Embryonic Stem Cell Gene Targeting and Chimera Production in Mice

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 themutation into livemice. 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 situationswhere the mutation has a phenotype in the form of a dominant effect on ES cells or in chimeras.