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| Status |
Public on Sep 02, 2017 |
| Title |
An efficient method for generation of bi-allelic null mutant mouse embryonic stem cell lines and its application for investigating epigenetic modifiers |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
H3K9me3 ChIP-Seq profiling of Setdb1 depleted mouse ES cells
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| Contributor(s) |
Marks H, Fisher CL, Cho LT, Andrews R, Wormald S, Carroll T, Iyer V, Tate P, Rosen B, Stunnenberg HG, Fisher AG, Skarnes WC |
| Citation(s) |
28981838 |
| Submission date |
Aug 31, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
h.marks@ncmls.ru.nl
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| Organization name |
Radboud University Nijmegen, RIMLS
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platforms (1) |
| GPL11002 |
Illumina Genome Analyzer IIx (Mus musculus) |
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| Samples (8)
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| Relations |
| SRA |
SRP008007 |
| BioProject |
PRJNA145283 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE31777_RAW.tar |
613.4 Mb |
(http)(custom) |
TAR (of BED, WIG) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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