm2.
FIG. 29 shows step 23 of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), and the construction of the plasmid ploxP-arm1-HPRT-chiA3R-arm2.
FIG. 30 shows the 24th step of the construction of the target vector ploxP-DT-arm1-HPRT-chiA3R-arm2 (24 steps in total), and the construction of the plasmid ploxP-DT-arm1-HPRT-chiA3R-arm2.
FIG. 31 shows a schematic diagram of the chimeric A3 adenosine receptor in the upper part, and the amino acid sequences of human and mouse A3 adenosine receptors in the lower part. The upper solid line shows the region of the human A3 adenosine receptor in the chimeric A3 adenosine receptor, and the dotted line shows the region of the mouse A3 adenosine receptor. The lower underline indicates the intracellular region of the A3 adenosine receptor, the asterisk indicates the amino acid sequence common to human and mouse, and the number indicates the number of the rightmost amino acid residue in each column.
FIG. 32 shows the first step of the construction of the human A3 adenosine receptor expression vector hA3R in pCAG (4 steps in total), the construction of the plasmid polyA in pBS.
FIG. 33 shows the second step of the construction of a human A3 adenosine receptor expression vector hA3R in pCAG (4 steps in total), the construction of plasmid hA3R in pBS.
FIG. 34 shows the third step in the construction of the expression vector hA3R 受 容 in pCAG for human A3 adenosine receptor (4 steps in total), construction of plasmid hA3R-polyA in pBS.
FIG. 35 shows the third step in the construction of a human A3 adenosine receptor expression vector hA3R in pCAG (4 steps in total), the construction of plasmid hA3R in pCAG.
FIG. 36 shows the first step of the construction of a chimeric A3 adenosine receptor expression vector cA3R in pCAG (3 steps in total), the construction of plasmid cA3R (N) in pBS.
FIG. 37 shows the second step in the construction of the chimeric A3 adenosine receptor expression vector cA3R in pCAG (3 steps in total), the construction of the plasmid cA3R (C) in pCAG.
FIG. 38 shows the third step in the construction of a chimeric A3 adenosine receptor expression vector cA3R in pCAG (all three steps), and the construction of plasmid cA3R in pCAG.
FIG. 39 shows the reactivity of mouse myeloma cell P3U1 transiently expressing human A3 adenosine receptor or chimeric A3 adenosine receptor to A3 adenosine receptor-specific agonist Cl-IB-MECA. (Intracellular Ca²⁺The figure shows the results of measurement of The horizontal axis is time (seconds), and Cl-IB-MECA was added to the cells at the time of the arrow. The vertical axis is the amount of luminescence of aequorin, and the intracellular Ca²⁺Represents the amount of increase. ● indicates P3U1 cells expressing human A3 adenosine receptor, ▲ indicates P3U1 cells expressing chimeric A3 adenosine receptor, △ indicates control P3U1 cells, □ indicates control P3U1 without addition of Cl-IB-MECA. It is a measurement result of a cell.
FIG. 40 shows the reactivity of the mouse myeloma cell line (total of 4 strains) stably expressing the human A3 adenosine receptor to the A3 adenosine receptor-specific agonist Cl-IB-MECA (intracellular Ca).²⁺The figure shows the results of measurement of The horizontal axis is time (seconds), and Cl-IB-MECA was added to the cells at the time of the arrow. The vertical axis is the amount of luminescence of aequorin, and the intracellular Ca²⁺Represents the amount of increase. ●, 、, black square and ◆ indicate P3U1 cells stably expressing human A3 adenosine receptor, and □ indicate control P3U1 cells.
