Abstract
Mammalian haploid cell lines 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 from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% 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 day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% 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. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.
Highlights
Most animal cells exist in the diploid condition, possessing two homologues of each chromosome
G-banding analysis confirmed that the haploid blastomeres did contain intact haploid genomes (Fig. 1C, D) [27,28]
This initial analysis suggested that authentic haploid cells could be obtained by culturing the parthenogenetically activated (PA) embryos containing haploid cells to generate haploid stem-like cells, or by transferring the haploid cell contained PA embryos into recipient gilts and collecting fetuses after 30 days to obtain haploid fetal fibroblasts
Summary
Most animal cells exist in the diploid condition, possessing two homologues of each chromosome. Haploid cell lines have advantages for forward and reverse genetic screens by avoiding allelic effects and exhibit an obvious phenotype in genetic modified individuals, especially when investigating polygenic traits [1]. Haploid cells may be useful to preserve genetic material of female mammals with a desired phenotype. Haploid cells are usually restricted to sperm and oocyte which are cells derived from primordial germ cells and that are unable to proliferate [4]. Mammalian haploid cells exhibiting genetic stability were successfully obtained from mouse [9,10,11], rat [12], and nonhuman primate [13] cells
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