Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.
Pubmed ID: 28981838 RIS Download
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
Consortium represents all publicly available gene trap cell lines, which are available on non-collaborative basis for nominal handling fees. Researchers can search and browse IGTC database for cell lines of interest using accession numbers or IDs, keywords, sequence data, tissue expression profiles and biological pathways, can find trapped genes of interest on IGTC website, and order cell lines for generation of mutant mice through blastocyst injection. Consortium members include: BayGenomics (USA), Centre for Modelling Human Disease (Toronto, Canada), Embryonic Stem Cell Database (University of Manitoba, Canada), Exchangeable Gene Trap Clones (Kumamoto University, Japan), German Gene Trap Consortium provider (Germany), Sanger Institute Gene Trap Resource (Cambridge, UK), Soriano Lab Gene Trap Resource (Mount Sinai School of Medicine, New York, USA), Texas Institute for Genomic Medicine - TIGM (USA), TIGEM-IRBM Gene Trap (Naples, Italy).
View all literature mentionsInternational functional genomics data collection generated from microarray or next-generation sequencing (NGS) platforms. Repository of functional genomics data supporting publications. Provides genes expression data for reuse to the research community where they can be queried and downloaded. Integrated with the Gene Expression Atlas and the sequence databases at the European Bioinformatics Institute. Contains a subset of curated and re-annotated Archive data which can be queried for individual gene expression under different biological conditions across experiments. Data collected to MIAME and MINSEQE standards. Data are submitted by users or are imported directly from the NCBI Gene Expression Omnibus.
View all literature mentionsCenter that produces knockout mice and carries out high-throughput phenotyping of each line in order to determine function of every gene in mouse genome. These mice will be preserved in repositories and made available to scientific community representing valuable resource for basic scientific research as well as generating new models for human diseases.
View all literature mentionsMus musculus with name B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J from IMSR.
View all literature mentions