| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1990: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
To establish a haploid cell line in mammal, the following experiments were carried out.
1. Production of haploid teratocarcinoma cells : Mouse unfertilized eggs were parthenogenetically activated and cultured for in vitro. Normally developed blastcysts were injected into testes of male mice. Production of teratomas and teratocarcinomas was observed. Although high frequency (70-80%) of production was seen by injection of normal fertilized eggs, none were appeared in the case of haploid embryos, probably because of limited developmental ability.
2. Isolation of haploid Embryonic Stem (ES) cells : Haploid parthenogenetic embryos were cultured on feeder cells in the medium containing fetal calf serum. Expansion of ES cells were very rare and differentiated cells were sometimes appeared. They were diploid cells in spite of their being derived from haploid embryos.
3. Analysis of chimeric mice between parthenogenetic haploid embryos and normal fertilized embryos : Two chimeric mice were analyze
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d to have parthenogenetic cells in brain, eyes and hair follicles at 10-30% and actually no contribution to other tissues. However almost all cells derived from the haploidembryos were diploid. When analyzing 8-10 day chimeric embryos, contribution of the parthenogeneic cells were high and in some cases, embryos were constituted only by parthenogenetic cells. It demonstrated that the parthenogenetic cells can be differentiated to all cell types in early stage of embryos. We also found that diploidization was occurred at early implantation stage.
4. Microinjection of oncogenes into haploid parthenogenetic embryos : Some oncogenes, such as c-myc, large T and E1A, were introduced into parthenogenetic haploid eggs by microinjection. The eggs were then cultured in vitro and expansion of ICM were checked. Actually no improvement on growth were observed by oncogenes themselves. However, in the case of DNA constructs that have oncogenes connected with enhancer-promoter region which functioned at pre- and postimplantation stage, improvement on haploid cell growth was observed. Now, we think it possible to establish the haploid cell lines by microinjection of the oncogenes which are constructed to function at early embryonic stages. Less
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