JP5543771B2 - Transgenic animals for analyzing CYP3A4 cytochrome P450 gene regulation - Google Patents
Transgenic animals for analyzing CYP3A4 cytochrome P450 gene regulation Download PDFInfo
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- JP5543771B2 JP5543771B2 JP2009297935A JP2009297935A JP5543771B2 JP 5543771 B2 JP5543771 B2 JP 5543771B2 JP 2009297935 A JP2009297935 A JP 2009297935A JP 2009297935 A JP2009297935 A JP 2009297935A JP 5543771 B2 JP5543771 B2 JP 5543771B2
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Description
本発明は、トランスジェニック動物の作製、および化合物(特に、生体異物(xenobiotics)またはステロイド(これらに限定されない))のヒトにおけるP450遺伝子の発現の調節に及ぼす影響を決定するための動物の使用に関する。 The present invention relates to the production of transgenic animals and the use of animals to determine the effect of compounds (especially, but not limited to xenobiotics or steroids) on the regulation of P450 gene expression in humans. .
多くの内因性および外因性化合物は、in vitroでの薬剤開発試験において治療的効果を有することが観察される。しかしながら、意図される治療効果はしばしば、例えば化合物が同時投与される場合、特定の化合物がCYP3A4遺伝子の発現を誘導するため、臨床的実施において実現されない。この誘導は、各化合物の意図される治療的効果が実現され得る前に化合物を代謝するCYP3A4シトクロムP450分子を生成する。従って、CYP3A4遺伝子の発現の誘導は、意図される投薬を妨害し、治療の失敗または最適以下の治療をもたらす。 Many endogenous and exogenous compounds are observed to have therapeutic effects in in vitro drug development trials. However, the intended therapeutic effect is often not realized in clinical practice because, for example, when compounds are co-administered, certain compounds induce expression of the CYP3A4 gene. This induction produces CYP3A4 cytochrome P450 molecules that metabolize the compound before the intended therapeutic effect of each compound can be realized. Thus, induction of CYP3A4 gene expression interferes with the intended medication, resulting in treatment failure or suboptimal treatment.
CYP3A4遺伝子発現の誘導は、時間、資源、および費用を特定の疾患状態の治療のための候補薬物の開発(これは、臨床的実施において最終的に失敗するか、または最適以下で行われるであろう)に浪費させるので、薬剤開発にとって重大な問題である。 Induction of CYP3A4 gene expression is a time, resource, and cost development of candidate drugs for the treatment of specific disease states (this will ultimately fail in clinical practice or be performed suboptimally. Is a serious problem for drug development.
薬剤開発の初期段階において、候補薬物がヒトにおいて意図される治療効果を達成するようであるか否かが決定され得る薬剤開発試験における使用のための動物モデルを有することは、有益である。 It would be beneficial to have an animal model for use in drug development trials that can determine whether a candidate drug appears to achieve the intended therapeutic effect in humans at an early stage of drug development.
このような動物モデルは、ヒトにおけるCYP3A4遺伝子発現(特に、組織特異的発現)の調節の少なくともいくつかの局面が再現されない限り、有用ではない。これは、ヒトにおいて、治療の目的のために投与された際に多くの化合物が必然的に接触する特定の組織(肝臓および小腸を含む)において、CYP3A4遺伝子が発現されるからである。従って、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物誘導された組織特異的発現を再現しないモデルでの臨床的実施において、候補となる薬物のバイオ−アベイラビリティーが意図される治療効果を達成するのに十分であるか否かを決定することはできない。 Such animal models are not useful unless at least some aspects of the regulation of CYP3A4 gene expression (especially tissue specific expression) in humans are reproduced. This is because, in humans, the CYP3A4 gene is expressed in certain tissues (including the liver and small intestine) that many compounds inevitably come into contact with when administered for therapeutic purposes. Therefore, in clinical practice in a model that does not reproduce the constitutive nature of the CYP3A4 gene observed in humans and xenobiotic-induced tissue-specific expression, the therapeutic effect intended for bio-availability of candidate drugs is achieved. It is not possible to decide whether it is enough to do.
WO99/61622およびGoodwinら、1999は、生体異物化合物に応答してCYP3A4遺伝子の転写を調節するCYP3A4遺伝子の転写部位のイニシエーションから8kb上流に局在する核酸分子を開示する。これらの文献は、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物的誘導性組織特異的および発生的発現を調節するためのエレメントを開示していない。 WO 99/61622 and Goodwin et al., 1999 disclose nucleic acid molecules located 8 kb upstream from the initiation of the transcription site of the CYP3A4 gene that regulates transcription of the CYP3A4 gene in response to xenobiotic compounds. These references do not disclose elements for regulating the constitutive and xenobiotic-induced tissue-specific and developmental expression of the CYP3A4 gene observed in humans.
ある化合物(例えば、薬剤開発試験において同定されたもの)が、CYP3A4を誘導し、それゆえ薬物−薬物相互作用または研究下における薬物の代謝の自己誘導を引き起こしそうであるか否かを決定するために、ヒトにおけるCYP3A4遺伝子の発現の少なくともいくつかの局面を再現する動物モデルの必要性が存在する。 To determine whether certain compounds (eg, those identified in drug development trials) are likely to induce CYP3A4 and hence self-induction of drug-drug interactions or drug metabolism under study There is a need for an animal model that reproduces at least some aspects of the expression of the CYP3A4 gene in humans.
WO99/61622 WO99 / 61622
発明の説明
本発明は、上記で特定された必要性に取り組むことに努め、また第1の局面において、以下:
(a)ヒトCYP3A4遺伝子の転写を調節し得、且つその遺伝子の転写部位のイニシエーションとその部位から少なくとも13,000ヌクレオチド上流に位置づけられる位置との間に局在するヒトCYP3A4遺伝子の配列と一致するヌクレオチド配列を含む、調節核酸分子;および
(b)該調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子、
(ここで、このレポーター核酸分子および調節核酸分子は、調節核酸分子がレポーター核酸分子の転写を調節し得るよう配置される)
を含む非−ヒト動物を提供する。
DESCRIPTION OF THE INVENTION The present invention seeks to address the needs identified above, and in a first aspect the following:
(A) It can regulate the transcription of the human CYP3A4 gene and matches the sequence of the human CYP3A4 gene located between the initiation of the transcription site of the gene and a position located at least 13,000 nucleotides upstream from the site. A regulatory nucleic acid molecule comprising a nucleotide sequence; and (b) a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule to indicate modulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;
(Wherein the reporter nucleic acid molecule and the regulatory nucleic acid molecule are arranged such that the regulatory nucleic acid molecule can regulate transcription of the reporter nucleic acid molecule)
A non-human animal is provided.
