Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.
Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.
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DOI：
10.1371/journal.pone.0097974
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Li R
中科院分区：
文献类型：
--
作者：
Liu Q;Zhang M;Hou D;Han X;Jin Y;Zhao L;Nie X;Zhou X;Yun T;Zhao Y;Huang X;Hou D;Yang N;Wu Z;Li X;Li R
哺乳动物单倍体细胞系为遗传学研究和转基因动物生产提供了有用的工具。为了获得猪单倍体细胞，进行了三组实验。首先，用染色体扩散分析法检测了猪孤雌激活（PA）8 - 16细胞胚胎卵裂球的基因组。48.15%的卵裂球保持了完整的单倍体基因组。基于该结果，测试了用于扩增单倍体细胞群体的两种主要方法。首先，从PA囊胚期胚胎培养胚胎干细胞样（ES样）细胞，第二，从植入30天PA胎儿的胎儿成纤维细胞进行培养。共6个ES样细胞系来自PA囊胚。没有发现染色体扩散到19条染色体（正常的单倍体），有4个细胞系显示出向多倍化发展的趋势（超过38条染色体）。胎儿成纤维细胞的核型显示出不同程度的异常。染色体数目以19-38条为主（59.48-60.91%）。其次为二倍体细胞（16.17%~ 22.73%）。虽然在18.52%的胎仔来源细胞系中检测到具有19条染色体的细胞的百分比较低（3.45-8.33%），但这些细胞不是真正的单倍体细胞，因为它们表现出一些染色体的随机丢失或获得。通过流式细胞术分选不能有效地富集单倍体成纤维细胞。相反，二倍体细胞得到有效富集。富集后的孤雌二倍体细胞核型正常，父系印迹基因表达水平极低。我们的结论是，只有有限数量的真正的单倍体细胞可以从猪卵裂阶段孤雌生殖胚胎。与小鼠不同，猪PA胚胎来源的单倍体细胞的核型不稳定，长期的孤雌胚胎培养，无论是在体内还是体外，都会导致染色体核型异常。分选富集的猪PA胚胎二倍体成纤维细胞可作为研究父系印迹基因的候选细胞。
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.
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影响因子：
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作者：
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