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Generation of a DKK1 homozygous knockout human embryonic stem cell line WAe001-A-21 using CRISPR/Cas9
Zhen Yang1, Fang Yuan2, Ning Wang2
1Institute of Public Health, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; University of Chinese Academy of Sciences, Beijing, China; Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China; Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
Abstract:
Dickkopf1 (DKK1) is a secreted inhibitor for the Wnt signalling, which is involved in cell proliferation, tissue regeneration and embryonic development. Using CRISPR/Cas9 editing, we established a homozygous mutant DKK1 human embryonic stem cell line (WAe001-A-21). It has a 41 bp deletion in exon 2 of DKK1, leading to its coding frame shift. The WAe001-A-21 cell line maintains a normal karyotype, pluripotency markers, typical stem cell morphology and the ability to differentiate into three germ layers.
Insights
Researchers created a Dickkopf1 (DKK1) mutant human embryonic stem cell line using CRISPR/Cas9. This DKK1-null cell line retains pluripotency and differentiation capabilities, valuable for Wnt signaling research.
Area of Science:
- Stem Cell Biology
- Molecular Genetics
- Developmental Biology
Background:
- Dickkopf1 (DKK1) is a crucial secreted inhibitor of the Wnt signaling pathway.
- Wnt signaling regulates fundamental biological processes including cell proliferation, tissue regeneration, and embryonic development.
Purpose of the Study:
- To generate a homozygous mutant Dickkopf1 (DKK1) human embryonic stem cell line using CRISPR/Cas9 gene editing.
- To characterize the created DKK1-null cell line for essential stem cell properties.
Main Methods:
- CRISPR/Cas9 gene editing was employed to introduce a homozygous deletion in the DKK1 gene.
- The resulting cell line (WAe001-A-21) was analyzed for karyotype, pluripotency markers, morphology, and differentiation potential.
Main Results:
- A homozygous mutant DKK1 human embryonic stem cell line (WAe001-A-21) was successfully established.
- The mutation involved a 41 bp deletion in exon 2 of DKK1, causing a coding frame shift.
- The WAe001-A-21 cell line exhibited a normal karyotype, maintained pluripotency markers, displayed typical stem cell morphology, and retained the ability to differentiate into three germ layers.
Conclusions:
- The established DKK1-null human embryonic stem cell line is a valuable tool for studying the role of DKK1 in Wnt signaling.
- This cell line provides a platform for investigating Wnt pathway functions in human development and disease models.
- The retention of pluripotency and differentiation capacity highlights the utility of this engineered stem cell line.
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