Study On Haploid Cells Derived From Pig Parthenogenetic Embryos

Mammalian haploid cell lines could provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from8-cell to16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by48.15%of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day30PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spreads with exactly19chromosomes (the normal haploid complement) were found. Four cell lines showed a tendency to develop to polyploidy (more than38chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with19-38chromosomes were the predominant cell type (59.48-60.91%).The diploid cells were the second cell type (16.17%-22.73%). Although a low percentage (3.4-8.3%) of cells with19chromosomes were detected in27%of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels.We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes or diploidization. The porcine PA embryo derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.