International Knockout Mouse Consortium - Knowledgebase

The Neo count qPCR assay uses a hydrolysis probe assay (eg Applied Biosystems TaqMan technology) to determine the copy number of the Neo cassette in a sample. Homozygotes will possess two copies of Neo, heterozygotes one copy and wild type mice wi...

Primers are designed to amplify sequences present only in the vector backbone. If a correct homologous recombination has occurred then no product should be amplified. If a product is detected it strongly suggests an incorrect targeting event or a ...

The Neo short range PCR detects the presence of the Neo reporter in the cassette. This can be used as a rapid screen on F1 chimera progeny samples to detect likely germ line transmission

The mutant-specific SR-PCR amplifies a product in the mutant form of the correct allele. This can be used in conjunction with a wild-type specific assay to determine the genotype of the mouse. Homozygous mice will produce no amplification in the w...

Confirmation of the loxP site downstream of the critical exon can be achieved by several methods. The TRPCR used by the EUCOMM screen uses universal primers to amplify across the critical exon region. Presence of a band (when accompanied by the p...

The wild type loss of allele (WTLoA) qPCR assay uses a hydrolysis probe assay (eg Applied Biosystems TaqMan technology) to determine the copy number of the wild type allele in a sample. Homozygotes will show no amplification, heterozygotes one cop...

The LacZ short range PCR detects the presence of the LacZ reporter in the cassette. This can be used as a rapid screen on F1 chimera progeny samples to detect likely germ line transmission. The WTSI genotyping assay is shown below

The 5’ cassette check used by WTSI confirms the presence of the upstream FRT site and that the 5’ end of the cassette is still present. The assay is shown below.