JP2008220289A - 非ヒトトランスジェニック動物 - Google Patents
非ヒトトランスジェニック動物 Download PDFInfo
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Images
Abstract
【解決手段】常染色体劣性高コレステロール血症(Autosomal Recessive Hypercholesterolemia:ARH)関連遺伝子であるARH遺伝子を過量発現させた非ヒトトランスジェニック動物による。
【選択図】図2
Description
J. Clin. Invest. 92:883-893, 1993 Proc. Natl. Acad. Sci. USA 91:4431-4435, 1994 Biochem. biopys. Res. Commun. 276:435-438, 2000 Nat. Med. 10:1067-73, 2004 Science 292: 1394-1398, 2001 Circulation reseach 95:945-952, 2004 The Lipid 16:316-322, 2005
1.常染色体劣性高コレステロール血症関連遺伝子であるARH遺伝子を過剰発現させてなる非ヒトトランスジェニック動物。
2.トランスジェニック動物が、低コレステロール血症モデル動物である前項1に記載の非ヒトトランスジェニック動物。
3.動物がマウスである、前項1又は2に記載の非ヒトトランスジェニック動物。
本発明の病理モデル動物を製造するには、まず、ARH遺伝子をクローニングする。このクローニング方法としては公知の様々な方法が可能である。例えば、ARHのmRNAからcDNAを作製し、そのcDNAをプラスミド等のベクターのDNAに組込み、更に該DNA組替えベクターを大腸菌等の宿主に組込んで、大腸菌を増殖させる。大量に産生された大腸菌からDNA組替えベクターを取りだし、ARH遺伝子をカラムクロマトグラフ法、あるいは電気泳動法等により分取、精製することによりクローニングすることができる。目的の塩基配列をもつ遺伝子が精製されていることの確認は、DNAシーケンシングなどにより確定することが望ましい。
マウス遺伝子ライブラリーから得られた本件ARHをコードするcDNA、あるいは、マウスmRNA から直接RT−PCR等の方法により得られた同cDNAをプラスミドベクター等にライゲーションにより挿入する。
クローニング用ベクターとしては、宿主内で特定遺伝子を増幅できる細菌プラスミド由来、酵母プラスミド由来、バクテリオファージ由来、トランスポゾン由来及びこれらの組合せに由来するベクター、例えば、コスミドやファージミドのようなプラスミドとバクテリオファージの遺伝的要素に由来するものを挙げることができる。
本件ARHタンパク質をコードする遺伝子は、好ましくはベクターを経由して宿主細胞により増殖する。本件ARHタンパク質をコードする遺伝子の宿主細胞への導入は、Davisら(BASIC METHODS IN MOLECULAR BIOLOGY, 1986)及びSambrookら(MOLECULAR CLONING:A LABORATORY MANUAL, 2nd Ed., Cold Spring Habor LABORATORY Press, Cold Spring Habor, NY., 1989)などの自体公知の実験室マニュアルに記載される形質転換や感染等により行うことができる。
そして、上記宿主細胞としては、大腸菌、ストレプトミセス、枯草菌、ストレプトコッカス、スタフィロコッカス等の細菌原核細胞や、酵母、アスペルギルス等の真菌細胞等を挙げることができる。
ARH過剰発現トランスジェニックマウスを製造するには、例えばマウスの受精卵の細胞核に上記方法で得たARHの遺伝子をマイクロインジェクトする。注入する遺伝子の量は1個の受精卵当り200〜1000コピーであることが好ましい。別に精管切断術を施した雄マウスと交尾させ偽妊娠状態にした仮親を用意して、この仮親の卵管内にこの受精卵を移植し、マウスを誕生させる。
本実施例において、ARHタンパク質をコードする遺伝子は、GenBank accession No. NM_015627(配列番号1)に示される。ARH遺伝子はRessourcenzentrum, Heubnerweg 6, 14059 Berlin-Chalottenburg, GermanyのRZPD社より購入した、クローンDKFZp586D0624はpSPORTのマルチクローニングサイトにクローニングされている。そのプラスミドからBamHI及びEcoRIで消化切断してDNAフラグメントとした。
本実験例においては、普通食を摂取させた場合のマウスにおける血清コレステロール値を対比した。
本実験例においては、高コレステロール血症脂肪食負荷前後のマウスにおける血清コレステロール値を対比した。
本実験例においては、高コレステロール血症脂肪食負荷前後のマウスにおけるVLDL−コレステロール値を対比した。対比は、血清中のコレステロールのうち、VLDL,LDL及びHDL画分のどの分画が上昇しているのかを調べることにより行った。血清50μlずつを採取し、カラムとしてTSKgel LipopropaxXL(TM)(東ソー)を使用したHPLCにより、血清中のコレステロールをVLDL,LDL及びHDLの各画分に分画した。本実験例では、得られたVLDLを、コレステロールエステラーゼとコレステロールオキシダーゼとを使用する酵素法の試薬、デタミナー(TM)LTC特注(協和メデックス)を使用し、製品に付属指示書に従って定量した。
Claims (3)
- 常染色体劣性高コレステロール血症関連遺伝子であるARH遺伝子を過剰発現させてなる非ヒトトランスジェニック動物。
- トランスジェニック動物が、低コレステロール血症モデル動物である請求項1に記載の非ヒトトランスジェニック動物。
- 動物がマウスである、請求項1又は2に記載の非ヒトトランスジェニック動物。
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