本明細書中に記載されるように、本発明者らは、遺伝子の転写部位のイニシエーションと転写部位のイニシエーションの13,000ヌクレオチド上流の位置との間に位置づけられるヒトCYP3A4遺伝子の領域の動物モデルへの取り込みが、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物的誘導された組織特異的発現を再現するために十分な遺伝情報をその動物に提供することを見出した。より具体的には、本発明者らは、この領域を含むトランスジーンを含む動物モデルを作製し、そしてこれらのモデルが、ヒトにおいて観察されるCYP3A4の組織特異的発現を再現する組織パターンにおけるトランスジーンの構成性および生体異物的誘導性発現を提供することを観察した。重要なことに、構成性発現のレベルは、化合物(例えば、生体異物またはステロイド)の動物への投与が組織特異的トランスジーン発現の調節に及ぼす影響を観察し得るのに十分である。 As described herein, we have an animal model of a region of the human CYP3A4 gene that is located between the initiation of the transcription site of the gene and the position 13,000 nucleotides upstream of the initiation of the transcription site. It has been found that incorporation into provides enough genetic information to the animal to reproduce the constitutive and xenobiotic-induced tissue-specific expression of the CYP3A4 gene observed in humans. More specifically, we have created animal models that contain transgenes containing this region, and these models trans in tissue patterns that reproduce the tissue-specific expression of CYP3A4 observed in humans. The gene was observed to provide constitutive and xenobiotic-induced expression. Importantly, the level of constitutive expression is sufficient to be able to observe the effect of administration of a compound (eg, xenobiotic or steroid) to an animal on the modulation of tissue-specific transgene expression.
さらに、本発明者らは、本明細書中に記載される動物モデルがまた、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物的誘導性の発生的発現の局面を再現することを観察した。 In addition, the inventors have observed that the animal models described herein also reproduce the constitutive and xenobiotic-induced developmental aspects of CYP3A4 gene observed in humans. .
本発明以前には、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物的誘導された組織特異的または発生的発現を刺激するのに必要な遺伝情報が遺伝子の転写部位のイニシエーションと転写部位のイニシエーションの13,000ヌクレオチド上流の位置との間のヒトCYP3A4遺伝子の領域内に含まれるという示唆が存在しなかったことから、これらの発見は予測不可能である。 Prior to the present invention, the constitutive nature of the CYP3A4 gene observed in humans and the genetic information necessary to stimulate xenobiotic-induced tissue-specific or developmental expression were determined by the initiation of the transcription site and the transcription site. These findings are unpredictable because there was no suggestion that they were contained within the region of the human CYP3A4 gene between positions 13,000 nucleotides upstream of the initiation.
さらに、本発明以前には、マウスCYP3A11およびヒトCYP3A4遺伝子の誘導プロフィールにおける相違が観察されており、さらにマウス転写因子(特に、PXRおよびCAR)およびヒトPXRおよびCARのリガンド結合プロフィールにおいてもまた、相違が観察されていた。従って、非−ヒト動物がヒトにおいて観察されるCYP3A4の構成性および生体異物的誘導された組織特異的または発生的発現を再現するためのCYP3A4遺伝子の領域と相互作用するのに十分な因子を有するという示唆は、存在しなかった。 Furthermore, prior to the present invention, differences in the induction profiles of the mouse CYP3A11 and human CYP3A4 genes were observed, as well as differences in the ligand binding profiles of mouse transcription factors (particularly PXR and CAR) and human PXR and CAR. Was observed. Thus, non-human animals have sufficient factors to interact with regions of the CYP3A4 gene to reproduce the constitutive and xenobiotic-induced tissue-specific or developmental expression of CYP3A4 observed in humans The suggestion did not exist.
さらに、本発明以前には、トランスジェニックモデルへ組み込まれる転写エンハンサーエレメントが、ヒトにおいて観察される遺伝子発現の調節を再現し得る程度を制限した遺伝子サイレンシングおよびモザイクトランスジーン発現のようなトランスジーンの組込みに関するメカニズムが観察されてきた。従って、ヒトCYP3A4遺伝子の領域がヒトにおいて観察されるCYP3A4遺伝子の発現の調節を再現し得るという示唆は、存在しなかった。しかしながら、本明細書中に記載されるように、本発明者らは、トランスジーンの発現がヒトにおいて観察されるCYP3A4遺伝子発現の局面を再現することを、2つの異なる初代系統(2 separate founder lines)において示した。 In addition, prior to the present invention, transcriptional enhancer elements incorporated into transgenic models have been limited to transgenes such as gene silencing and mosaic transgene expression to a degree that can reproduce the regulation of gene expression observed in humans. Mechanisms for integration have been observed. Thus, there was no suggestion that the region of the human CYP3A4 gene could reproduce the regulation of CYP3A4 gene expression observed in humans. However, as described herein, we have found that the expression of the transgene reproduces the aspect of CYP3A4 gene expression observed in humans in two different founder lines (2 separate founder lines). ).
従って、第2の局面において、本発明は、以下;
(a)遺伝子の転写部位のイニシエーションから約13,000ヌクレオチド上流に及ぶヒトCYP3A4遺伝子のヌクレオチド配列と同一であるヌクレオチド配列を含む調節核酸分子;および
(b)調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子、
(ここで、このレポーター核酸分子および調節核酸分子は、この調節核酸分子がレポーター核酸分子の転写を調節し得るよう配置される。)
を含む非ヒト哺乳動物を提供する
1つの実施形態において、この調節核酸分子は、配列番号1に示される配列を含む。
Accordingly, in a second aspect, the present invention provides the following:
(A) a regulatory nucleic acid molecule comprising a nucleotide sequence identical to the nucleotide sequence of the human CYP3A4 gene spanning about 13,000 nucleotides upstream from the initiation of the transcription site of the gene; and (b) transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule A reporter nucleic acid molecule for producing a detectable amount of a reporter molecule to indicate modulation;
(Wherein the reporter nucleic acid molecule and the regulatory nucleic acid molecule are positioned such that the regulatory nucleic acid molecule can regulate transcription of the reporter nucleic acid molecule.)
In one embodiment, the regulatory nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 1.
さらに、本明細書中に記載されるように、本発明者らは、転写部位のイニシエーションと転写部位のイニシエーションの約3,200ヌクレオチド上流の位置との間のヒトCYP3A4遺伝子の領域を含むトランスジェニック動物を作製し、そしてこれらの動物においてトランスジーンが生体異物により誘導可能でないか、または構成性に発現されないことを観察した。従って、本発明者らは、ヒトにおいて観察されるCYP3A4の構成性および生体異物的誘導された組織特異的および発生的発現を再現するために必要な遺伝情報が、該遺伝子の転写部位のイニシエーションの約3,200ヌクレオチド上流に位置づけられる位置と転写部位のイニシエーションの13,000ヌクレオチド上流の位置との間のヒトCYP3A4遺伝子の領域内に含まれることを見出した。 In addition, as described herein, the inventors have included a transgenic comprising a region of the human CYP3A4 gene between the transcription site initiation and a position about 3,200 nucleotides upstream of the transcription site initiation. Animals were created and observed in these animals that the transgene was not inducible by xenobiotics or was not constitutively expressed. Thus, the inventors have found that the genetic information necessary to reproduce the constitutive and xenobiotically induced tissue-specific and developmental expression of CYP3A4 observed in humans is the transcription site initiation of the gene. It was found to be contained within the region of the human CYP3A4 gene between a position located approximately 3,200 nucleotides upstream and a position 13,000 nucleotides upstream of the transcription site initiation.
