简介：
Overview
This article presents a detailed protocol for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and Cas9 nickase mutant. The method successfully introduces two point mutations into the HNF4α locus in human embryonic stem cells.
Key Study Components
Area of Science
- Gene Editing
- Stem Cell Biology
- Genomics
Background
- Gene editing in human pluripotent stem cells is complex.
- Paired Cas9 nickase provides a reliable editing method.
- Seamless genomic editing is advantageous for precise modifications.
- Efficient genotyping screening is crucial for successful editing.
Purpose of Study
- To develop a method for precise gene editing in human pluripotent stem cells.
- To introduce specific mutations in the HNF4α gene.
- To enhance the efficiency of selecting edited cells.
Methods Used
- Maintenance of stem cells on recombinant laminin surfaces.
- Nucleofection of cells with Cas9 and targeting vector plasmids.
- Use of puromycin selection to isolate successfully edited cells.
- Genotyping and sequencing to confirm successful edits.
Main Results
- Two point mutations were successfully introduced into the HNF4α locus.
- Cells maintained pluripotent characteristics post-editing.
- Efficient selection and genotyping methods were established.
- Edited cells expressed key pluripotent markers.
Conclusions
- The protocol enables precise gene editing in human pluripotent stem cells.
- It provides a foundation for studying gene function and differentiation.
- Future applications include investigating specific gene roles in development.
What is the main advantage of this gene editing protocol?
The main advantage is the seamless genomic editing, which allows for precise modifications without leaving unwanted sequences.
How are the edited cells selected?
Edited cells are selected using puromycin, which helps isolate cells that have successfully integrated the desired mutations.
What markers are used to confirm pluripotency?
Markers such as Nanog, Oct4, SSEA-4, and TRA-160 are used to confirm that the edited cells maintain pluripotent characteristics.
What is the role of ROCK inhibitor in the protocol?
ROCK inhibitor is used to enhance cell survival and promote cell-to-cell contact during the early stages post-nucleofection.
How is the success of gene editing verified?
Success is verified through genotyping and Sanger sequencing of the edited regions to confirm the introduction of the desired mutations.
Can this method be applied to other genes?
Yes, this method can be adapted to target other genes of interest in human pluripotent stem cells.
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