Generating Knock Out mice
Strategy 1: Frameshift KO mice (fsKO)
A Genome Editing Agent (GEA) based on CRISPR technology will be designed by SAGE to target the gene of interest. GEAs will target exonic sequences near the start of the open reading frame. Injection of the GEA into a mouse one cell embryo (zygote) will generate a double stranded break at the target sequence. Repair of the double stranded break by Non Homologous End Joining will result in small deletions (or insertions). A frameshift mutation occurs when the number of nucleotides that are deleted or inserted is not a multiple of three, which will likely result in a dysfunctional protein. Injected zygotes are transferred to psudopregnant females and the resulting pups are screened for indels using PCR and sequencing.
The process for Strategy 1 includes:
- Design and generation of GEAs: SAGE will design a GEA to target the customer's gene of interest. Once the design has been approved by the customer, SAGE will prepare the custom GEA reagent for injection into mouse zygotes. GEAs will be designed to maximise the efficiency of GEA binding at the intended target site and to minimise off target binding.
- Injection: For most projects, one injection session will be sufficient to generate the fsKO mouse line. If necessary, two additional injection sessions will be performed. GEAs will be injected into C57Bl/6 mouse zygotes and the surviving embryos transferred into pseudopregnant females.
- Screening/genotyping: SAGE will perform the molecular screening assays to identify founders with GEA-induced mutations. This will involve extraction of genomic DNA from biopsy tissue, PCR across the putative mutation site, assaying for mutations by detection of heteroduplex formation, and identifying mutations by Sanger sequencing of PCR protects. Founders with frameshift mutations will then be transferred to the client's animal facility (cost not included).
Strategy 2: Deletion KO mice (delKO)
Two GEAs that are complementary to intronic sequences flanking a critical exon or exons of the gene of interest will be injected into mouse zygotes. Simultaneous cleavage of the DNA by both GEAs followed by NHEJ-repair will result in the deletion of the critical exon(s).
Design and generation of the GEAs, injection and screening/genotyping for delKO projects will be performed as for fsKO mice (see above).
It is anticipated KO mice will be generated in 3-4 months, although this make take longer during very busy periods.