従って第3の局面において、本発明は、以下;
(a)遺伝子の転写部位のイニシエーションより約3,000ヌクレオチド上流の位置から約8,000ヌクレオチド上流に及ぶヒトCYP3A4遺伝子の配列と同一であるヌクレオチド配列を含む調節核酸分子;および
(b)調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子、
(ここで、このレポーター核酸分子および調節核酸分子は、この調節核酸分子がレポーター核酸分子の転写を調節し得るよう配置される。)
を含む非−ヒト哺乳動物を提供する
1つの実施形態において、この調節核酸分子は、配列番号2に示される配列を含む。
Accordingly, in a third aspect, the present invention provides the following:
(A) a regulatory nucleic acid molecule comprising a nucleotide sequence identical to the sequence of the human CYP3A4 gene extending from about 3,000 nucleotides upstream to about 8,000 nucleotides upstream from the initiation of the transcription site of the gene; and (b) a regulatory nucleic acid A reporter nucleic acid molecule for producing a detectable amount of the reporter molecule to indicate modulation of transcription of the reporter nucleic acid molecule by the molecule;
(Wherein the reporter nucleic acid molecule and the regulatory nucleic acid molecule are positioned such that the regulatory nucleic acid molecule can regulate transcription of the reporter nucleic acid molecule.)
In one embodiment, the regulatory nucleic acid molecule comprises the sequence shown in SEQ ID NO: 2.
第4の局面において、本発明は、以下:
(a)ヒトCYP3A4遺伝子の転写を調節し得、且つ該遺伝子の転写部位のイニシエーションの約7,200ヌクレオチド上流の位置から約600ヌクレオチド上流に及ぶヒトCYP3A4遺伝子の配列と同一であるヌクレオチド配列を含む、調節核酸分子;および
(b)該調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子、
(ここで、レポーター核酸分子および調節核酸分子は、この調節核酸分子がレポーター核酸分子の転写を調節し得るよう配置される。)
を包含する非−ヒト動物を提供する。
In a fourth aspect, the present invention provides the following:
(A) a nucleotide sequence that can regulate transcription of the human CYP3A4 gene and is identical to the sequence of the human CYP3A4 gene extending from about 7,200 nucleotides upstream to about 600 nucleotides upstream of initiation of the transcription site of the gene A regulatory nucleic acid molecule; and (b) a reporter nucleic acid molecule for producing a detectable amount of the reporter molecule to indicate modulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule;
(Wherein the reporter nucleic acid molecule and the regulatory nucleic acid molecule are positioned such that the regulatory nucleic acid molecule can regulate transcription of the reporter nucleic acid molecule.)
A non-human animal is provided.
1つの実施形態において、この調節核酸分子は、配列番号3に示される配列を含む。 In one embodiment, the regulatory nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3.
別の実施形態において、調節核酸分子はCYP3A4遺伝子の以下のフラグメントのいずれか1つの配列を有する:
(i)ヌクレオチド位置−13,000〜+53から成るフラグメント;
(ii)−8,000〜+53に隣接するヌクレオチド位置−13,000〜−12,700からなるフラグメント;
(iii)−1,200〜+53に隣接するヌクレオチド位置−13,000〜−5,100からなるフラグメント;
(v)−362〜+53に隣接するヌクレオチド位置−7,800〜−6,000からなるフラグメント;
(vi)−362〜+53に隣接するヌクレオチド位置−7,500〜−6,000からなるフラグメント;。
In another embodiment, the regulatory nucleic acid molecule has the sequence of any one of the following fragments of the CYP3A4 gene:
(I) a fragment consisting of nucleotide positions -13,000 to +53;
(Ii) a fragment consisting of nucleotide positions -13,000 to -12,700 adjacent to -8,000 to +53;
(Iii) a fragment consisting of nucleotide positions -13,000 to -5,100 adjacent to -1,200 to +53;
(V) a fragment consisting of nucleotide positions −7,800 to −6,000 adjacent to −362 to +53;
(Vi) a fragment consisting of nucleotide positions −7,500 to −6,000 adjacent to −362 to +53;
−362〜+53に隣接するヌクレオチド位置−7836〜−7207からなるフラグメントの配列を有する調節核酸分子は、この構築物がヒトCYP3A4遺伝子の転写を調節するために必要な最小配列(より具体的には、生体異物に応答するエレメント(「生体異物応答エレメントモジュール」または「XREM」)およびCYP3A4遺伝子の近位のプロモーター)を含む場合、特に好ましい。 A regulatory nucleic acid molecule having the sequence of a fragment consisting of nucleotide positions -7836 to -7207 adjacent to -362 to +53 is a minimal sequence (more specifically, required for this construct to regulate transcription of the human CYP3A4 gene) It is particularly preferred if it contains elements that respond to xenobiotics (“Xenobiotic Response Element Module” or “XREM”) and a promoter proximal to the CYP3A4 gene).
本発明の調節核酸分子は、代表的に、核レセプターと接触する際にヒトCYP3A4遺伝子の転写を調節し得る少なくとも1つのエンハンサーを含む。このようなエンハンサーの例は、生体異物またはステロイドのようなリガンドへ結合する核レセプターと接触する際に、ヒトCYP3A4遺伝子の転写を調節し得るものである。他の例は、PXR(プレグナンXレセプター(別名としては、SXR(ステロイドおよび生体異物レセプター)として知られる))およびRXR(9−cis レチノイン酸レセプター)、またはCAR(構成性アンドロスタンレセプター−β)およびRXRのへテロダイマーからなる核レセプターと接触する際に、ヒトCYP3A4遺伝子の転写を調節し得るものである。 The regulatory nucleic acid molecules of the invention typically comprise at least one enhancer that can regulate transcription of the human CYP3A4 gene when contacted with a nuclear receptor. Examples of such enhancers are those that can regulate transcription of the human CYP3A4 gene when contacted with nuclear receptors that bind to ligands such as xenobiotics or steroids. Other examples are PXR (pregnane X receptor (also known as SXR (steroids and xenobiotic receptors))) and RXR (9-cis retinoic acid receptor), or CAR (constitutive androstane receptor-β) And the transcription of the human CYP3A4 gene upon contact with a nuclear receptor consisting of the heterodimer of RXR.
本発明者らは、本発明の調節核酸分子と実質的に同じヌクレオチド配列を有する特定の核酸分子もまた、ヒトにおいて観察されるCYP3A4遺伝子の構成性および生体異物的誘導された組織特異的および発生的発現を再現するための十分な遺伝情報を有すると考えている。従って、ヒトCYP3A4遺伝子の転写を調節する分子の能力を著しく制限することなく、ヌクレオチドが調節核酸分子の領域(より具体的には、上記のもののようなエンハンサーを含まない領域)において改変または欠失され得ることは理解されるであろう。 The inventors have also found that certain nucleic acid molecules having substantially the same nucleotide sequence as the regulatory nucleic acid molecules of the invention are also constitutive and xenobiotically induced tissue specific and developmental of CYP3A4 genes observed in humans. It is considered that it has sufficient genetic information to reproduce the expression. Thus, without significantly limiting the ability of the molecule to regulate transcription of the human CYP3A4 gene, nucleotides are altered or deleted in regions of the regulatory nucleic acid molecule (more specifically, regions that do not contain an enhancer such as those described above). It will be understood that this can be done.
本発明者らは、他のヒト遺伝子(具体的には、治療のために使用される外因性および内因性化合物の治療活性を改変または調節し、薬物−薬物相互作用を引き起こす産物をコードするそれらの遺伝子(例えば、シトクロムP450遺伝子またはABCトランスポータースーパーファミリー遺伝子(例えば、P−グリコプロテイン(別名として、MDR−1として知られる))の発現をさらに再現し得る動物モデルを提供することが有益であることを認識している。これらの遺伝子のいくつかの構成性および生体異物誘導される組織特異的発現を調節する領域が知られ、そしていくつかの場合において、非−ヒト動物モデルが作製された。本発明者らは、これらの動物の遺伝的バックグラウンドが、例えば慣用的な交配技術によって、本発明の非−ヒト哺乳動物へ組み込まれ得ることを認識している。 The inventors have identified other human genes, specifically those that encode products that alter or regulate the therapeutic activity of exogenous and endogenous compounds used for therapy and cause drug-drug interactions. It would be beneficial to provide an animal model that can further reproduce the expression of the genes of (eg, cytochrome P450 genes or ABC transporter superfamily genes (eg, P-glycoprotein, otherwise known as MDR-1)) It is recognized that some constitutive and xenobiotic-induced tissue-specific expressions of these genes are known, and in some cases, non-human animal models have been created. We have determined that the genetic background of these animals is, for example, by conventional mating techniques. - recognizes that can be incorporated into human mammal.
従って第5の局面において、本発明は、本発明の第1から第4の局面のいずれか1つの非−ヒト哺乳動物を提供し、さらに以下:
(c)ヒト遺伝子の転写を調節し得るさらなる調節核酸分子;および
(d)さらなる調節核酸分子によるさらなるレポーター核酸分子の転写の調節を示すための検出可能な量のさらなるレポーター分子を産生するためのさらなるレポーター核酸分子、
(ここで、このさらなるレポーター核酸分子および調節核酸分子は、さらなる調節核酸分子がさらなるレポーター核酸分子の転写を調節し得るよう配置される。)
を包含する。
Accordingly, in a fifth aspect, the invention provides a non-human mammal of any one of the first to fourth aspects of the invention, and further:
(C) additional regulatory nucleic acid molecule capable of regulating transcription of a human gene; and (d) to produce a detectable amount of an additional reporter molecule to indicate modulation of transcription of the additional reporter nucleic acid molecule by the additional regulatory nucleic acid molecule. An additional reporter nucleic acid molecule,
(Wherein the additional reporter nucleic acid molecule and the regulatory nucleic acid molecule are arranged such that the additional regulatory nucleic acid molecule can regulate transcription of the additional reporter nucleic acid molecule.)
Is included.
1つの実施形態において、少なくとも1つのさらなる調節核酸分子は、配列番号4に示される配列を有する。別の実施形態において、少なくとも1つのさらなる調節核酸分子は、配列番号5に示される配列を有する。 In one embodiment, the at least one additional regulatory nucleic acid molecule has the sequence shown in SEQ ID NO: 4. In another embodiment, the at least one additional regulatory nucleic acid molecule has the sequence shown in SEQ ID NO: 5.
本明細書中に記載される本発明の調節核酸分子は、ヒトにおいて観察されるCYP3A4遺伝子の構成性組織特異的および発生的発現を再現するために十分であるが、本発明者らは、ヒトCYP3A4遺伝子の転写を調節するための調節核酸分子と相互作用し得る少なくとも1つのヒト転写因子を組み込むことによって、遺伝子の生体異物誘導性(inducibility)の局面が動物においてより良く再現し得ることを認識している。このような因子の例は、核レセプターである。これらのレセプターは、該レセプターが生体異物またはステロイドのようなリガンドへ結合される際に、ヒトにおいてCYP3A4遺伝子転写を調節し得るものであり得る。このようなレセプターの1つの例は、ヒトPXR(プレグナンXレセプター(別名としては、SXR(ステロイドおよび生体異物レセプター)として知られる))である。別の適切なレセプターは、ヒトCAR(構成性アンドロスタンレセプター−β)である。ヒトPXRまたはCARレセプターを含む非−ヒト動物が知られている。本発明者らは、これらの動物の遺伝的バックグラウンドが、例えば慣用的な交配技術によって、本発明の非−ヒト哺乳動物へ組み込まれ得ることを認識している。 Although the regulatory nucleic acid molecules of the invention described herein are sufficient to reproduce the constitutive tissue specific and developmental expression of the CYP3A4 gene observed in humans, we have Recognizing that inducibility aspects of genes can be better reproduced in animals by incorporating at least one human transcription factor that can interact with regulatory nucleic acid molecules to regulate transcription of the CYP3A4 gene doing. An example of such a factor is the nuclear receptor. These receptors can be those that can regulate CYP3A4 gene transcription in humans when the receptor is bound to a ligand such as a xenobiotic or steroid. One example of such a receptor is human PXR (pregnane X receptor (also known as SXR (steroid and xenobiotic receptor))). Another suitable receptor is human CAR (constitutive androstane receptor-β). Non-human animals containing human PXR or CAR receptors are known. The inventors recognize that the genetic background of these animals can be incorporated into the non-human mammals of the invention, for example, by conventional mating techniques.
従って第6の局面において、本発明の非−ヒト動物はさらに、ヒトCYP3A4遺伝子の転写を調節するための少なくとも1つのヒト転写因子を含む。好ましくは、この転写因子は核レセプターである。好ましくは、この核レセプターは、ヒトPXR(プレグナンXレセプター(別名としては、SXR(ステロイドおよび生体異物レセプター)として知られる))およびヒトRXR(9−cis レチノイン酸レセプター)、またはヒトCAR(構成性アンドロスタンレセプター−β)およびヒトRXRのへテロダイマーである。 Accordingly, in a sixth aspect, the non-human animal of the present invention further comprises at least one human transcription factor for regulating transcription of the human CYP3A4 gene. Preferably, this transcription factor is a nuclear receptor. Preferably, the nuclear receptor is human PXR (pregnane X receptor (also known as SXR (steroids and xenobiotic receptors))) and human RXR (9-cis retinoic acid receptor), or human CAR (constitutive Androstane receptor-β) and human RXR heterodimer.
