標的遺伝子組換えによる突然変異体の作成とその生体機能の研究

1994 Fiscal Year Annual Research Report

• Project/Area Number: 05275102
• Research Institution: Kyushu University
• Principal Investigator: 勝木 元也 九州大学, 生体防御医学研究所, 教授 (20051732)
• Co-Investigator(Kenkyū-buntansha): 前田 秀一郎 山梨医科大学, 教授 (10117244) ; 林崎 良英 理化研, ライフ・筑波研究センター, 主任研究員 (70192705) ; 金田 安史 大阪大学, 細胞生体工学センター, 助教授 (10177537) ; 糸原 重美 京都大学, ウイルス研究所, 助教授 (60252524) ; 相沢 慎一 熊本大学, 医学部, 教授 (60073011)
• Keywords: 標的遺伝子組換え / ジーンターゲッティング / 中枢神経系 / 免疫系 / 形態形成 / グルタミン酸受容体 / ドーパミン受容体 / H-ras

Research Abstract

本研究は標的遺伝子組換え法(ジーンターゲッティング法)を用いて、固体レベルでの解析が必須であると考えられる中枢神経系、免疫系、形態形成等に関与する遺伝子を計画的に破壊し、それぞれの遺伝子の生体での機能を解析することを目的としている。計画班員は、きわめて緊密に連携し、独立を保ちながら、多種の遺伝子の突然変異体の作成に成功した。例えば林崎班員が単離したマウスのreeler遺伝子は、さっそく勝木班員に送られ、すぐにターゲッティングにとりかかるといった状況は、重点領域研究の計画研究として初めて出来ることである。
本年度においてホモ型にまで進んで突然変異マウスを列挙すれば次の如くである。グルタミン酸受容体(NMDARタイプ5種、およびmGluR4種):ホモ型突然変異体受容体6(mGR6)破壊マウスは光のON刺激に対して反応しないことが確かめられた。ドーパミン受容体:ホモ型変異体は、不稔であるが、他の性状はみかけ上正常である。今後薬理学的行動実験を行う。H-ras遺伝子:ホモ型突然変異体は正常に生まれ、驚くべきことに正常と変わらない。その他、N-ras、K-ras、ジストロフィン、Otx2、Brn2、HGF、リアノジン受容体などの遺伝子が破壊された。それぞれの遺伝子破壊によって認められる生体の特徴は、細胞レベルやタンパク質化学レベルの既存の成果から容易に予測されものは少なく、それぞれの遺伝子産物が多くの生体機能に関わっていることが示された。
本年度は以上の他にも、各班員それぞれに1個以上の遺伝子破壊に成功し多くの大きな成果が得られたと評価できる。

