PAX8-mCherry-Puro 人胚胎干细胞系的建立及验证

In Objective: To track and enrich target cells during the differentiation of human pluripotent stem cells(hPSCs) into thyroid cells, we utilized CRISPR/Cas9 technology to achieve site-specific insertion of the mCherry-Puro sequence immediately before the stop codon of the PAX8 gene in the human embryonic stem cell line H1, thereby enabling tracking and enrichment of cells expressing PAX8.Methods: Two sgRNAs were designed near the stop codon of the human PAX8 gene. These sgRNAs were inserted into a Cas9 expression vector and tested for cutting efficiency in 293T cells. The optimal sgRNA-Cas9 plasmid was selected, and a donor plasmid was constructed containing the left homology arm, mCherry, puro, and right homology arm. The constructed sgRNA-Cas9 expression plasmid and donor plasmid were introduced into the human embryonic stem cell line H1 via electroporation. Following puromycin selection, single colony genotyping, and immunofluorescence staining were performed to validate the accuracy of the cell line.Results: We successfully constructed a human embryonic stem cell line with precise insertion of the mCherry-puro sequence into the PAX8 genomic locus. Immunofluorescence staining demonstrated colocalization of PAX8 and mCherry, validating the accuracy of this cell line.