該レポーター核酸分子が転写される場合、結果として該レポーター核酸分子は、検出され得る任意の分子であり得る。例えば、該レポーター核酸分子は、CYP3A4シトクロム、またはシトクロムを産生するよう翻訳されるmRNA転写物であり得る。ホタルルシフェラーゼ、β−ガラクトシダーゼ、アルカリホスファターゼ、緑色蛍光タンパク質またはクロラムフェニコールアセチルトランスフェラーゼを含む市販のレポーター分子が使用され得る。 When the reporter nucleic acid molecule is transcribed, as a result, the reporter nucleic acid molecule can be any molecule that can be detected. For example, the reporter nucleic acid molecule can be CYP3A4 cytochrome, or an mRNA transcript that is translated to produce cytochrome. Commercial reporter molecules including firefly luciferase, β-galactosidase, alkaline phosphatase, green fluorescent protein or chloramphenicol acetyltransferase can be used.
それゆえ、1つの実施形態において、このレポーター核酸分子は、ホタルルシフェラーゼ、β−ガラクトシダーゼ、アルカリホスファターゼ、緑色蛍光タンパク質またはクロラムフェニコールアセチルトランスフェラーゼからなるレポーター分子の群より選択されるレポーター分子を生産し得る。 Thus, in one embodiment, the reporter nucleic acid molecule produces a reporter molecule selected from the group of reporter molecules consisting of firefly luciferase, β-galactosidase, alkaline phosphatase, green fluorescent protein or chloramphenicol acetyltransferase. obtain.
以下に例示される本発明の非−ヒト哺乳動物はマウスであるが、本発明者らは、任意の他の非−ヒト哺乳動物(特に、標準的なトランスジェニック技術が開発されているもの(例えば、ラットおよびウサギを含む))が本発明において使用され得ると考えている。しかしながら、代表的には、非−ヒト哺乳動物はマウスである。 Although the non-human mammals of the present invention exemplified below are mice, the present inventors have made any other non-human mammals (especially those for which standard transgenic techniques have been developed ( For example, it is contemplated that rats and rabbits) can be used in the present invention. Typically, however, the non-human mammal is a mouse.
別の局面において、本発明は、本発明の非−ヒト哺乳動物の組織を提供する。 In another aspect, the present invention provides a non-human mammalian tissue of the present invention.
1つの実施形態において、この組織は、本発明の非−ヒト哺乳動物を生産し得る胚である。 In one embodiment, the tissue is an embryo that can produce a non-human mammal of the invention.
さらなる局面において、本発明は、ある化合物がヒトCYP3A4遺伝子の転写に影響を及ぼし得るか否かを決定する方法を提供し、この方法は以下の工程を包含する:
(a)該化合物を本発明に従う非ヒト哺乳動物へ投与すること、および
(b)レポーター分子が哺乳動物におけるレポーター核酸分子によって産生されるか否かを決定すること。
In a further aspect, the present invention provides a method for determining whether a compound can affect transcription of the human CYP3A4 gene, which method comprises the following steps:
(A) administering the compound to a non-human mammal according to the invention, and (b) determining whether the reporter molecule is produced by a reporter nucleic acid molecule in the mammal.
1つの実施形態において、レポーター分子の産生は、結合する化合物がヒトCYP3A4遺伝子の転写に影響し得ることを示唆する。 In one embodiment, production of the reporter molecule suggests that the compound that binds can affect transcription of the human CYP3A4 gene.
あらゆる化合物がこの方法において試験され得るが、好ましい化合物は生体異物またはステロイド化合物である。 Although any compound can be tested in this method, preferred compounds are xenobiotics or steroidal compounds.
本発明者らは、ある化合物がヒトCYP3A4遺伝子の転写を調節し得るか否かを決定するための方法において、CYP3A4遺伝子の5’フランキング領域を含むが−7836から−7207までの領域について欠失する非ヒト動物がネガティブコントロールとして有用であると認識している。 In a method for determining whether a compound can regulate transcription of the human CYP3A4 gene, the inventors include a 5 ′ flanking region of the CYP3A4 gene but lack a region from −7836 to −7207. We recognize that non-human animals that lose are useful as negative controls.
ここで、本発明の実施形態は、以下の実施例(これは、単に例示するためであり、本発明の範囲を制限するためでないと理解されるであろう)において記載される。 Embodiments of the invention will now be described in the following examples, which will be understood to be merely illustrative and not limiting the scope of the invention.
物質および方法
トランスジーン構築物。E.coli lacZレポーター遺伝子に結合されるヒトシトクロムP450 CYP3A4遺伝子の上流5’フランクを用いて、2つのトランスジーン構築物を合成した(図1)。第1の構築物(−3CYP3A4/lacZと称する)は、転写開始部位に関係する−3213bpにおけるHindIII部位から転写開始部位の下流ヌクレオチド+56bpまでのCYP3A4遺伝子の領域を含んでいた。他の構築物(−13CYP3A4/lacZと称する)は、転写開始部位の上流−12,926bpにおけるKpnI部位から下流+56bpまでのCYP3A4遺伝子の領域を含んでいた。これは、CYP3A4遺伝子の近位プロモーターに加え、−7836と−7208との間に局在するXREM領域のDNA配列を含む。−10468bpと+906bpとの間のCYP3A4遺伝子のDNA配列を決定し、アクセス番号AF185589のもとにGenBank/EMBL/DDJBデータベースに寄託した。公にアクセス可能なGenbankファイルから、−10,469bpから−12,926bpまでの領域をカバーするさらなる配列情報を得た。E.coli lacZ レポーター遺伝子は、真核生物翻訳開始および終結シグナル、SV40転写終結およびポリアデニル化シグナルならびにイントロンについてのDNA配列に隣接する細菌酵素β−ガラクトシダーゼについてのコーディング領域を含む。CYP3A4/lacZトランスジーン構築物を、ベクター配列から離し、アガロースゲルにて精製し、マイクロインジェクションした。
Materials and Methods Transgene constructs. E. Two transgene constructs were synthesized using the 5 ′ flank upstream of the human cytochrome P450 CYP3A4 gene linked to the E. coli lacZ reporter gene (FIG. 1). The first construct (designated −3CYP3A4 / lacZ) contained a region of the CYP3A4 gene from the HindIII site at −3213 bp related to the transcription start site to the downstream nucleotide of the transcription start site +56 bp. The other construct (referred to as -13CYP3A4 / lacZ) contained a region of the CYP3A4 gene from the KpnI site up to +56 bp downstream at −12,926 bp upstream of the transcription start site. This includes the DNA sequence of the XREM region located between -7836 and -7208 in addition to the proximal promoter of the CYP3A4 gene. The DNA sequence of the CYP3A4 gene between -10468 bp and +906 bp was determined and deposited in the GenBank / EMBL / DDJB database under access number AF185589. Additional sequence information covering the region from -10,469 bp to -12,926 bp was obtained from publicly accessible Genbank files. E. The E. coli lacZ reporter gene contains the coding region for the bacterial enzyme β-galactosidase flanking the eukaryotic translation initiation and termination signal, the SV40 transcription termination and polyadenylation signal and the DNA sequence for the intron. The CYP3A4 / lacZ transgene construct was separated from the vector sequence, purified on an agarose gel, and microinjected.
