|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥1,200,000 (Direct Cost : ¥1,200,000)
To clarify the possible role of deltaEF1 in embryogenesis, we undertook the disruption of deltaEF1 gene by gene targeting using mouse embryonic stem (ES) cells. We expect the affects of those results on embryogenesis which should give us insight into the function of deltaEF1 in vivo.
First, we cloned the mouse deltaEF1 homologue using chicken cDNA by cross-hybridization to the mouse counterpart. The cloned mouse genomic and cDNAs were partially characterized. We found that the deltaEF1 is well conserved between the two species and that the mouse deltaEF1 has also two clustered zinc finger regions in N-terminal and C-terminal proximal region and the homeodomain-like sequence in its middle portion.
Second, we constructed a targeting vector so that the C-terminal zinc finger portion, which has the essential function to bind to the DNA, can be truncated, resulting in making the targeted gene nonfunctional. Using E14 mouse embryonic stem ES cells, we performed electroporation with the targeting vector. We have obtained one ES cell clone which has a disrupted allele of deltaEF1 gene on one of the two homologous chromosomes.
Finally, we produced mouse chimeras from blastocysts (derived from F1 of C57BL/6 and C3H mice) which were injected with the targeted ES cells. One of the chimeras could generate offspring which has the disrupted allele of deltaEF1 gene derived from the ES cells. These heterozygous animals look normal and fertile. In order to understand the in vivo-function of deltaEF1 protein, we are now going to get the homozygous animals by intercrossing the heterozygous mice and to see how the embryonic development can be affected.