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.