FIG. 41 shows the reactivity of the mouse myeloma cell line (9 strains in total) stably expressing the chimeric A3 adenosine receptor to the A3 adenosine receptor-specific agonist Cl-IB-MECA (intracellular Ca).²⁺The figure shows the results of measurement of The horizontal axis is time (seconds), and Cl-IB-MECA was added to the cells at the time of the arrow. The vertical axis is the amount of luminescence of aequorin, and the intracellular Ca²⁺Represents the amount of increase. ●, ○, ▲, black square, boxed ×, Δ, Δ, ×, and Δ indicate the measurement results of P3U1 cells stably expressing human A3 adenosine receptor, and □ indicates the measurement results of control P3U1 cells.
FIG. 42 shows the genomic DNA content of mouse contained in plasmid pBS-probe5 ′.SacI-SmaThe position of the I fragment is schematically shown.
FIG. 43 shows the genomic DNA content of mouse contained in plasmid pBS-probe3 ′.NheThe position of the I fragment is schematically shown.
FIG. 44 shows the mouse genomic structure around the A3 adenosine receptor gene and the mouse genomic structure when the mouse A3 adenosine receptor gene was replaced by DNA encoding a chimeric A3 adenosine receptor by homologous recombination. Is shown.
FIG. 45 shows the mouse genomic structure when the mouse A3 adenosine receptor gene was replaced by DNA encoding the chimeric A3 adenosine receptor by homologous recombination, and the locus between loxP and loxP sequences by Cre recombinase. The genomic structure of the mouse when the existing drug resistance markers (PGK promoter and hprt gene) are removed is shown.
FIG. 46 shows the use of a 5 ′ probe for genomic DNA of C57BL / 6J mice, heterozygous mice in which the A3 adenosine receptor gene was replaced with DNA encoding chimeric A3 adenosine receptor, and homozygous mice. 2 shows the results of Southern hybridization analysis. Lane 1 shows the results of C57BL / 6J mice, lane 2 shows the results of heterozygous mice, and lane 3 shows the results of homozygous mice.
FIG. 47 shows the use of a 3 ′ probe for genomic DNA of a C57BL / 6J mouse, a heterozygous mouse in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor, and a homozygous mouse. 2 shows the results of Southern hybridization analysis. Lane 1 shows the results of C57BL / 6J mice, lane 2 shows the results of heterozygous mice, and lane 3 shows the results of homozygous mice.
FIG. 48 shows FACS analysis of C57BL / 6J mouse-derived BMMC using TNP-specific IgE and anti-c-kit antibody. a) shows the result of FACS analysis of unstained BMMC, and b) shows the result of FACS analysis of BMMC stained with an antibody. The vertical axis indicates the fluorescence intensity of cells stained with the PE-labeled anti-c-kit antibody, and the horizontal axis indicates the fluorescence intensity of cells stained with FITC-labeled TNP-specific IgE.
FIG. 49 shows FACS analysis of homozygous mouse-derived BMMC in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor using TNP-specific IgE and anti-c-kit antibody. Is shown. a) shows the result of FACS analysis of unstained BMMC, and b) shows the result of FACS analysis of BMMC stained with an antibody. The vertical axis indicates the fluorescence intensity of cells stained with the PE-labeled anti-c-kit antibody, and the horizontal axis indicates the fluorescence intensity of cells stained with FITC-labeled TNP-specific IgE.
FIG. 50 shows intracellular Ca of BMMC derived from C57BL / 6J mouse.²⁺Shows the variation of the amount. a) the result of adding only the calcium measurement buffer, b) the result of adding the final concentration of 1 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA, and c) the result of adding the final concentration of 1 μmol / L of the A3 adenosine receptor. Ten minutes before the addition of the agonist Cl-IB-MECA, the human A3 adenosine receptor antagonist KF26777 was added, and d) shows the result of adding 10 ng / mL final concentration of TNP-BSA. The vertical axis is Ca²⁺The fluorescence amount of Fluo-3 at a wavelength of 530 nm, which is excited by the above, is shown as a relative value with the value before adding the drug being 1.0. The horizontal axis shows the time after drug addition in seconds.
FIG. 51 shows intracellular Ca of mouse-derived BMMC in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor.²⁺Shows the variation of the amount. a) the result of adding only the calcium measurement buffer, b) the result of adding the final concentration of 1 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA, and c) the result of adding the final concentration of 1 μmol / L of the A3 adenosine receptor. Ten minutes before the addition of the agonist Cl-IB-MECA, the human A3 adenosine receptor antagonist KF26777 was added, and d) shows the result of adding 10 ng / mL final concentration of TNP-BSA. The vertical axis is Ca²⁺The fluorescence amount of Fluo-3 at a wavelength of 530 nm, which is excited by the above, is shown as a relative value with the value before adding the drug being 1.0. The horizontal axis shows the time after drug addition in seconds.
FIG. 52 shows degranulation of BMMC derived from C57BL / 6J mice. The vertical axis indicates β-hexaminidase activity (total activity is 100%) released extracellularly. The black bar shows the case where the final concentration of 1 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA was added, and the white bar shows the case where no A3 adenosine receptor agonist was added.
FIG. 53 shows degranulation of mouse-derived BMMC in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor. The vertical axis indicates β-hexaminidase activity (total activity is 100%) released extracellularly. The black bar shows the case where the final concentration of 1 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA was added, and the white bar shows the case where no A3 adenosine receptor agonist was added.
FIG. 54 shows the change in the number of cells of C57BL / 6J mouse-derived BMMC during culture in a medium without PWM-SCM. The vertical axis indicates the number of cells, and the horizontal axis indicates the time (hour) from the start of culture. ▲ indicates the case where the human A3 adenosine receptor antagonist KH26777 at a final concentration of 1 μmol / L was added, and ● indicates the case where it was not added.
FIG. 55 shows the change in the number of cells of mouse-derived BMMC in which the A3 adenosine receptor gene has been replaced with DNA encoding a chimeric A3 adenosine receptor during culture in a medium without PWM-SCM. The vertical axis indicates the number of cells, and the horizontal axis indicates the time (hour) from the start of culture. ▲ indicates the case where the human A3 adenosine receptor antagonist KH26777 at a final concentration of 1 μmol / L was added, and ● indicates the case where it was not added.
FIG. 56 shows the apoptosis rate of cells in C57BL / 6J mouse-derived BMMC cultured in BMMC medium and BMMC medium without PWM-SCM for 48 hours, as measured by TUNEL staining. The vertical axis indicates the percentage (%) of cells showing apoptosis. The white bar shows the results in the BMMC medium and the black bar shows the results in the PWM-SCM-free BMMC medium.
FIG. 57 shows BMMCs derived from mice in which the A3 adenosine receptor gene cultured in a BMMC medium and a BMMC medium without PWM-SCM for 48 hours was replaced with DNA encoding a chimeric A3 adenosine receptor, as measured by TUNEL staining. 5 shows the apoptosis rate of the cells in FIG. The vertical axis indicates the percentage (%) of cells showing apoptosis. The white bar shows the results in the BMMC medium and the black bar shows the results in the PWM-SCM-free BMMC medium.
FIG. 58 shows intracellular Ca of pancreatic cells derived from C57BL / 6J mice.²⁺Shows the variation of the amount. a) The result of adding only the calcium measurement buffer, b) the result of adding the final concentration of 100 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA, and c) the result of adding the final concentration of 100 μmol / L of the A3 adenosine receptor. The result of adding the human A3 adenosine receptor antagonist KF26777 at a final concentration of 100 μmol / L 10 minutes before the addition of the agonist Cl-IB-MECA is shown. The vertical axis is Ca²⁺The wavelength of Fluo-3 excited by is shown as a relative value with the value before drug addition being 1.0. The horizontal axis shows the time after drug addition in seconds.
