CRISPR/Cas9

{slide=Complete CRISPR model design and creation}

• Guide design and preparation
• Blastocyst assay to assess activity of guides
• Establishment of genotyping strategy
• Repair template design
• Compatible with most efficient guide(s)
• Unique restriction site for downstream screening of founders
• Pronuclear microinjection of 150 embryos
• Transfer of embryos to pseudopregnant recipients
• Optional screening of founder animals
• Transfer of founder litters at weaning

{/slide}

Rodent Model Generation

{slide=Pronuclear Microinjection}

CRISPR/Cas9

• Loss of function alleles
• Knockout
• Large genomic deletions
• Hypomorphic

Targeted Insertions

• Patient mutations
• SNPs
• Conditional alleles
• ROSA26 insertions
• Reporter tags

Simple Transgenics

• Reporters
• Overexpression vectors

{/slide}

{slide=ES Cell Microinjection}

ES Cell Targeting

• Conventional
• CRISPR induced recombination
• CRISPR guides and compatible ROSA26 targeting vector

{/slide}

Preparation of commercially available ES cells (EUCOMM / KOMP)

Assisted Reproductive Techniques

{slide=Cryopreservation}

Mouse

• Sperm
• Embryos
• Oocytes
• Ovaries

Rat

• Sperm
• Embryos

{/slide}

{slide=Rederivation}

Mouse

• In vitro fertilization
• Transfer of cryopreserved embryos
• Pathogen-free
• Ovarian transplant

Rat

• Transfer of cryo-preserved embryos

{/slide}

Consultation

• Design of transgenic constructs
• Screening strategies
• Breeding schemes

Maintenance of Stock Lines

• Cre stains (ex. Ella)
• Reporters (ex TdTomato, GFP)
• Male/female breeders and controls (ex. C57B6J)