Abstract
The identification of correctly targeted embryonic stem (ES) cell clones from among the large number of random integrants that result from most selection paradigms remains an important hurdle in the generation of animals bearing homologously targeted transgenes. Given the limitations inherent to Southern blotting and standard PCR, we utilized quantitative real-time polymerase chain reaction (qPCR) to rapidly identify murine ES cell clones containing insertions at the correct genomic locus. Importantly, this approach is useful for screening ES clones from conditional/insertional “knock-in” strategies in which there is no loss of genetic material. Simple validation avoids the generation of assays prone to false negative results. In this method, probe and primer sets that span an insertion site detect and quantify the unperturbed gene relative to an irrelevant reference gene, allowing ES cell clones to be screened for loss of detection of one copy of the gene (functional loss of homozygousity (LOH)) that occurs when the normal DNA is disrupted by the insertion event. Simply stated, detected gene copy number falls from two to one in correctly targeted clones. We have utilized such easily designed and validated qPCR LOH assays to rapidly and accurately identify insertions in multiple target sites (including the Lepr and mTOR loci) in murine ES cells, in order to generate transgenic animals.
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Acknowledgements
Thanks to Bin Zhang and David Ginsburg for sharing unpublished results. The University of Michigan transgenic core is supported by the Michigan Diabetes Research and Training Center (NIH DK20572), the Michigan Comprehensive Cancer Center (NIH P30CA046592), and Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000815). Supported by NIH K01 DK60654 and AHA 0750060Z (to GS); by ADA 1-06-JF-16 and ACS RSG-07-091-01-TBE (to DCF); and by NIH-R01 DK56731, NIH-R01 DK57768, and grants from the American Diabetes Association (to MGM).
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Soliman, G.A., Ishida-Takahashi, R., Gong, Y. et al. A simple qPCR-based method to detect correct insertion of homologous targeting vectors in murine ES cells. Transgenic Res 16, 665–670 (2007). https://doi.org/10.1007/s11248-007-9110-2
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DOI: https://doi.org/10.1007/s11248-007-9110-2