FIG. 59 shows intracellular Ca of pancreatic cells derived from homozygous mice in which the A3 adenosine receptor gene was replaced with DNA encoding a chimeric A3 adenosine receptor.²⁺Shows the variation of the amount. a) The result of adding only the calcium measurement buffer, b) the result of adding the final concentration of 100 μmol / L of the A3 adenosine receptor agonist Cl-IB-MECA, and c) the result of adding the final concentration of 100 μmol / L of the A3 adenosine receptor. The result of adding the human A3 adenosine receptor antagonist KF26777 at a final concentration of 100 μmol / L 10 minutes before the addition of the agonist Cl-IB-MECA is shown. The vertical axis is Ca²⁺The wavelength of Fluo-3 excited by is shown as a relative value with the value before drug addition being 1.0. The horizontal axis shows the time after drug addition in seconds.

Claims (37)

A DNA encoding a chimeric seven-transmembrane receptor in which the entire intracellular region of the human seven-transmembrane receptor is replaced by a corresponding portion of a non-human mammalian ortholog of the receptor; And a transgenic non-human mammal expressing the chimeric seven transmembrane receptor. A receptor in which any part of the intracellular region of a human seven-transmembrane receptor is substituted with a corresponding part of a non-human mammal ortholog of said receptor, wherein said human receptor is expressed in cells of said non-human mammal. A transgenic non-human mammal having in its genome a DNA encoding a chimeric seven transmembrane receptor that exhibits a cellular response to an agonist acting on the body, and expressing the chimeric seven transmembrane receptor. The transgenic non-human mammal according to claim 2, wherein any part of the intracellular region is a part containing at least one of the following (a) to (d).
(A) the second intracellular region from the N-terminus; (b) the third intracellular region from the N-terminus; (c) the palmitic acid binding site of the C-terminal intracellular region; and (d) the phosphorylation by the kinase in the intracellular region. Part The DNA encoding the chimeric seven-transmembrane receptor is inserted in both homologous chromosomes, replacing the region encoding the seven-transmembrane receptor of the gene of its own ortholog. The transgenic non-human mammal according to any one of claims 1 to 3. The transgenic non-human mammal according to any one of claims 1 to 4, wherein the seven-transmembrane receptor is the receptor according to any one of the following (a) to (l).
(A) A3 adenosine receptor (b) 5-HT1B serotonin receptor (c) NK1 tachykinin receptor (d) B2 bradykinin receptor (e) β3 adrenergic receptor (f) calcitonin receptor (g) M2 muscarinic acetylcholine Receptor (h) α2A adrenergic receptor (i) β1 adrenergic receptor (j) CCKB cholecystokinin receptor (k) neurotensin receptor (l) DP prostanoid receptor The non-human mammal according to any one of claims 1 to 5, wherein the non-human mammal is a non-human mammal selected from the group consisting of mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, goats, cows and monkeys. 2. The transgenic non-human mammal according to claim 1. A transgenic mouse having in its genome a DNA encoding a chimeric A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 32 and expressing the chimeric A3 adenosine receptor. Both homologous chromosomes in which the DNA encoding the chimeric A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 32 has been inserted by replacing the region encoding the A3 adenosine receptor of the own A3 adenosine receptor gene, The transgenic mouse according to claim 7, which is present in the genome. The method for producing a transgenic non-human mammal according to any one of claims 1 to 3, comprising the following steps (a) to (d).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor (B) introducing the expression vector into a fertilized egg of a non-human mammal; and (c) introducing the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal. (D) breeding the non-human mammal and selecting, from the offspring, an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor The method for producing a transgenic non-human mammal according to any one of claims 1 to 3, comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor (B) introducing the expression vector into a non-human mammal embryonic stem cell; and (c) introducing the embryonic stem cell into a fertilized egg of a non-human mammal. Step (d) Transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal (e) Rearing the non-human mammal and culturing the chimeric seven transmembrane receptor from the offspring Step of selecting an individual having the DNA to encode in the genome The method for producing a transgenic non-human mammal according to any one of claims 1 to 3, comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor Constructing an expression vector containing a DNA encoding the body, (b) incorporating the expression vector into sperm, (c) fertilizing an unfertilized egg of a non-human mammal with the sperm, and (d) transforming the fertilized egg. Transplanting into the oviduct or uterus of a pseudopregnant female non-human mammal (e) breeding the non-human mammal, and having a genomic DNA encoding the chimeric seven-transmembrane receptor from the offspring The process of selecting individuals The method for producing a transgenic non-human mammal according to any one of claims 1 to 3, comprising the following steps (a) to (f).