- Our facility is available to UW investigators as well as the greater Seattle academic research community.
- Customers outside of the UW are subject to UW institutional overhead fee.
- The UWMCC is a full-service facility that generates novel mouse models using CRISPR/Cas9 technology.
Consultation and Design
- Meet with the PI to discuss the project and discuss the strategy.
- UWMCC will design the gRNA and donor DNA.
- UWMCC will design and test PCR primers that will be used for genotyping assays.
Test of gRNA efficiency
- UWMCC will test the gRNA (up to 4) in mESC or zygotes cultured to blastocysts to select the guide with best cutting efficiency and closest as possible to intended editing site
Design and construction of DNA donor
Introduction of gRNA/Cas9 and donor DNA into mouse embryos
Micro-injection or electroporation of gRNA + Cas9 + repair DNA into the nucleus or cytoplasm of mouse embryos depending on experiment. Alternatively, large inserts or loxp sites may be introduced into mouse ES cells first. The targeted mouse ES cells will be injected into mouse blastocysts to create chimeras.
Surgical transfer of injected embryos into surrogate mothers
Weaning, ear-tagging, and tissue biopsy of pups
Screen pups for founders carrying desired mutation
- Pups will be genotyped by PCR followed by Sanger sequencing to identify founder mice with desired genome editing.
- Screening to determine mono-allelic or bi-allelelic