State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), School of Life Sciences, Fudan University, Shanghai, China.
Biol Reprod. 2019 Sep 1;101(3):538-548. doi: 10.1093/biolre/ioz087.
For years, extensive efforts have been made to use mammalian sperm as the mediator to generate genetically modified animals; however, the strategy of sperm-mediated gene transfer (SMGT) is unable to produce stable and diversified modifications in descendants. Recently, haploid embryonic stem cells (haESCs) have been successfully derived from haploid embryos carrying the genome of highly specialized gametes, and can stably maintain haploidy (through periodic cell sorting based on DNA quantity) and both self-renewal and pluripotency in long-term cell culture. In particular, haESCs derived from androgenetic haploid blastocysts (AG-haESCs), carrying only the sperm genome, can support the generation of live mice (semi-cloned, SC mice) through oocyte injection. Remarkably, after removal of the imprinted control regions H19-DMR (differentially methylated region of DNA) and IG-DMR in AG-haESCs, the double knockout (DKO)-AG-haESCs can stably produce SC animals with high efficiency, and so can serve as a sperm equivalent. Importantly, DKO-AG-haESCs can be used for multiple rounds of gene modifications in vitro, followed by efficient generation of live and fertile mice with the expected genetic traits. Thus, DKO-AG-haESCs (referred to as 'artificial spermatids') combed with CRISPR-Cas technology can be used as the genetically tractable fertilization agent, to efficiently create genetically modified offspring, and is a versatile genetic tool for in vivo analyses of gene function.
多年来，人们一直致力于利用哺乳动物精子作为介导物来产生基因修饰动物；然而，精子介导基因转移（SMGT）策略无法在后代中产生稳定和多样化的修饰。最近，从携带高度特化配子基因组的单倍体胚胎中成功衍生出了单倍体胚胎干细胞（haESCs），并且可以通过基于 DNA 数量的定期细胞分选来稳定地维持单倍体以及自我更新和多能性，在长期细胞培养中。特别是，从雄核单倍体囊胚（AG-haESCs）衍生的仅携带精子基因组的 haESCs，可以通过卵母细胞注射支持活鼠（半克隆，SC 鼠）的产生。值得注意的是，在去除 AG-haESCs 中的印迹控制区 H19-DMR（DNA 差异甲基化区域）和 IG-DMR 后，双敲除（DKO）-AG-haESCs 可以以高效率稳定地产生 SC 动物，因此可以作为精子的等效物。重要的是，DKO-AG-haESCs 可以在体外进行多次基因修饰，然后有效地产生具有预期遗传特征的活产和可育的小鼠。因此，与 CRISPR-Cas 技术相结合的 DKO-AG-haESCs（称为“人工精子细胞”）可用作可遗传修饰的受精剂，以有效地产生基因修饰的后代，并且是用于体内基因功能分析的多功能遗传工具。
