Journal
BIOLOGY OF REPRODUCTION
Volume 101, Issue 3, Pages 538-548Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/biolre/ioz087
Keywords
haploid embryonic stem cells; androgenetic haESCs; 'artificial spermatids'; semi-cloned mouse (SC mouse); CRISPR-Cas9; gene editing
Categories
Funding
- Genome Tagging Project
- Chinese Academy of Sciences [XDB19010204]
- Shanghai Municipal Commission for Science and Technology [17411954900, 17JC1400900, 17JC1420102, 16JC420500, 19QA1407500, 18ZR1432300]
- National Natural Science Foundation of China [31530048, 81672117, 31730062, 31821004]
- China Postdoctoral Science Foundation [2018T110340, 2017M621349]
- Shanghai Industrial Technology Institute [17CXXF003]
- Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation
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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.
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