Technical Report

Transgenic Research

, Volume 16, Issue 5, pp 665-670

A simple qPCR-based method to detect correct insertion of homologous targeting vectors in murine ES cells

  • Ghada A. SolimanAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical School
  • , Ryoko Ishida-TakahashiAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical School
  • , Yusong GongAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical School
  • , Justin C. JonesAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical School
  • , Rebecca L. LeshanAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical School
  • , Thomas L. SaundersAffiliated withDivision of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School
  • , Diane C. FingarAffiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical SchoolDepartment of Cell and Developmental Biology, University of Michigan Medical School
  • , Martin G. MyersJr.Affiliated withDivision of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan Medical SchoolDepartment of Molecular and Integrative Physiology, University of Michigan Medical School Email author 

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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.

Keywords

qPCR ES cell Gene targeting