Large-scale, saturating insertional mutagenesis of the mouse genome

Abstract

We describe the construction of a large-scale, orderly assembly of mutant ES cells, generated with retroviral insertions and having mutational coverage in >90% of mouse genes. We also describe a method for isolating ES cell clones with mutations in specific genes of interest from this library. This approach, which combines saturating random mutagenesis with targeted selection of mutations in the genes of interest, was successfully applied to the gene families of G protein-coupled receptors (GPCRs) and nuclear receptors. Mutant mouse strains in 60 different GPCRs were generated. Applicability of the technique for the GPCR genes, which on average represent fairly small targets for insertional mutagenesis, indicates the general utility of our approach for the rest of the genome. The method also allows for increased scale and automation for the large-scale production of mutant mice, which could substantially expedite the functional characterization of the mouse genome.G protein-coupled receptor ͉ retroviral vector ͉ ES cell ͉ knockout mice

Large-scale gene trapping in C57BL/6N mouse embryonic stem cells

“…Endogenous gene expression was eliminated or drastically reduced in all lines. Unlike the results reported by Gragerov et al (2007), we did not observe incomplete gene inactivation for insertions located within the 5Ј-untranslated region of trapped genes (six of 18 lines). This is probably because all available transcript data present in public databases are reviewed to select insertions located downstream from alternate promoters/TSSs.…”

“…To date, all such mixed clones have been successfully separated and confirmed by both IPCR and QPCR. No individual clones bearing more than one insertion per genome have been identified in OmniBankII or the original 129S5/SvEvBrd-derived OmniBank library, although multiple retroviral insertions per ES cell clone have been reported for other library resources (Gragerov et al 2007).…”

“…In an attempt to overcome these limitations, a recent study produced 10 million clones, and potentially 22.7 million retroviral insertion mutations in an unsequenced but screenable format (Gragerov et al 2007). Based on analysis of capture rate of 403 GPCR and nuclear receptor genes, the mutation collection was estimated to contain 90% coverage of mouse genes, with a mutation density per gene equivalent to that achieved in the present study.…”

An Inducible and Reversible Mouse Genetic Rescue System

“…Three mouse strains were established from ES clones T1, T2 and T3, and crossed to particular case of ApoE, the virus is inserted in the third intron. The vector contains splice acceptor, stop codons, polyA signal and transcriptional terminator to ensure gene inactivation, which we confirmed to be the case by showing that ,90% of isolated genespecific ES clones were null alleles [18]. (The remaining were mostly knock-downs, and in nearly all these cases the retrovirus was inserted into 59 UTR (exon or intron), suggesting that retroviral insertions upstream of the coding regions of genes should be avoided.)…”

“…The 10-million clone ES cell library we utilized [18] has been estimated to contain insertional mutations for .90% of genes, and individual ES clones with retroviral insertions in a specific target gene can be rapidly identified through a PCR pooling strategy and subsequently isolated in a streamlined process. The identification and modification of the TIGRE locus allows rapid insertion of any gene of interest via co-transfection with Cre.…”