![]() The KO-first allele overcomes the limitations of constitutively-expressed mutations, flexibly producing reporter knockouts, conditional knockouts, and null alleles through exposure to site-specific cyclization recombination (Cre) and flippase (FLP) recombinases – such as in crosses to FLP and cre mice. Conditional alleles, including the KO-first allele, allow for analysis of gene function in a tissue-specific or temporal manner during development. The KO-first strategy has implications for functional genomics and proteomics across many model systems, including mouse, rat, and human pluripotent stem cells – serving as a functional genomics platform enabling systematic, genome-scale programs for proteomic mapping. Advantages of the Knockout-First (KO-First) Conditional Allele Strategy ES cell-based gene targeting has been used to establish the KO-first strategy for creating a multipurpose allele for both knockout and conditional applications by retaining the complete target gene. Gene targeting in embryonic stem (ES) cells remains one of the primary technologies for creating genetically modified animal models due to its accuracy and versatility. The IMPC has utilized CRISPR/Cas9 technology to generate knockout genes for several years, however, increasing concerns regarding off-target effects has cast doubts upon using CRISPR/Cas9 without thorough gene sequencing & analysis to validate the precision of the tool. Gene trapping has provided random insertional mutations in more than half of the protein-coding genes in mice, but this technique is limited by the inability to precisely engineer the gene-trap alleles, making it unsuitable for the generation of a complete set of gene knockouts. Some initial approaches to deciphering gene function include genome-wide mutagenesis strategies or gene trapping in mouse embryonic stem (ES) cells. The complete functional analysis of protein-coding genes remains an important goal, and technical challenge, for geneticists to-date. The complete sequencing of the human and murine genomes has not only provided insights for research, but has also raised further questions regarding the understanding of gene function. Functional Analysis of Protein-Coding Genes The strategies used by the IMPC have complementary properties to produce knockout alleles, but commonly “rely on the identification of a ‘critical’ exon common to all transcript variants that, when deleted, disrupts gene function.” 1 Herein, we discuss the advantages provided by the KO-first allele design for elucidation of functional genomics and proteomics, as well as its contribution to generating lacZ-tagged null mutations in every protein-coding gene as part of the IMPC program. Herein, we provide a brief introduction to the ‘ knockout-first’ (KO-first) conditional allele strategy that forms the basis for generation of lacZ-tagged conditional alleles as part of the International Knockout Mouse Consortium (IKMC) effort organized by the International Mouse Phenotyping Consortium (IMPC). Since the early 2000s, large-scale knockout consortia have been established with the goal of generating a complete resource of reporter-tagged null mutations in C57BL/6 mouse ES cells.
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