The CRI Mouse Genome Engineering Core Facility offers CRISPR/Cas9 services for direct production of mutant mice by pronuclear injection. We use the Easi-Crispr method as described in the Nature Protocols 2017 publication “Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.”
This technology allows production of genetically modified mice in as little as three to four months. A number of mutations can be introduced, including point mutations such as SNPs, LoxP sites, reporter genes, protein tags, deletions, etc.
Embryonic stem (ES) cell gene targeting services
Targeting embryonic stem cells can produce knock-out mice, conditional knockout mice and knock-in mice.
Targeting Vector Design
We recommend consulting with the facility’s scientific staff prior to beginning construction of the targeting vector and positive control plasmid.
ES Cell Targeting and Screening
The client must demonstrate the ability to detect the targeted locus by PCR and Southern hybridization assays prior to project initiation.
ES Cell Microinjection
The Core will inject up to three different clonal mouse embryonic stem cell lines into host embryos and transfer them into pseudopregnant females to produce chimeric animals.
Initial Breeding of Chimeras
When the ES cells are implanted into host embryos, some may migrate to the genital ridge and become germ cells. A chimera with a high proportion of ES cell derived fur has a high probability of transmitting the targeted gene to its offspring. So, if your animal space is limited, we recommend to mate only males above 40 precent and females above 80 percent chimerism to wild-type (B6) animals.
Transgenic mouse services
We offer a full range of pronuclear injection services for production of transgenic mice. We routinely perform transgenic mouse production in C57BL/6, and other strains are available upon request.
Submission of DNA
For transgenes less than 20 kb in size, the CRI Mouse Genome Engineering Core Facility creates transgenic mice from DNA the client provides. For an optimum number of founders, the transgene plasmid should be purified using an endotoxin-free system. The plasmid backbone should be cut from the transgene DNA with restriction enzymes in such a way that the transgene DNA is easily separated from the plasmid backbone by agarose gel electrophoresis. The client should supply the facility with 20 µg ofthe digested DNA.
All clients are required to complete and submit a request through PPMS before the initiation of project work.
When the project is accepted by the facility, please arrange to drop off your DNA or other materials to NL12.110C with Tripti Sharma.
The transgene DNA is injected into the pronuclei of embryos and implanted into pseudopregnant recipient females. Pups are born approximately three weeks after the injections. We transfer mice to the client as soon as animals are weaned.
The facility’s pronuclear microinjection service includes injection of up to 500 embryos or production of three transgenic founders, whichever is achieved first. Clients are responsible for genotyping founder mice. Different founder lines may display distinct expression levels; some founders may not transmit the transgene to their offspring, and some lines may not express the transgene at all. The production of three founders gives a reasonable chance to obtain at least one good transgenic line. The facility makes no guarantees with respect to founder production, germline transmission or gene expression in the founders and their offspring. A deleterious transgene may result in an inability to obtain founders or to obtain germline-transmission orexpression in the offspring. If no transgenic animals are obtained after injection of 500 embryos, the client will be billed for the service, and we advise a consultation with the facility scientific director before attempting additional microinjection projects with that transgene.
Colony management Services
The Mouse Genome Engineering Core Facility offers a variety of cost-effective colony management solutions that provide clients with flexibility in the management of their rodent colonies.
Our colony management service offerings include:
- Sperm cryopreservation – We will freeze a minimum of ten and a maximum of 25 straws of sperm for each line. You will need to supply three to five males that have undergone previous mating, and that are between 10 weeks to 6 months of age. All frozen sperm is tested by IVF to assure competency after thaw [competency = a fertilization rate of greater than, or equal to 20%].
- Rederivation- We will mate homozygotes to obtain embryos for homozygous lines, or mate homozygous or heterozygous males to wild type females for lines that may be rederived as heterozygotes. All pups produced will be transferred to the investigator for genoytping. Please note: it is in the investigator’s best interest to genotype any animals imported for this procedure prior to the rederivation procedure. It is not at all unusual for the animals received to be incorrect.
- In Vitro Fertilization – We will use fresh or previously frozen sperm to fertilize oocytes obtained from up to 10 superovulated females. Embryos surviving to the two-cell stage will be implanted into pseudopregnant females for gestation or frozen for later implant. All surviving pups will be transferred to the contracting investigator.
- Intracytoplasmic sperm injection (ICSI).