|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1993 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Positional cloning approach has been successful in isolation of mutated genes of various kinds, including genes responsible for tumorigenesis. However, despite such successes and recent progress in gene mapping and genomic cloning technologies, the identification and isolation of mutated genes still remains formidable task. In cases no gross genomic rearrangements e.g.translocation or deletion are found, it is very difficult to pinpoint a responsible gene within a large genomic segment. Yhus, there is an increasing demand for a method to functionally characterize the cloned genomic DNA.In this study, a method that allows rapid recovery of transcribed sequences from large cloned genomic DNA has been established. Also, in order to functionally characterize large genomic DNA,a technique to generate transgenic mice carrying YAC DNA is being developed.
The T/t complex of mouse is a large genetic region on proximal half of chromosome 17 that carry a number of loci affecting embryogenesis and
germ cell functions. We have been analyzing this region, attempting to ultimately isolate genes responsible for these mutations. Among these, we have special interests in mutations such ast^<omega5>, a recessive postimplantation lethal, showing defects in embryonic ectoderm formation ; qk (quaking), a neurological mutant. We have mapped t^<omega5> within mouse MHC region, H-2 complex. t^<omega5> was found to be very close to the H-2K gene with a possible genetic distance less than 0.1cM.We have cloned genomic region spanning -200kb around the K gene into cosmid contigs and have searched genes within the cloned region. Now, we extended the cloned area by isolating several YAC clones that cover -800kb from the K-gene toward CryA1 marker. Genetic analysis suggests that the mutation lies within the cloned DNA,while no structural alterations are found so far in this region. Twenty two novel genes have been found in this cloned genomic area by using our' screening in solution' method. Also, to find a responsible gene, a technique to introduce YAC DNA into germ line has been applied. We first established a protocol for isolation and purification of large DNA fragment without breakage, and thus isolated 650 kb YAC from t^<omega5> region was injected into mouse fertilized eggs. After transferring 320 injected eggs to pseudopregnant foster, 39 pups were born. Of which 3 mice carry at least a part of the YAC vector sequence, suggesting transgene integration. Genomic DNA from these putative transgenic mice are now being characterized in detail. Less