Research

Generation of Knockout Mice using Electroporation

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General Description

Content

CRISPR/Cas9-mediated NHEJ in mouse embryos will be used to generate founder animals harboring insertion/deletion (indel) or interval deletion knockout alleles. At this time, the Core can perform genome editing with S.p. Cas9 or A.s. Cas12a/Cpf1.

What Will Happen

  1. The User Investigator will initiate a project in iLabs. An IACUC protocol number and account) must be provided at this time.
  2. A consultation meeting between the Core and User Investigator will be held to discuss the desired genome modification. 
  3. Based on this discussion the Core will:
    • Design a genome editing approach to produce and detect the desired allele, including selection of a critical exon, selection of guide RNAs, and a PCR-based genotyping scheme. The design will be reviewed with the User and modifications made if necessary.
    • Review genome editing approaches designed by the User Investigator, including verification that the approach will produce a null allele, independent computational assessment of guide RNA quality, and review of the genotyping approach. The requested information must be provided to the Core and the Core must approve the approach prior to proceeding to the next steps.
  4. The Core will have guide RNA(s) synthesized by an approved vendor. Lab produced guide RNAs will not be used.
  5. Guide RNA(s) will be checked for concentration and degradation after resuspension.
  6. User investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [see Guide RNA testing in mouse zygotes].
  7. The guide RNA(s) and Cas9 protein will be complexed into RNPs and an electroporation mix in nuclease-free buffer will be prepared at concentrations determined by the Core.
  8. 80 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Please contact the GERM Core to discuss the necessary steps involved to use other strains.
  9. Live-born founder animals will be held by the GERM Core until 14 days of age and subsequently transferred to the User Investigator.
  10. Genotyping:
    • It is recommended that the Core perform genotyping of live-born offspring for the desired genome editing event [see Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at additional costs.
    • If the User conducts their own genotyping, Core Staff can review genotyping results at an additional cost.
  11. If the Core performs genotyping, they will report back to the User the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the user conducts their own genotyping, the same information will be reported back to the Core.

What to Expected

  1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
    • Design completion or verification
    • Reagent ordering
    • Embryo electroporation
  2. A minimum of 10 live-born offspring. If 10 live-born offspring are not produced, the electroporation will be repeated for 80 embryos.
  3. At least one live-born animal with the desired genome editing (one founder animal).
  4. If one founder is not identified:
    • If the guide RNAs were (i) tested in mouse embryos by the Core and the Core approved their use based on the genotyping results and (ii) the Core conducted the founder genotyping, the Core can review results, redesign the approach and reagents if needed, and the electroporation can be repeated. Project costs associated with re-design and additional embryo electroporation will be reduced by 50%. Costs associated with purchase of new guide RNAs and donor DNAs will not be reduced.
    • If the guide RNAs were not tested in mouse embryos or the user conducts their own genotyping, there will be no reduced costs for additional work.
  5. Indel mutations are often too small to detect by conventional PCR. Sanger sequencing and trace analysis will need to be performed to identify and characterize alleles produced.
  6. Exon deletion alleles are typically sufficient in size to detect by conventional PCR. The size of the interval deletion often varies between animals because NHEJ repair is semi-random. Sanger sequencing of deletion allele PCR products is necessary to verify the deletion and sequence at the NHEJ repair site.
  7. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped and Sanger sequenced to assess which alleles were inherited by which animals. A colony should be established from N1 animals harboring the same NHEJ allele (i.e. the same sequence at the repair junction). We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended.