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Efficient SNP editing in haploid human pluripotent stem cells

机译:单倍体人多能干细胞中高效的SNP编辑

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摘要

Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP.
机译:对从单性衍生的胚胎干细胞系(PES12)提取的DNA进行外序测序。选择影响胆汁酸转移的SLC10A2基因突变作为对该概念研究证明,以对人类多能单倍体细胞进行尝试校正的概念研究证明的突变。验证了突变的确认,并设计了在制备进行CRISPR方面设计了RNA和校正模板。单倍体细胞接受串联荧光活性细胞分选(FACS),Hoechst 33342产生越来越多的富含富集的培养物。在p上进行核。 37然后将细胞与+ GFP分选1N DNA含量,以鉴定表达用GFP标记的Cas9的单倍体细胞。

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