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Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Published on: May 28, 2015
Generation of a LEUTX-2A-mCherry knock-in H1 human embryonic stem cell line using CRISPR/Cas9 system
Xiu Yu1, Zezhong Su2
1School of Medicine, Jiaying University, Meizhou 514015, China; Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou 510080, China.
Abstract:
LEUTX is exclusively expressed at the 8-cell stage and serves as a key regulator of human embryonic genome activation. Induced 8-cell-like cells (8CLCs) derived from pluripotent stem cells offer a tractable model to dissect the molecular transition from pluripotency to totipotency. However, current 8CLC induction protocols are hampered by low efficiency and cellular heterogeneity. To overcome these limitations, we developed a fluorescent reporter system that dynamically monitors endogenous LEUTX expression, facilitating the purification and functional characterization of bona fide 8CLCs. This tool enables systematic interrogation of the regulatory networks underlying totipotency acquisition.
