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Nuclease-Mediated Knockout Rodent

  • It is an alternative to the conventional knockout which is faster, because it skips the need for ES cell work and chimera production.
  • This approach can be done in ~ 2 months.
  • Main strain available for nuclease-mediated knock out production are; C57BL/6 (B6) or FVB for mouse and Sprague-Dawley (SD) for Rat, although we can use any per your request.
  • Several types of artificially constructed nucleases (e.g., TALEN, Cas9 and ZFN) can be engineered to recognize and cleave arbitrary sequences. When such nucleases (or their DNA or mRNA precursors) designed to target a certain site in the mouse/rat genome are microinjected into fertilized mouse/rat eggs, cleavage at the site followed by imperfect repair can result in small deletions (and more rarely insertions) of one or more base pairs. If the cut site is in the coding region of a gene, the result can be frameshift mutations downstream of the site.

Diagram Nuclease

Transgenic Strategy Design
  • This includes the selection of target sites in the gene based on our optimized algorithm that maximizes on-target nuclease activity and minimizes off-target activity
  • Genotyping assays based on PCR and sequencing will also be designed for the screening of knockout founder mice
Nuclease Expression Vector Construction
  • DNA vectors that express the desired nucleases will be constructed and the efficacy of these vectors will be tested in cell culture
  • For each gene to be knocked out, we will construct vectors against at least two target sites in the gene to ensure success
Nuclease Injection into Mouse Eggs
    Preparation of Nuclease mRNA: 1-2 Weeks
    • Nuclease expression vectors will be transcribed in vitro
    • The resulting mRNA will be artificially capped and polyadenylated to facilitate its proper translation into protein in mammalian cells
    Nuclease Injection to Obtain Founders: 6-12 Weeks
    • The nuclease mRNA will be injected into fertilized mouse/rat eggs, followed by implantation of the eggs into surrogate mothers to obtain offspring We guarantee a minimum of 2 knockout founders with frameshift mutations, if vector constructed by us
    • In cases where the nuclease expression vectors are provided by customer, we will inject a min. of 200/300 eggs per target site
Knockout Founder Screening
  • Pups will be screened by PCR and sequencing to identify knockout founder mice. Specifically, the site targeted by the nucleases will be PCR-amplified, followed by sequencing of the PCR product to reveal any mutations that might have occurred
  • Mice carrying frameshift deletions/insertions on at least one allele are considered knockout founders
Optional: Breeding founders to obtain F1
  • We will breed up to 5 founders to wildtype mice/rats of matching strain background, and genotype their offspring to obtain F1 mice bearing the knockout allele
Generation of Transgenic Founder Mice/Rats
  • We guarantee a minimum of 3 transgenic founders in case of plasmid injection or 2 transgenic founders in case of BAC injection
  • Pups will be genotyped by PCR to identify founder mice

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