|Budget Amount *help
¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥2,900,000 (Direct Cost : ¥2,900,000)
After the application of this grant, Dr. Kimura (Oosaka-Univ) published the sequence of mouse OTR gene. Based on that sequence, we got OTR cDNA from brain derived cDNA of 1295 vEv mouse. With this cDNA and pGKhprt/MCA1tk vector, we generated OTR-targeting vector. Just after the completion of this vector, we got an important information from my friend in USA, saying that knock-out project for OTR gene, carried out at a Lab. in NIH, USA, gave them unexpected result : the early embryonic death of OTR deficient homozygous mice in utero. With this information, we changed four initial strategy of "conventional knockout of OTR gene" to "conditionalknockout". The conditional knockout are dependent of combination of specificelement loxP and the gene coding bacterial recombinase cre, derived from P1 phage. After establishing of ES cell line, whose targeted gene is homologousely substituted by "fox gene", the ES cells are used to generate the chimeric, and successively the homozygous mutant mice.
Crossing of floxP mice with the other trasgenic mice, which is harboring exogenous cre gene under control of Tc-inducible promoter, or tissue specific enhancer/promoter, can generate new mutant mice, whose targeted gene is lost and inactivated in specific tissue, or specific stage in embryogenesis. Because of the alteration of experimental situation about OTR gene knockout, I and Dr. Kimura decided to begin this project as a collaboration from April, 1998. We gotlambda phage harboring mouse OTR gene from Dr. KimuRa, and made detailed restriction maps of OTR gene, and generated new OTR knockout vector. This vector comprises neomycin resistant gene, 3 loxP elements, 5'arm and 3'arm of OTR gene, the fragment coding for exon 2 and 3, HSV-tk gene, and vector encoding ori andampicillin resistant gene. The total length of this new vector is 18.5 kb. Now we are introducing this vector to E14TG2a ES cell and screening the cell, whose OTR gene was correctly targeted. In near future, we will use new inducible cre system, in which mutated steroid receptor-cre fusion protein will be activated by the administration of steroid derivative. This mutant mouse system is being developed by Dr. Brown, C.in Baylor College of Medicine, Houston, Texas, USA.For uterus specific OTR geneknock out, we will obtain a mutant mouse which is harboring cre-gene under control of smooth muscle specific actin promoter. This mutant mouse was generated by Dr. Miwa, Osaka university. Less