title: Efficient Generation of Gene-Modified Mice by Haploid Embryonic Stem Cell-Mediated Semi-cloned Technology.
abstract: Haploid embryonic stem cells can be derived from androgenetic embryos produced by injection of sperm into enucleated oocytes or by removal of the female pronucleus from zygotes. These cells, termed AG-haESCs, can be used in place of sperm to produce the so-called semi-cloned (SC) mice. Importantly, AG-haESCs carrying H19-DMR and IG-DMR knockouts (DKO-AG-haESCs) can efficiently and stably support the generation of SC mice via intracytoplasmic AG-haESCs injection (ICAHCI), which provides a new route to obtain genetically modified mice. In this chapter, we describe the procedures for AG-haESCs culturing, enrichment of haploid cells by FACS, genomic manipulation in DKO-AG-haESCs by CRISPR/Cas9 and generation of live SC mice with gene-modified DKO-AG-haESCs.
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.|Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
authors.forename: Cuiqing
authors.initials: C
authors.lastname: Zhong
authors.affiliation: Group of Epigenetic Reprogramming, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. jsli@sibcb.ac.cn.|Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China. jsli@sibcb.ac.cn.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D053595:Embryonic Stem Cells / Q000502:physiology*; D005260:Female; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics; D006238:Haploidy; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D015053:Zygote
keywords: CRISPR-Cas9; Fluorescence-activated cell sorting (FACS); Genetic manipulation; Haploid ES cells (haESCs); Intracytoplasmic AG-haESCs injection (ICAHCI); Semi-cloned mice (SC mice)