トランスジェニックマウス系統の作製。FVB/N株マウスから回収した接合体の前核へのDNA構築物のマイクロインジェクションによって、CYP3A4/lacZトランスジーンを有するマウスを作製した。マイクロインジェクションおよび胚の操作を標準的な技術により行った。サザン分析によって同定したトランスジェニック初代(founders)から繁殖させることによって、安定なトランスジェニックマウス系統を確立した。 Generation of transgenic mouse lines. Mice carrying CYP3A4 / lacZ transgenes were generated by microinjection of DNA constructs into the pronuclei of zygotes recovered from FVB / N strain mice. Microinjection and embryo manipulation were performed by standard techniques. Stable transgenic mouse lines were established by breeding from transgenic founders identified by Southern analysis.
マウスへの生体異物の投与。−3CYP3A4/lacZおよび−13CYP3A4/lacZトランスジーンについての8−10週齢の雄および雌マウスの半接合体を使用して、ある範囲の生体異物およびホルモンのトランスジーン−由来β−ガラクトシダーゼの発現を活性化する能力を試験した。4日間毎日1回の腹腔内注射によって、マウスへ以下の試薬およびビヒクルを投与した:リファンピシン/コーン油;デキサメタゾンホスフェート/H2O;プレグネノロン 16α−カルボニトリル/H2O中の2% Tween20;フェノバルビタール/H2O;クロトリマゾール/2% Tween20;フェニトイン/2% Tween20;17α−OH プロゲステロン/2% Tween20;プレグネノロン/2% Tween20。Faulding(Mulgrave, Australia)から得られたデキサメタゾンホスフェートおよびUpjohn Co.(Kalamazoo, MI)からのプレグネノロン 16α−カルボニトリルを除いて、全ての試薬はSigma Chemical Co.(St Louis, MO)によって供給された。トランスジーンの誘導について試験するために全ての試薬について使用された用量は、100mg/kg体重であった。雄半接合体トランスジェニックマウスを用いて、1−100mg/kgの範囲において、用量応答研究を行った。 Xenobiotic administration to mice. The expression of a range of xenobiotic and hormonal transgene-derived β-galactosidases using hemizygotes of 8-10 week old male and female mice for the -3CYP3A4 / lacZ and -13CYP3A4 / lacZ transgenes The ability to activate was tested. Mice were administered the following reagents and vehicle by intraperitoneal injection once daily for 4 days: rifampicin / corn oil; dexamethasone phosphate / H 2 O; pregnenolone 16α-carbonitrile / 2% Tween 20 in H 2 O; pheno Barbital / H 2 O; clotrimazole / 2% Tween 20; phenytoin / 2% Tween 20; 17α-OH progesterone / 2% Tween 20; pregnenolone / 2% Tween 20. Dexamethasone phosphate obtained from Faulding (Mulgrave, Australia) and Upjohn Co. All reagents were from Sigma Chemical Co. except for pregnenolone 16α-carbonitrile from (Kalamazoo, MI). (St Louis, MO). The dose used for all reagents to test for induction of the transgene was 100 mg / kg body weight. Dose response studies were performed in the range of 1-100 mg / kg using male hemizygous transgenic mice.
トランスジーンおよびマウスCyp3a遺伝子発現の分析。X−gal(5−ブロモ−4−クロロ−3−インドリル−β−D−ガラクトピラノシド)を用いる染色により、肝臓および他の組織のスライス(slices)および凍結切片(sections)において、β−ガラクトシダーゼ活性を視覚化した。組織を、0.25% グルタルアルデヒド、0.1M リン酸緩衝液(pH7.3)、5mM EGTA、2mM MgCl2中で固定化し:0.1Mリン酸緩衝液(pH7.3)、0.01%デオキシコール酸ナトリウム、0.025%NP40、2mM MgCl2中で洗浄し、そして1mg/ml X−gal、5mM フェリシアン化カリウム、および5mM フェロシアン化カリウムを補った洗浄溶液中での37℃におけるインキュベーションによって染色した。標準的な技術に従って、O−ニトロフェニル−β−D−ガラクトピラノシド(ONPG)アッセイを用いて、全肝臓ホモジェネート[100mg 新鮮な組織/ml 0.25M Tris−HCl(pH7.3)]において、β−ガラクトシダーゼ活性のレベルを測定した。適切な希釈の後、ホモジェネートをβ−ガラクトシダーゼアッセイ試薬(0.1M リン酸ナトリウム緩衝液(pH7.3)/1mM MgCl2/50mmol β−メルカプトエタノール/0.88mg/ml ONPG)と共に37℃にてインキュベーションし、1M Na2CO3の添加によってクエンチし(quenched)、そして420nmでの吸光度を測定した。β−ガラクトシダーゼ活性の単位は、A420/mg肝臓/分として示される。 Analysis of transgene and mouse Cyp3a gene expression. By staining with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), β- in liver and other tissue slices and frozen sections. Galactosidase activity was visualized. Tissues were fixed in 0.25% glutaraldehyde, 0.1 M phosphate buffer (pH 7.3), 5 mM EGTA, 2 mM MgCl 2 : 0.1 M phosphate buffer (pH 7.3), 0.01 Stain by washing at 37 ° C. in wash solution supplemented with 1 mg / ml X-gal, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide, washed in% sodium deoxycholate, 0.025% NP40, 2 mM MgCl 2 did. In whole liver homogenate [100 mg fresh tissue / ml 0.25 M Tris-HCl (pH 7.3)] using the O-nitrophenyl-β-D-galactopyranoside (ONPG) assay according to standard techniques. The level of β-galactosidase activity was measured. After appropriate dilution, at 37 ° C. with a beta-galactosidase assay reagent (0.1M sodium phosphate buffer (pH7.3) / 1mM MgCl 2 / 50mmol β- mercaptoethanol /0.88mg/ml ONPG) homogenate Incubated, quenched by the addition of 1M Na 2 CO 3 and the absorbance at 420 nm was measured. Units of β-galactosidase activity are shown as A 420 / mg liver / min.
