The Transgenic and Knockout Core can develop mice with targeted mutations such as deletions, small insertions or point mutations directly in fertilized embryos. It takes about two months to generate a gene-targeted founder mouse with CRISPR technology. Typically, the positive founders are transferred to the investigator.
CRISPR reagents are injected into fertilized one-cell-stage embryos, and the microinjected embryos are transferred into pseudopregnant surrogate mothers that develop, deliver and nurse mutant offspring.
The service includes:
- Design of guide RNAs (gRNAs) and single-stranded oligo donors (ssODNs)
- Ordering of Cas9/gRNAs
- Microinjection of reagents into zygotes and transfer microinjected embryos into surrogate mothers
- ID tagging and tail DNA isolation from pups
- Transfer of founder mice to investigator
The following is each investigator's responsibility:
- Confirming targeting design
- Screening of founder pups
- Breeding of founders
- Mutant genotyping design and establishment
All the necessary assistance for procedures such as screening, founder breeding planning and genotyping can be provided upon request. Please feel free to contact the core for any assistance.
Some important notes about the CRISPR/Cas9 targeting service:
- We have successfully generated mutants and offer mutagenesis in inbred C57BL/NHsd and FVB/NHsd (from Envigo). We have also modified existing mutants or other strains provided by investigators. Please contact the core for gene editing in other strains.
- Although gRNAs will be designed to have minimal off-targets in coding regions, off-target mutations can occur in founder mice. Backcrossing is highly recommended to eliminate or reduce off-target mutations.
- Founder mice can be mosaic (having different mutations in different tissues or cells) and they may not transmit the mutation found in screening. Assistance in screening and breeding strategies can be provided upon request.
Although the above services are set up to deliver the mutant mice to investigators as efficiently as possible, the core can perform screening, breeding or other services on a fee basis as our schedule allows. Please contact us with any request and concern.