کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2093549 | 1081965 | 2015 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells](/preview/png/2093549.png)
• A selection-free knockin strategy was established for making reporter hESC lines
• OCT4-mOrange, OCT4-eGFP, and PDX1-eGFP hESC reporter lines were created
• Faithful reporter activities were established
SummaryThe development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.
Journal: - Volume 4, Issue 6, 9 June 2015, Pages 1103–1111