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor Step (b) of constructing a retroviral vector containing a DNA encoding the body; Step (c) of producing a recombinant retrovirus using the retroviral vector; and (c) applying the recombinant retrovirus to an unfertilized egg of a non-human mammal. Infecting (d) In vitro fertilizing the unfertilized egg (e) Transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal (f) Raising the non-human mammal Selecting an individual having a DNA encoding the chimeric seven-transmembrane receptor in the genome from the offspring The method for producing a transgenic non-human mammal according to claim 4, comprising the following steps (a) to (h).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor Constructing a target vector containing the DNA encoding the body (b) introducing the target vector into a non-human mammalian embryonic stem cell; and (c) DNA encoding the chimeric seven-transmembrane receptor, A step of selecting an embryonic stem cell having a chromosome inserted by substituting the region encoding the seven-transmembrane receptor of the gene of the own ortholog (d) a step of introducing the selected embryonic stem cell into a fertilized egg ( e) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human mammal; (f) breeding the non-human mammal and selecting a germ-line chimera from the offspring (g). series A chromosome into which the DNA encoding the chimeric seven-transmembrane receptor has been substituted and replaced with a region encoding the seven-transmembrane receptor of the self-orthologous gene from the offspring of the mela, The individuals obtained in steps (h) and (g) of selecting individuals having the following are crossed, and the DNA encoding the chimeric seven-transmembrane receptor is expressed on both homologous chromosomes from the offspring. For selecting a homozygote that has been inserted in place of the region encoding the seven-transmembrane receptor of the ortholog gene The method for producing a transgenic non-human mammal according to any one of claims 1 to 3, comprising the following steps (a) to (e).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor Step (b) of constructing an expression vector containing a DNA encoding the body. Step (c) of introducing the constructed expression vector into cells of a non-human mammal. (C) Non-human cell enucleated from the cell nucleus obtained in (b). Introducing the unfertilized egg into a non-fertilized egg of a mammal and initiating the development; (d) transplanting the unfertilized egg having started the development into the oviduct or uterus of a pseudopregnant female non-human mammal; and (e) the non-human mammal. And selecting an individual having in its genome a DNA encoding the chimeric seven-transmembrane receptor from pups born The method for producing a transgenic non-human mammal according to claim 4, comprising the following steps (a) to (g).
(A) Chimeric seven-transmembrane receptor in which the entire intracellular region or any part of the intracellular region of the human seven-transmembrane receptor is replaced by the corresponding portion of the non-human mammalian ortholog of the receptor (B) introducing the constructed target vector into cells of a non-human mammal; and (c) introducing a DNA encoding the chimeric seven-transmembrane receptor into the target cell. (C) selecting a cell having a chromosome inserted by substituting the region encoding the seven-transmembrane receptor of the ortholog gene of (d) unfertilized non-human mammal whose nucleus of the selected cell is enucleated (E) transplanting the unfertilized egg that has started to develop into the oviduct or uterus of a pseudopregnant female non-human mammal, and (f) rearing the non-human mammal, Child A step of selecting an individual having a chromosome inserted by replacing the DNA encoding the chimeric seven-transmembrane receptor with the region encoding the seven-transmembrane receptor of the gene of its own ortholog ( g) The individuals obtained in (f) are bred to each other, and the DNA encoding the chimeric seven-transmembrane receptor is expressed on both homologous chromosomes of the offspring of the offspring by the seven-fold membrane of the gene of its own ortholog. A step of selecting a homozygote inserted in place of the region encoding the penetrating receptor A cell derived from the transgenic non-human mammal according to any one of claims 1 to 8. 