CRISPR Microinjection
Genome Editing via CRISPR/Cas9 System
Click here to see the background on Genome Editing via CRISPR/Cas9 System.
Resources and recommendations
Project initiation
Before initiating a CRISPR/Cas9 mouse project please
- Consult with the TMM Core regarding the preparation or purchase of these components and the targeting strategy under consideration.
- Receive approval of the Animal Care and Use Committee (IACUC) and the Institute Biosafety Committee (IBC) for your animal use and recombinant DNA protocols. Then fill out, sign and submit the Project Request Form to TMM.
Designing and generation of CRISPR/cas9 components
Design of CRISPR targets, synthesis and purification of RNA and DNA components is performed by the customer or company. TMM requests that customers bring high-purity CRISPR/cas9 components (contact the TMM director about amount and concentration since this varies for each project).
Mouse strains
Inbred C57BL/6N transgenic mice: Currently majority of our customers request transgenic mice on the C57BL/6N inbred background because the genome and phenotype of this mouse line is well known. This background is also advantageous if the transgenic line is crossed with induced mutants that are maintained as congenic strains on this background. We offer the production of CRISPR/cas9 KO or transgenic mice on the C57BL/6N inbred background with either plasmid or BAC transgenes. However, because the overall efficiency is lower in C57BL/6, we charge higher fees than for the F1-hybrid transgenic mice.
F1-hybrid mouse strain: The (C57BL/6xDBA/2)F1 hybrid strain has been used in this core for standard transgene microinjections. Under the terms of the agreement, we will produce at least 30 offspring from the injections of pronuclear stage embryos. These animals will be transferred to the PI's colony at 3 weeks of age (weaning age).
Responsible parties and timeline
| Step | Timeframe | Protocol | Responsible party |
|---|---|---|---|
| 1 | Variable | Project design, generation of CRISPR/cas9 components | Customer/Company |
| 2 | Variable | Facility schedules injection | TMM |
| 3 | Week 1 | Microinjection of CRISPR/cas9 components into 200 zygotes | TMM |
| 4 | Week 4 | Offspring born | TMM |
| 5 | Week 7 | Transfer potential offspring to customer | Customer |
| 6 | Variable | Genotyping of pups | Customer |
| 7 | Variable | Breeding of founders and genotyping o F1s | Customer |
Service fees include
- Purchase of virgin C57BL6/NJ or (B6xDBA-2)F1 females (donors of oocytes).
- Superovulation of the virgin females.
- Microinjection into 200 C57BL/6NJ or (B6xDBA-2)F1 zygotes.
- Transplantation of the microinjected embryos into pseudopregnant recipient females.
- Animal housing charges for recipients and their offspring until weaning.
- TMM guarantees the generation of 30 pups and at least one CRISPR/cas9 genetically modified mouse. TMM does not guarantee the generation of "founders" with desired KO-in of gene.
Service fees do not include
- Animal housing charges for litters after weaning.
- Ear/tail tissue collection for DNA isolation for genotyping.
- Health testing of the foster mother.
- Shipping charges.
- Customers will be charged per diem while mice remaining within the TMM's mouse room after weaning.
Additional services
According to the customers' request, TMM can purchase and use other mouse lines for the generation of genetically modified mice. Moreover, TMM can tag, collect tail samples, extract DNA and genotype the offspring by PCR. For this purpose the animals can be housed in our colony beyond the usual 3 weeks (weaning age). These additional services will be charged as extra fees including the extra costs for the cage(s) per diem.