マウスCyp3a11 cDNAのヌクレオチド852−1061に相補的なリボプローブを用いるノーザン分析によって、内因性マウスCyp3a mRNA発現のレベルを測定した。フィルターをはがし、そして負荷量(loading)を標準化するために18S rRNAオリゴヌクレオチドを用いて再プローブした。 Endogenous mouse Cyp3a mRNA expression levels were measured by Northern analysis using a riboprobe complementary to nucleotides 852-1061 of mouse Cyp3a11 cDNA. Filters were stripped and reprobed with 18S rRNA oligonucleotides to normalize loading.
結果
lacZへ結合するヒトCYP3A4遺伝子の−3.2kb領域を含む構築物を用いて、4つのトランスジェニック系統を作製した。トランスジーン−由来β−ガラクトシダーゼ活性は、マウス由来の腎臓、大および小腸、脾臓、肺および肝臓組織において、ビヒクルまたは生体異物で処理した全ての4つの−3CYP3A4/lacZトランスジェニック系統について、検出されなかった(表1)。対照的に、−13CYP3A4/lacZ構築物を有する4系統のうち3つにおいて、トランスジーンの発現が容易に検出された。系統9/4は肝臓において非常に低い構成性レベルを有し、β−ガラクトシダーゼは主要な血管に隣接した単離肝細胞においてのみ検出された。生体異物の投与は、中心静脈のまわりの細胞の領域において強い発現をもたらした(図2)。9/4系統の未処理マウスにおけるトランスジーン発現の基本レベルが極めて低いので、誘導は明らかであり、そして本質的にオフ/オン・プロセス(off/on process)である。9/4系統に由来するマウスにおける他の組織内での発現は、消化管(大部分は、小腸の絨毛において)に制限された。
Results Four transgenic lines were generated using a construct containing the -3.2 kb region of the human CYP3A4 gene that binds to lacZ. Transgene-derived β-galactosidase activity is not detected in kidney, large and small intestine, spleen, lung and liver tissue from mice for all four -3CYP3A4 / lacZ transgenic lines treated with vehicle or xenobiotics (Table 1). In contrast, transgene expression was readily detected in 3 out of 4 lines with the -13CYP3A4 / lacZ construct. Line 9/4 has a very low constitutive level in the liver and β-galactosidase was only detected in isolated hepatocytes adjacent to the main blood vessels. Xenobiotic administration resulted in strong expression in the area of cells around the central vein (FIG. 2). Since the basal level of transgene expression in 9/4 line naïve mice is very low, induction is obvious and is essentially an off / on process. Expression in other tissues in mice derived from the 9/4 strain was restricted to the gastrointestinal tract (mostly in the small intestinal villi).
9/4系統由来のマウスの肝臓溶解物におけるトランスジェニックβ―ガラクトシダーゼ活性を測定することによって、ある範囲の生体異物に対する誘導の相対的程度を分析した(図3A)。デキサメタゾンおよびプレグネノロン 16α−カルボニトリルは最も強力な誘導物質であり、一方リファンピシンはトランスジーンを相対的に大きくないレベルまで活性化した。フェノバルビタール、クロトリマゾールおよびフェニトインは中程度の誘導物質であった。9/4系統におけるトランスジーンの誘導プロフィールは、同マウスにおける内因性Cyp3a11遺伝子について観察されたものと類似しており(図3B)、ヒトPXRよりもむしろマウスの活性化プロフィールを反映するようである。天然に存在するプレグネノロンおよび17α−プロゲステロンのようなステロイドを用いてトランスジーンの活性化が観察されたが、その誘導は生体異物と比べて弱かった。肝臓トランスジーン発現において著しい性差があり、殆どの試薬について、雄よりも雌においてより低いレベルが観察された。このような雄−優性パターンは、マウスCyp3a11遺伝子の誘導プロフィールにおいては顕著でなかった。実際に、リファンピシンおよびプレグネノロン 16α−カルボニトリルを用いた処理の後に、雄よりも雌においてより高いレベルのCyp3a11 mRNAを観察した。性−関連トランスジーン発現パターンにおけるこの明らかな逆転(reversal)についての理由は、知られていない。しかしながら、Cyp3a11 mRNAは、マウスのFVB/N株の雄においてのみ唯一検出可能であり、これは、雌に比べて雄におけるマウスCyp3a11遺伝子の誘導の程度が相対的に大きかったことに帰するかも知れない(図3B)。 The relative extent of induction against a range of xenobiotics was analyzed by measuring transgenic β-galactosidase activity in liver lysates of mice from 9/4 strain (FIG. 3A). Dexamethasone and pregnenolone 16α-carbonitrile were the most potent inducers, while rifampicin activated the transgene to relatively low levels. Phenobarbital, clotrimazole and phenytoin were moderate inducers. The induction profile of the transgene in the 9/4 strain is similar to that observed for the endogenous Cyp3a11 gene in the same mouse (FIG. 3B) and appears to reflect the activation profile of the mouse rather than the human PXR . Activation of the transgene was observed with steroids such as naturally occurring pregnenolone and 17α-progesterone, but the induction was weak compared to xenobiotics. There were significant gender differences in liver transgene expression, with lower levels observed in females than in males for most reagents. Such a male-dominant pattern was not significant in the induction profile of the mouse Cyp3a11 gene. Indeed, after treatment with rifampicin and pregnenolone 16α-carbonitrile, higher levels of Cyp3a11 mRNA were observed in females than in males. The reason for this apparent reversal in the sex-related transgene expression pattern is unknown. However, Cyp3a11 mRNA can only be detected in male FVB / N strain males, which may be attributed to the relatively high degree of induction of the mouse Cyp3a11 gene in males compared to females. No (FIG. 3B).
有意なトランスジーン発現を示した他の系統−15/10は、未処理マウスにおける肝臓および小腸の両方において、より高い構成性レベル(constitutive level)を有した。発現は他の臓器において検出されず、9/4系統において観察される組織特異性を確かなものにした。同じセットの試薬が、9/4系統由来のマウスと同じレベルまで、肝臓および腸のトランスジーン発現を増大させ得た。しかしながら誘導の全体的な程度は、15/10系統における高い基本レベルに起因して、9/4系統において観察された程大きくなかった。誘導プロフィールも類似しており、デキサメタゾンが最も強力な活性化剤であり、またリファンピシンが最も弱かった(データを示さない)。 Another line-15 / 10, which showed significant transgene expression, had higher constitutive levels in both liver and small intestine in untreated mice. Expression was not detected in other organs, confirming the tissue specificity observed in the 9/4 line. The same set of reagents could increase liver and intestinal transgene expression to the same level as mice from 9/4 strain. However, the overall degree of induction was not as great as observed in the 9/4 line due to the high baseline level in the 15/10 line. The induction profiles were similar, dexamethasone was the most potent activator and rifampicin was the weakest (data not shown).