17. The cell according to claim 16, wherein the cell is an egg, sperm or embryonic stem cell. A method for producing a transgenic non-human mammal, further comprising introducing a foreign gene into the cell according to claim 16 or 17. A transgenic non-human mammal produced by the method of claim 18. A method for producing a knockout non-human mammal, comprising introducing a foreign gene into the cell according to claim 16 or 17 to delete a specific gene. A knockout non-human mammal produced by the method of claim 20. A disease model which expresses a chimeric seven-transmembrane receptor, wherein the transgenic non-human mammal according to any one of claims 1 to 8 is crossed with a disease model animal of the same or different strain. A method for producing a non-human mammal. A disease model non-human mammal expressing the chimeric seven transmembrane receptor produced by the method according to claim 22. 9. Preparation of a disease state model non-human mammal expressing a chimeric seven-transmembrane receptor, wherein the disease is induced in the transgenic non-human mammal according to any one of claims 1 to 8. Method. A disease model non-human mammal expressing the chimeric seven-transmembrane receptor produced by the method according to claim 24. A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) and (b):
(A) a step of administering a test substance to the non-human mammal according to any one of claims 1 to 8, 19, 21, 23 and 25 to examine a pharmacological effect; and (b) a test substance to the non-human mammal. Examining the pharmacological effect of the test substance on the non-human mammal in comparison with the pharmacological effect of the case where no is administered. A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) and (b):
(A) a step of administering a test substance to the cell according to claim 16 or 17 and examining a pharmacological effect; and (b) comparing the pharmacological effect obtained when the test substance is not administered to the cell with the test substance. Examining the pharmacological effects on cells A method for evaluating the pharmacological effect of a test substance on a seven-transmembrane receptor, comprising the following steps (a) to (c):
(A) a step of inducing differentiation of the embryonic stem cells according to claim 17 and obtaining the differentiated cells (b) a step of administering a test substance to the differentiated cells obtained in (a) and examining a pharmacological effect ( c) examining the pharmacological effect of the test substance given to the cells by comparing the pharmacological effect obtained when the test substance was not administered to the differentiated cells obtained in (a) The test substance is an agonist whose agonist activity on a human seven-transmembrane receptor is at least 10 times that of a non-human mammal ortholog of the receptor or antagonist activity on a human seven-transmembrane receptor. 29. The method for evaluating a pharmacological effect according to any one of claims 26 to 28, wherein is an antagonist having 10 times or more the antagonist activity of the receptor to an ortholog of a non-human mammal. The method for evaluating a pharmacological effect according to any one of claims 26 to 28, wherein the test substance is an agonist or an antagonist of the receptor according to any one of the following (a) to (l).
(A) A3 adenosine receptor (b) 5-HT1B serotonin receptor (c) NK1 tachykinin receptor (d) B2 bradykinin receptor (e) β3 adrenergic receptor (f) calcitonin receptor (g) M2 muscarinic acetylcholine Receptor (h) α2A adrenergic receptor (i) β1 adrenergic receptor (j) CCKB cholecystokinin receptor (k) neurotensin receptor (l) DP prostanoid receptor A DNA encoding a chimeric A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 33. A DNA having the nucleotide sequence of SEQ ID NO: 32. DNA encoding a mouse A3 adenosine receptor having the amino acid sequence shown in SEQ ID NO: 4. A DNA having the nucleotide sequence of SEQ ID NO: 3. A DNA having the nucleotide sequence of SEQ ID NO: 11. A target vector comprising the DNA according to claim 31. DNA consisting of a continuous sequence of 500 bases or more of the sequence of bases 1 to 637 of the base sequence shown in SEQ ID NO: 11 and a sequence of 500 bases or more of the base sequence of 3735 to 5270 of the base sequence shown in SEQ ID NO: 11 The target vector according to claim 36, comprising a DNA consisting of:

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