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Mouse embryonic stem cells (ESCs) are powerful tools for functional analysis of stem cell-related genes; however, complex gene manipulations, such as locus-targeted introduction of multiple genes and conditional gene knockout conditional knockout, are technically difficult. Here, we review recent advances in technologies aimed at generating cKO clones in ESCs, including two new methods developed in our laboratory: the simultaneous or sequential integration of multiple genes system for introducing an unlimited number of gene cassettes into a specific chromosomal locus using reciprocal recombinases; and the all-in-one cKO system, which enables introduction of an EGFP reporter expression cassette and FLAG-tagged gene of interest under an endogenous promoter. In addition, methods developed in other laboratories, including conventional approaches to establishment of cKO cell clones, inducible Cas9-mediated cKO generation, and cKO assisted by reporter construct, invertible gene-trap cassette, and conditional protein degradation. Finally, we discuss the advantages of each approach, as well as the remaining issues and challenges.

Multiple Gene Transfer and All-In-One Conditional Knockout Systems in Mouse Embryonic Stem Cells for Analysis of Gene Function