Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai, China.
Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China.
J Intern Med. 2016 Sep;280(3):236-45. doi: 10.1111/joim.12503. Epub 2016 May 3.
Haploid cells contain one set of chromosomes and are amenable for genetic analyses. In mammals, haploidy exists only in gametes. An intriguing question is whether haploid cells can be derived from gametes. Recently, by application of haploid cell enrichment using fluorescence-activated cell sorting, stable haploid embryonic stem cells (haESCs) have been successfully derived from oocyte-derived parthenogenetic and sperm-derived androgenetic embryos from several species. Whilst both parthenogenetic and androgenetic (AG)-haESCs enable whole-genome genetic screening at the cellular level, such as screening of drug resistance or disease-related genes, AG-haESCs, after intracytoplasmic injection into oocytes, can also be used to produce alive semi-cloned mice. Nevertheless, one major drawback associated with wild-type AG-haESCs is the very low birth rate of healthy semi-cloned mice. Of interest, after inhibiting the expression of two paternally imprinted genes (H19 and Gtl2) in AG-haESCs by removal of their differentially DNA methylated regions, double-knockout AG-haESCs can efficiently and stably support the generation of healthy semi-cloned pups. Importantly, double-knockout AG-haESCs are feasible for multiple genetic manipulations, followed by efficient generation of semi-cloned mice carrying multiple genetic traits; thus they could be used to validate candidate loci that have been identified in genome-wide association studies of multigenic diseases by generation of mouse models carrying multiple alterations. Of note, by combining a CRISPR-Cas9 library and double-knockout AG-haESCs, semi-cloned mice carrying different mutant genes can be efficiently generated in one step, enabling functional mutagenic screening in mice. HaESCs, therefore, provide a powerful tool for genetic analyses in mammals at both the cellular and organismal levels.
单倍体细胞只含有一组染色体，适合进行遗传分析。在哺乳动物中，单倍体仅存在于配子中。一个有趣的问题是，配子是否可以衍生出单倍体细胞。最近，通过应用荧光激活细胞分选对单倍体细胞进行富集，已经成功地从几种物种的卵母细胞来源的孤雌生殖和精子来源的雄核胚胎中衍生出稳定的单倍体胚胎干细胞（haESCs）。虽然孤雌生殖和雄核（AG）-haESCs 能够在细胞水平上进行全基因组遗传筛选，例如筛选药物抗性或疾病相关基因，但 AG-haESCs 在细胞质内注射到卵母细胞中后，也可用于产生活的半克隆小鼠。然而，与野生型 AG-haESCs 相关的一个主要缺点是健康半克隆小鼠的出生率非常低。有趣的是，通过去除 AG-haESCs 中两个父源印记基因（H19 和 Gtl2）的差异 DNA 甲基化区域来抑制其表达后，双敲除 AG-haESCs 可以有效地稳定支持健康半克隆幼仔的产生。重要的是，双敲除 AG-haESCs 可进行多次基因操作，随后高效生成携带多个遗传特征的半克隆小鼠；因此，它们可用于通过生成携带多个改变的小鼠模型来验证在多基因疾病的全基因组关联研究中已确定的候选基因座。值得注意的是，通过结合 CRISPR-Cas9 文库和双敲除 AG-haESCs，可以在一步中高效生成携带不同突变基因的半克隆小鼠，从而能够在小鼠中进行功能诱变筛选。因此，haESCs 为哺乳动物在细胞和机体水平上的遗传分析提供了一种强大的工具。
