A conditional knockout resource for the genome-wide study of mouse gene function

Abstract

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

“…Targeting was confirmed by long-range PCR (Skarnes et al 2011) and zygosity for the targeted allele was confirmed by a qPCR assay specific for the lacZ sequence. Mutants and WT littermates were housed in an environmentally controlled animal facility on a 12:12-h light:dark cycle with lights on at 07:00 h. Mice were fed Harlan Teklad global rodent diet #2918 with a composition of 18% protein and 6% fat.…”

“…See Skarnes et al (2011) for a description of the targeting strategy and cell lines produced, and visit the International Knockout Mouse Consortium (IKMC; www.mousephenotype.org) or the KOMP Repository (www.komp.org) and the project webpage (www.kompphenotype.org) for a description of the specific alleles for each of the mutants with LacZ staining in this survey. For all alleles, the lacZ reporter was designed to be under the control of the native promoter of the targeted gene.…”

“…The targeting vectors contain a bacterial beta-galactosidase reporter (lacZ), which, when inserted into the target gene, is expressed under the control of the target gene promoter (Skarnes et al 2011). We completed LacZ staining analysis in 66 tissues using whole mount (WM) and frozen section (FS) histochemical staining in male and female adult mice for each of the mutant lines.…”

Protein Interactome and Its Application to Protein Function Prediction

“…Such identification has focused on a specific target gene or protein, or a small set of protein complexes. Gene knockout, knockdown of gene expression, and targeted mutations are some methods for protein function identification (Recillas-Targa, 2006;Skarnes et al, 2011). Such low-throughput experiments were replaced by high-throughput experiments including genome sequencing and determination of the protein interactome.…”

Establishing Three Dimensional High Throughput Imaging Pipeline for Deep Phenotyping Mouse Embryonic Development

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The mission of the International Mouse Phenotyping Consortium (IMPC) is to systematically phenotype mice resulting from null alleles in the approximately 20,000 known or predicted protein encoded genes [1] to determine the function of each gene and the relationship to human disease. With a prediction of more than 30% of the mouse lines will either be lethal (no homozygous pups were recovered at postnatal day 14) or sub-viable (less then 12.5% of homozygous pups at postnatal day 14), an embryonic lethal pipeline including an assessment of the approximate stage of lethality, followed by 3D imaging to further analyze and archive the phenotype has been established [2].

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