生体異物誘導の用量応答。デキサメタゾンによる9/4系統におけるトランスジーン発現の活性化は、範囲1〜100mg/kgにわたり用量−依存的であった(図4A)。増大する用量のデキサメタゾンで処理されたマウスに由来する肝臓ホモジェネートにおけるより高いトランスジーン−由来β−ガラクトシダーゼ活性は、X−galによって染色された細胞の拡大された領域に関連していた。低用量のデキサメタゾンでは、中心静脈のまわりのわずか1−2細胞厚の肝細胞の輪(a ring of hepatocyte)がトランスジーンを発現した(図4B)。100mg/kg デキサメタゾンを用いると、X−gal陽性肝細胞の領域が10細胞に至るまで(中心静脈と門脈三管との間のほぼ中程)増大した。トランスジーンを発現する肝細胞の類似する用量−依存的拡大(expansion)が、他の試薬を用いて、また−13CYP3A4/lacZ構築物も含む15/10系統においても観察された。 Xenobiotic-induced dose response. Activation of transgene expression in line 9/4 by dexamethasone was dose-dependent over the range 1-100 mg / kg (FIG. 4A). The higher transgene-derived β-galactosidase activity in liver homogenates from mice treated with increasing doses of dexamethasone was associated with an expanded area of cells stained with X-gal. At low doses of dexamethasone, a ring of hepatocytes only 1-2 cells thick around the central vein expressed the transgene (FIG. 4B). With 100 mg / kg dexamethasone, the area of X-gal positive hepatocytes increased to 10 cells (approximately midway between the central vein and the portal tract). Similar dose-dependent expansion of hepatocytes expressing the transgene was observed using other reagents and also in the 15/10 line containing the -13CYP3A4 / lacZ construct.
Claims (13)
(a)ヒトCYP3A4遺伝子の構成性及び生体異物誘導性発現のために提供され得、且つ該遺伝子の転写部位のイニシエーション(initiation)から13,000ヌクレオチド上流に及ぶヒトCYP3A4遺伝子のヌクレオチド配列と同一であるヌクレオチド配列を含む、調節核酸分子;および
(b)該調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子
(ここで、該レポーターおよび調節核酸分子は、該調節核酸分子が該レポーター核酸分子の転写を調節し得るよう配置される)
を含むマウス及びラットから選択される哺乳動物:
ここで該哺乳動物は、ある化合物がヒトCYP3A4遺伝子を誘導し得るかどうかを決定するために、マウス又はラットの肝臓及び消化管において検出可能な該レポーター分子の構成性及び生体異物誘導性組織発現パターンを有することを特徴とし、
該調節核酸分子が配列番号3に示される配列を含む。 Less than:
(A) the nucleotide sequence of the human CYP3A4 gene that can be provided for constitutive and xenobiotic-inducible expression of the human CYP3A4 gene and extends 13,000 nucleotides upstream from the initiation of the transcription site of the gene; A regulatory nucleic acid molecule comprising a nucleotide sequence; and (b) a reporter nucleic acid molecule for producing a detectable amount of a reporter molecule to indicate modulation of transcription of the reporter nucleic acid molecule by the regulatory nucleic acid molecule, wherein The reporter and regulatory nucleic acid molecule are positioned such that the regulatory nucleic acid molecule can regulate transcription of the reporter nucleic acid molecule)
Mammals selected from mice and rats containing:
Here, the mammal is able to determine whether a compound is capable of inducing the human CYP3A4 gene, the constitutive and xenobiotic-induced tissue expression of the reporter molecule detectable in mouse or rat liver and gastrointestinal tract. Characterized by having a pattern ,
The regulatory nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3 .
(a)ヒトCYP3A4遺伝子の構成性及び生体異物誘導性発現のために提供され得、且つ配列番号1に示される配列を含む、調節核酸分子;および
(b)該調節核酸分子によるレポーター核酸分子の転写の調節を示すための検出可能な量のレポーター分子を産生するためのレポーター核酸分子
(ここで、該レポーターおよび調節核酸分子は、該調節核酸分子が該レポーター核酸分子の転写を調節し得るよう配置される)
を含む、マウス及びラットから選択される哺乳動物:
ここで該哺乳動物は、ある化合物がヒトCYP3A4遺伝子を誘導し得るかどうかを決定するために、マウス又はラットの肝臓及び消化管において検出可能な該レポーター分子の構成性及び生体異物誘導性組織発現パターンを有することを特徴とする。 Less than:
(A) a regulatory nucleic acid molecule that can be provided for constitutive and xenobiotic-inducible expression of the human CYP3A4 gene and comprises the sequence shown in SEQ ID NO: 1; and (b) A reporter nucleic acid molecule for producing a detectable amount of a reporter molecule to indicate the regulation of transcription, wherein the reporter and regulatory nucleic acid molecules allow the regulatory nucleic acid molecule to regulate transcription of the reporter nucleic acid molecule Placed)
A mammal selected from mice and rats, comprising:
Here, the mammal is able to determine whether a compound is capable of inducing the human CYP3A4 gene, the constitutive and xenobiotic-induced tissue expression of the reporter molecule detectable in mouse or rat liver and gastrointestinal tract. It has a pattern.
ヒト遺伝子の転写を調節し得るさらなる調節核酸分子;および
さらなる調節核酸分子によるさらなるレポーター核酸分子の転写の調節を示すための検出可能な量のさらなるレポーター分子を産生するためのさらなるレポーター核酸分子、
ここで、該さらなるレポーターおよびさらなる調節核酸分子は、該さらなる調節核酸分子が該さらなるレポーター核酸分子の転写を調節し得るよう配置される。 A mammal according to any one of 請 Motomeko 1-3, further comprising:
An additional regulatory nucleic acid molecule capable of regulating transcription of the human gene; and an additional reporter nucleic acid molecule for producing a detectable amount of an additional reporter molecule to indicate modulation of transcription of the additional reporter nucleic acid molecule by the additional regulatory nucleic acid molecule;
Here, the additional reporter and additional regulatory nucleic acid molecule are positioned such that the additional regulatory nucleic acid molecule can regulate transcription of the additional reporter nucleic acid molecule.
(a)該化合物を請求項1〜8のいずれかに記載の哺乳動物へ投与すること、および
(b)レポーター分子が該哺乳動物におけるレポーター核酸分子によって産生されるか否かを決定すること。 A method for determining whether a compound can affect the transcription of the human CYP3A4 gene, comprising the following steps:
(A) administering the compound to a mammal according to any of claims 1-8 , and (b) determining whether a reporter molecule is produced by a reporter nucleic acid molecule in the mammal.
(b)レポーター分子が該哺乳動物におけるレポーター核酸分子によって産生されるか否かを決定すること。 Use of a mammal according to any of claims 1 to 8 for determining whether a compound can affect the transcription of the human CYP3A4 gene by a method comprising the following steps: (a) Administering said compound to a mammal according to any of claims 1-8 , and (b) determining whether a reporter molecule is produced by a reporter nucleic acid molecule in said mammal.
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