Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.
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.
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Qiang
Q
Liu
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Manling
M
Zhang
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Dongxia
D
Hou
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Xuejie
X
Han
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Yong
Y
Jin
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Lihua
L
Zhao
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Xiaowei
X
Nie
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Xin
X
Zhou
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Ting
T
Yun
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Yuhang
Y
Zhao
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Xianghua
X
Huang
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Daorong
D
Hou
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Ning
N
Yang
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Zhaoqiang
Z
Wu
The Key Laboratory of the National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, Inner Mongolia, China.
Xueling
X
Li
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, Jiangsu, China; Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, China.
Rongfeng
R
Li
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D001757:Blastomeres; D002460:Cell Line; D002478:Cells, Cultured; D002871:Chromosome Banding; D016000:Cluster Analysis; D004624:Embryo Transfer; D053595:Embryonic Stem Cells; D005333:Fetus; D005347:Fibroblasts; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006238:Haploidy; D066298:In Vitro Techniques; D059785:Karyotype; D007621:Karyotyping; D009865:Oocytes / Q000378:metabolism; D010312:Parthenogenesis / Q000235:genetics*; D013552:Swine