Embryonic Stem Cell Gene Targeting and Chimera Production in Mice
- 1Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032, USA
- 2Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
- ↵3Correspondence: vep1{at}columbia.edu
Abstract
Producing a custom gene mutation in embryonic stem (ES) cells, whether through homologous recombination or CRISPR–Cas gene editing, is the first step along the way to getting the mutation into live mice. However, there are a number of additional steps along the way, each presenting technical challenges. Here, we provide a guide for troubleshooting when the results are not as expected and to distinguish technical problems from possible biological effects of the mutation. From the isolation of clonal lines of targeted ES cells through the production of ES cell chimeras with the targeted ES cell clone, we discuss common technical problems and their most likely causes and solutions. We also provide guidance for situations where the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.
Footnotes
-
From the Mouse Phenotypes collection by Virginia E. Papaioannou and Richard R. Behringer.