Research Products

• [Publications] Tsunematsu,S.,et al.: "Hepatic tumors induced by carbon tetrachloride in transgenic mice carrying human c-H-ras proto-oncogene without mutations." Int J Cancer. 59. 554-559 (1994)
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• [Publications] Lafaille,JJ.,et al.: "High incidence of spontaneous autoimmune encephalomyelitis in immuno-deficient anti-myelin basic protein Tcell receptor trasgenic mice." Cell. 78. 399-408 (1994)
• [Publications] Gondo,Y.,et al.: "Gene replacement of the p53 gene with the lacZ gene in mouse embryonic stem cells and mice by using two steps of homologous recombination." Biochem Biophys Res Commun. 202. 830-837 (1994)
• [Publications] Doi,ST.,et al.: "Tissue-specific tumorigenesis Induced by Dimethylbenzanthracene in transgenic mice carrying human c-Ha-ras proto-oncogenes." Jpn J Can Res. 85. 801-807 (1994)
• [Publications] Miyado,K.,et al.: "Isolation of a yeast cDNA clone that encodes a novel trans-membrane protein having a c-terminal highly basic region." Biochem Biophys Res Commun. 199. 1363-1371 (1994)
• [Publications] Nada,S.,et al.: "Identification of major tyrosine-phosphorylatedproteins in Csk-deficient cells." Oncogene. 9. 3571-3578 (1994)
• [Publications] Umemori,H.,et al.: "Initial events of myelination involve Fyn tyrosine kinase signalling." Nature. 367. 572-576 (1994)
• [Publications] Kubo,S.,et al.: "A novel form of self tolerance dictated in the thymus of transgenic mice with autoreactive TCRα and βchain genes." Int Immunol. 6. 593-602 (1994)
• [Publications] Hashimoto,N.,et al.: "Parthenogenetic activation of oocytes,but normal spermatogenesis and somatic cell growth,by c-mos-deficiency in mice." Nature. 370. 68-70 (1994)
• [Publications] Yagi,T.,et al.: "Fyn expression during neurogenesis in mouse embryos." Oncogene. 9. 2433-2440 (1994)
• [Publications] Tanaka,N.,et al.: "Cellular commitment to oncogene-induced transformation or apoptosisis dependent on the transcription factor IRF-1." Cell. 77. 829-839 (1994)
• [Publications] Kohmura,N.,et al.: "A novel non-receptor tyrosine kinase,srm;cloning and targeted disruption." Mol Cell Biol. 14. 6915-6925 (1994)
• [Publications] Ishizaki,K.,et al.: "Increased UV-induced SCEs in p53-deficient mouse cells." Int J Cancer. 58. 254-257 (1994)
• [Publications] Tamemoto,H.,et al.: "Targeted disruption of the insulin receptor substrate-1 gene results in insulin resistance and growth retardation." Nature. 372. 182-186 (1994)
• [Publications] Gomi,H.,et al.: "Prion protein (PrP) is not involved in the pathogenesis of spongiform encephalopathy in zitter rats." Neurosci Lett. 166. 171-174 (1994)
• [Publications] Takahama,Y.,et al.: "Entry of CD4^-CD8^- immature thymocytes into the CD4/CD8 developmental pathway is controlled by thymidine kinase signals that can be provided throught Tcell receptor components." Int Immunol. 6. 1505-1514 (1994)
• [Publications] Morishita,R.,et al.: "Intimal hyperplasia after vascular injury is inhibited by antisense cdc2 kinase oligonucleotides." J Clin Invest. 93. 1458-1464 (1994)
• [Publications] Tomita,N.,et al.: "A novel gene transfer technique mediated by HVJ(Sendai virus),nuclear protein and liposomes." Cancer Diagno Prev. 18. 485-491 (1994)
• [Publications] Naora,H.,et al.: "Transgenic mouse model of hemifacial microsomia:cloning and characterization of insertional mutation region on Chromosome 10." Genomics. 23. 515-519 (1994)
• [Publications] Takagi,S.,et al.: "An efficient expression vector for transgenic medaka construction." Mol Marine Biol Biotechnol. 3. 192-199 (1994)
• [Publications] Okuizumi,H.,et al.: "Application of the RLGS method to large-size genomes using restriction trapper." DNA Research. 1. 99-102 (1994)
• [Publications] Sasaki,N.,et al.: "The gene responsible for LEC hepatitis,located on rat chromosome 16,is the homolog to the human Wilson disease gene." Biochem Biophys Res Commun. 202. 512-518 (1994)
• [Publications] Kalcheva,I.,et al.: "Comparative mapping of the imprinted U2afbpL gene on mouse chromosome II and human chromosome 5." Cytogenet Cell Genet. 68. 19-24 (1995)
• [Publications] Maki,K.,et al.: "Targeted pituitary tumorigenesis using the human thyrotropin β-subunit chain promoter in trnasgenic mice." J Mol Cell Endocrinol. 105. 147-154 (1994)
• [Publications] Kawai,J.,et al.: "Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning." Mol Cell Biol. 14. 7421-7427 (1994)
• [Publications] Imoto,H.,et al.: "Direct determination of NotI cleavage sites inthe genomic DNA of adult mouse kidney and human trphoblast using whole range restriction landmark genomic scanning." DNA Res. 1. 239-243 (1994)
• [Publications] Hayashizaki,Y.et al.: "A genetic linkage map of the mouse using Restriction Landmark Genomic Scanning(RLGS)." Genetics. 138. 1207-1238 (1994)
• [Publications] Tada,M.,et al.: "Localization of mouse imprinted gene U2afbp-rsto A3.2-4 band of Chromosome 11 by FISH." Mammalian Genome. 5. 655-657 (1994)
• [Publications] Hirotsune,S.,et al.: "Spot mapping onthe standard profile of restriction landmark genomic scanning(RLGS) of sorted chromosome 20 using methylation-insensitive enzyme." Genomics. 24. 593-596 (1994)
• [Publications] Hayashizaki,Y.: "Systematic screening of endogenous imprinted genes using RLGS and identification of a new imprinted gene U2af binding protein related" sequence." RIKEN Review. 6. 41-43 (1994)
• [Publications] Hayashizaki,Y.,et al.: "Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method." Nature Gen. 6. 33-40 (1994)
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• [Publications] Nagata,Y.,et al.: "A6-kb upstream region of the human trasthyretin gene can direct developmental,tissue-specific and quantitatively normal expression in transgenic mouse." J Biochem. 117. 169-175 (1994)
• [Publications] 勝木元也: "Molecular Medicine(臨時増刊)マニュアル疾患モデルマウス" 中山書店, 397 (1994)
• [Publications] Imai,E.,et al.: "Extracellular Matrix in the Kidney" Basel,Karger, 226 (1994)