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A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells
Published on: March 3, 2016
Identifying genes preferentially expressed in undifferentiated embryonic stem cells
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. xli@genetics.med.harvard.edu
BMC Cell Biology
|August 30, 2007
Summary
We developed a novel retroviral gene trap vector to identify genes crucial for maintaining undifferentiated embryonic stem (ES) cells. This tool helps uncover key genes involved in stem cell biology.
Area of Science:
- Stem cell biology
- Molecular biology
- Genetics
Background:
- The mechanisms governing embryonic stem (ES) cell maintenance and differentiation are not fully understood.
- Identifying genes critical for maintaining the undifferentiated state of ES cells is essential for developmental biology research.
Purpose of the Study:
- To develop and validate a novel retroviral gene trap vector for identifying genes preferentially expressed in undifferentiated ES cells.
- To investigate the utility of this vector in uncovering novel genes involved in ES cell regulation.
Main Methods:
- Development of a retroviral gene trap vector containing GFP and Neo reporter genes.
- Selection of ES cell clones with vector integration into transcriptionally active loci using G-418 drug resistance.
- Utilizing GFP Fluorescence-Activated Cell Sorting (FACS) profiling to identify clones with reduced GFP fluorescence upon differentiation.
- Confirmation of gene down-regulation during differentiation using Northern blot analysis.
Main Results:
- Successfully screened 300 ES clones and identified six clones with down-regulated GFP expression during differentiation, representing four distinct genes.
- Identified integration sites within Zfp-57 (known to be enriched in undifferentiated ES cells), a novel CSL/RBP-J kappa isoform (key LIN-12/Notch pathway transcription factor), a potential noncoding RNA gene, and a potential novel gene locus.
- Validated the down-regulation of reporter gene expression in selected clones via Northern blot analysis.
Conclusions:
- Demonstrated the effectiveness of a novel retroviral gene trap vector for identifying genes preferentially expressed in undifferentiated ES cells.
- The identified genes, including Zfp-57 and a novel CSL/RBP-J kappa isoform, provide new insights into ES cell maintenance and differentiation pathways.
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