International Knockout Mouse Consortium - Knowledgebase

As the mutant mouse lines generated by WTSI MGP pass through the WTSI MGP primary phenotypic characterisation studies the information generated may be viewed through the IMPC Portal where the gene target in question may be searched for. A Heat Map...

The long range PCR (LR-PCR) genotyping primers may be obtained from the through the International Knockout Mouse Consortium (IKMC) website (www.knockoutmouse.org), searching for the gene target in question and following the links for details on th...

The mouse model the MGP sends will be supplied with an information sheet relating to the genetic background. This details contributing elements such as the Embryonic Stem Cell origin, Test Cross for germ line transmission and subsequent backcrossi...

SR-PCR genotyping uses a combination of separate PCR reactions that detect the presence/absence of the cassette lacZ (confirms the presence of the cassette), the gene-specific wild type allele and the gene-specific mutant allele by short range PCR.

We use a qPCR reaction to count the neo gene marker present in the targeting cassette; heterozygotes will have one copy of the Neo gene, a homozygote will have two copies and a wild type will have no copies.

One of the methods used in the WTSI MGP QC to confirm targeting of the intended gene by the mutation cassette is LR-PCR. WTSI MGP uses 3’ LR-PCR to confirm targeting. The primers used are a the 3’ arm FRTL Universal primer (5’ primer) and either t...

The following document outlines the genotyping strategies for detecting tm1a conversion to the b, c and d forms After treatment with a source of Cre recombinase to excise the critical exon and neo cassette (in promoter-driven lines), the Tm1a all...

Currently this depends on whether the mice were derived from EUCOMM or KOMP ES cell resources and where the mice are available from: For Mice sourced through EMMA: No.

The LacZ short range PCR detects the presence of the LacZ reporter in the cassette in the tm1a form of the EUCOMM / KOMP allele. This can be used as a rapid screen on F1 chimera progeny samples to detect likely germ line transmission. The WTSI gen...

An MTA is required before mice can be shipped. The MTA required depends on whether the mice were derived from EUCOMM or KOMP ES cell resources. Currently: For Mice sourced through EMMA:

In designing a strategy for the introduction of FlpeR and Cre lines consideration to the mutation type should be contemplated. Allele maps may be viewed at the HTGT website and searching for the gene in question and viewing the details .The clone ...

As the WTSI MGP generates the mutant mouse lines for phenotypic analysis we are keen to offer available mice whilst on the shelf to the scientific community who have expressed an interest. However WTSI MGP is not a distribution centre that breeds ...

Taking some Acot6 mutant mice as an example:

The clones labelled as “Knockout first” are expected to contain all the loxP sites as shown in the allele map. The knockout is obtained by introduction of a splice acceptor/reporter- cassette with a strong polyA site into an endogenous intron upst...

This information if available may be found through the IMPC Portal where the gene target in question may be searched for. This information may also be found on the Heat Map available to view.

If the cassette has been successfully flipped out the internal LacZ PCR would be expected to fail, but the mutant-specific PCR would be expected to still amplify a band in mutant mice, as it is 5’ to the first FRT site. In homozygous mutant mice t...

After treatment with a source of Flp recombinase to excise the selection cassette, the Tm1a allele is converted to the Tm1c form. Detection of this event and subsequent genotyping of mouse lines requires a modified strategy to that of Tm1a genot...

After treatment with a source of Cre recombinase to excise the critical exon and neo cassette (in promoter-driven lines), the Tm1a allele is converted to the Tm1b form. Detection of this event and subsequent genotyping of mouse lines requires a ...

WTSI MGP generates mutant mouse lines from the KOMP and EUCOMM ES cell resources. The structure of the targeted mutation in the ES cells used to generate these mutant mice is not verified by the WTSI MGP. It is recommended therefore that the recip...

The mutant mouse lines generated by WTSI MGP are used for primary phenotypic characterisation studies. As these lines are generated, WTSI is eager to provide mice directly to the scientific community should mice be available. However, WTSI does no...

Availability of ES cells may be investigated and sourced through the International Mouse Phenotyping Consortium (IMPC www.mousephenotype.org) website by searching for your target of interest and following the links to the appropriate resource.

Details of the targeted allele may be viewed through the International Mouse Phenotyping Consortium (IMPC) website (https://www.mousephenotype.org), searching for the gene target in question and following the links for details on the appropriate p...

A. WTSI MGP does not supply ovocytes, sperm or embryos. As the WTSI MGP generates the mutant mouse lines for phenotypic analysis WTSI MGP is keen to offer available mice whilst on the shelf to the scientific community who have expressed an intere...

For lines registered through EMMA. Current costing may be found at the EMMA website.

Currently:

Confirmation of the targeted mutation is determined by either long-range PCR (LR-PCR; Please see ‘What is LR-PCR?’) or by the detection of homozygous mice by gene specific short-range PCR (SR-PCR; Please see: ‘What is SR-PCR?’). If we observe homo...

Summary of all IKMC allele types The wild-type is represented as a transcript with three exons. The ‘floxed’ exon (the exon that will be deleted for the null allele) is exon number 2.

The WTSI MGP generates mutant mouse lines using ES cell resources from the European Conditional Mouse Mutagenesis programme (EUCOMM www.eucomm.org ) and Knockout Mouse Project (KOMP www.komp.org ). ES cells may be obtained by the scientific commun...