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Open Access Highly Accessed Methodology article

Identifying genes preferentially expressed in undifferentiated embryonic stem cells

Xiajun Li and Philip Leder

Author affiliations

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

Citation and License

BMC Cell Biology 2007, 8:37  doi:10.1186/1471-2121-8-37

Published: 28 August 2007

Abstract

Background

The mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood.

Results

To address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-Jkappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene.

Conclusion

Taken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells.