We offer CRISPR/Cas9 nuclease services for direct production of mutant mice and rats by pronuclear injection. This technology allows production of genetically modified mice and rats in as little as 3-4 months. Many different kinds of mutations can be introduced, including point mutations such as SNPs, LoxP sites, reporter genes, protein tags, deletions, etc. Genetic modification can be performed in any strain for which pronuclear injection is possible, thus eliminating the need for ES cells and backcrossing. Genes on the same chromosome can be mutated simultaneously, avoiding the need for double targeting of ES cells. Contact the core director to see if CRISPR/Cas9 technology can be used for your project.
For projects where CRISPR/Cas9 modification is not optimal we also provide ES cell gene targeting services for creation of genetically modified mice. Gene targeting services are available in ES cells from C57BL/6, 129P2/Ola and BALB/c background strains. Gene targeting vectors can be generated by the Core or provided by the client. Targeting vectors for many genes may be available from the knockout mouse consortium (KOMP/EUCOMM/NorCOMM). Targeted ES cells for many mouse genes are also available from KOMP/EUCOMM, allowing the production of conditional (floxed) alleles in less time than a full targeting project. The Core can perform validation and microinjection of KOMP/EUCOMM cells when available.
Targeting Vector Design: General Considerations and Client/Facility Responsibilities
We highly recommend letting the core provide vector construction and ES cell screening for most efficient project progression.
Clients who want to construct their own targeting vectors should consult with the facility director prior to beginning construction of their targeting vector and positive control plasmid. The "short" arm of homology should generally be at least 1.5 kb long. A positive control plasmid for PCR screening would contain the entire a fragment of genomic DNA encompassing the short arm of homology, but slightly longer, to allow for a targeted locus-specific set of primers to be used in the screening of ES cells. The long arm can be up to 10 kb long, but a length of 5-10 kb is acceptable for a reasonable frequency of targeting at most loci. The source of the arms of homology should be considered in constructing a targeting vector. In some instances, isogenic DNA may be required for efficient targeting. BAC clones constructed from C57BL/6 DNA are available through CHORI for vectors to be targeted in C57BL/6 ES cells. BMQ BAC clones are available from CHORI as a source of 129 strain DNA for vectors intended for targeting in E14 ES cells (strain 129P2/Ola). C57BL/6 or E14 ES cell DNA is also available from the facility for PCR cloning. The deletion size should also be considered. Deletions of 10 kb or less are easily obtained following positive-negative selection.
For deletions larger than 10kb, we recommend BAC-based targeting vectors constructed by the Core. BAC vectors allow constitutive deletion of large regions as well as insertion of LoxP sites for conditional deletion of large genes or gene regions. Contact the Core director for additional information about BAC targeting vector construction.
Submitting Targeting DNA
Clients submitting gene targeting vectors should follow the following guidelines: Targeting DNA should be supplied to the facility after it has been linearized with a restriction endonuclease. The client should provide a minimum of 100 µg of linearized targeting vector and a picture of a gel demonstrating that the DNA has been linearized. The endonuclease should be heat-inactivated if possible (consult with the endonuclease supplier for specific information on heat-inactivation). The core will purify and precipitate the DNA prior to electroporation.
ES Cell Targeting & Screening
For vectors produced by the Core, the core will provide all ES cell screening including positive control development, PCR and Southern blot assay development, and ES cell screening. Clients may also request ES cell screening services for vectors provided by the client or purchased from KOMP/EUCOMM.
The following instructions apply to clients providing their own ES cell screening: The client must demonstrate the ability to detect the targeted locus by PCR and Southern hybridization assays prior to project initiation. The primary ES cell screen is normally by PCR using one primer outside the arm of homology and one primer specific to the introduced mutation (e.g. neo primer). To develop the PCR screen, 10 fg of the positive control plasmid is mixed with 5 µl of ES lysate (available from the core) in the PCR reaction. The facility will provide ES cell lysate DNA for assay development. Clients wishing to perform primary screening by mini-Southern blot may request the plates of cells for extraction of DNA. The Core can provide ES cell screening by PCR and Southern blot upon request. ES cells testing positive for the targeted locus by PCR must be confirmed by a Southern hybridization assay. PCR-positive clones are expanded, frozen in liquid nitrogen and grown in large quantities for Southern hybridization to confirm correctly targeted ES clones. The facility will provide purified DNA to the client for this purpose. Clones testing positive by Southern blot may then be advanced for blastocyst microinjection. The facility is obligated to provide the client with up to four 96-well plates to screen by PCR. In signing the "Service Request Form", the client accepts these conditions and agrees to pay for services whether or not a targeted ES cell line has been obtained. The client is not liable for negative results due to failures on the part of the facility (such as clone loss due to incubator malfunction). However, clients are responsible for paying for negative experiments due to errors on their part (such as providing the wrong targeting DNA). If recombinant ES cells have not been obtained following 2 rounds of electroporation, selection and screening, it is advisable to meet with the facility director to discuss options. The frequency of PCR-positive clones being correctly targeted by Southerns typically ranges from 50 to 100%. The facility can provide ES cell DNA to the client for them to establish their Southern hybridization assay.
Generating Transmitting Chimeras
Before any animal work can begin, including blastocyst microinjections of the targeted ES cells, the client must provide the facility with the IACUC number of an approved Application to Use Live Vertebrate Animals from their institutional animal welfare committee. This number assures the facility that the proposed experiments have been approved and that proper animal handling and care protocols are in place. Gene-targeted mice are produced by microinjection of gene-targeted ES cells into strain C57BL/6 or C57BL/6-albino mouse blastocysts. The ES cells can either be those provided by the client (e.g. from KOMP/EUCOMM) or those made by the facility. Those provided by the client must be expanded and stored in liquid nitrogen in the facility and this will incur an additional charge. Client-provided ES cells also must be screened for mouse pathogens and mycoplasma contamination before microinjection. There is no guarantee on the production of chimeras or transmission from targeted ES cells made outside the facility. Targeted ES cells made within the facility are expanded for injection without an additional charge and carry a qualified guarantee of at least 4 high (> 75%) chimera males, or transmission of the intended genetic modification, whichever comes first. This guarantee applies only to the standard ES cells used in the facility. At approximately 3 weeks of age (weaning) all chimeras are transferred into the care of the client unless breeding services have been requested. Animal space must be arranged by the client within their institution to receive the animals. At UNC, animal space can be obtained only by contacting the DLAM office. If mice cannot be moved out of the facility because of a failure to secure cage space in a timely manner, a colony maintenance fee will be charged to the client.
Suggestions for Initial Breeding of Chimeras
C57BL/6 ES cells are injected into C57BL/6-albino blastocysts, producing chimeras with black fur from the ES cells on the white host background. 129P2/Ola ES cells are injected into C57BL/6 blastocysts, producing agouti (brown) fur from the ES cells on the black host background. When the ES cells are implanted into host embryos, some of the ES cells migrate to the genital ridge and become germ cells. Because germ cells are derived from the same cell lineage that gives rise to the skin (primitive ectoderm), the probability of germ cells originating from the implanted ES cells can be roughly equated with the degree of chimerism. In other words, a chimera with a high proportion of ES cell derived fur has a high probability of transmitting the targeted gene to its offspring. High chimera females are rarely produced, but may also transmit the intended genetic modification and should be mated. If space is not an issue, all chimeras should be mated to wild-type (B6) animals. If space is an issue, then only males above ~40% and females above ~80% chimerism should be mated. Germline transmission may be ascertained by breeding chimeras back to the host blastocyst strain. For C57BL/6 ES cells injected into C57BL/6-albino blastocysts, chimeras are typically mated to C57BL/6-albino females. Black pups indicate germline transmission and should be genotyped for the mutant allele, which should be found in about 50% of black pups. White pups are derived from the host blastocyst. For 129P2/Ola ES cells injected into C57BL/6 blastocysts, chimeras are typically mated to C57BL/6 females. Agouti pups indicate germline transmission and should be genotyped for the mutant allele, which should be found in about 50% of agouti pups. Black pups are derived from the host blastocyst. We recommend that selectable marker cassettes be removed to avoid the risk of their contributing to mutant allele phenotypes. Most targeting vectors have the selection cassette (usually a PGK-promoter and neomcyin resistance gene) flanked by FRT sites to allow Flp-mediated removal. Chimeras or F1 animals can be mated to Flpe mice and PCR screening can be used to detect animals with the cassette removed. These animals are then backcrossed and pups screened for removal of the Flpe transgene.
Extended Breeding Options
There are several breeding options that can be adopted. The option chosen may be critical, as there are a number of phenotypic differences among the various strains of mice used for genetic research and it behooves the client to become familiar with strain differences as they relate to their gene and expected phenotype(s).
A. Establishing an Isogenic Strain. If there are no known differences in the gene or expected phenotype, the best option is to create an isogenic mouse line to avoid potential influences that a mixed genotype might have on the phenotype. The only way to generate an isogenic mouse line is to mate a transmitting chimera with a strain identical to that from which the ES cells used to make the chimera were derived (for example, strains 129/Ola). A common approach is to first mate the chimeras to strain B6 mates to determine which of the chimeras are transmitting the 129 genome. However, when crossed to an isogenic animal, coat color cannot be used to identify germline transmission and all of the offspring originating from the chimera must be genotyped to determine transmission of the targeted allele. This is where establishing a genotyping protocol by PCR becomes very important in reducing the time and costs of experiments. Heterozygotes can then be interbred to produce genetically uniform animals that vary among themselves only at the targeted locus.
B. Establishing a Congenic Strain. When strain differences are suspected to influence the phenotype of the gene-targeted mouse, it is advisable to reduce strain-related variability by establishing a congenic mouse strain. This is accomplished through multiple back-crosses onto a genetic background that is favorable to the phenotype. Back-crossing is achieved by mating the chimera or its agouti (+/-) offspring with the strain of animal needed to support the phenotype (for example, strain Balb/C). The offspring from this mating are considered back-cross 1 or N1. In the example, heterozygous N1 offspring would then be mated with wild-type Balb/C animals to produce back-cross 2 (N2) offspring. Again, heterozygous pups would be mated with wild-type Balb/C animals to produce N3 offspring. The cycle of selecting heterozygous animals and mating these with wild-type animals continues until back-cross 9 (N9) is achieved, at which time a "negligible" amount of the 129 genome remains associated with the modified allele and the strain is considered congenic and genetically homogeneous. It is in the interest of the client to begin back-crossing their mutant mouse strain as soon as possible. If an interesting phenotype is found in their animal, but its penetrance is less than 100% in the first group of homozygous animals produced for the study (typically mixed genotypes), it is not unusual for a reviewer to request that the experiments be repeated in a congenic strain. Since back-crossing to N9 can take more than a year and there are likely to be competitors making similar animals, a late start in back-crossing can make the difference between a high profile and a low profile publication. It is possible to develope a congenic strain more rapidly through the use of strain-specific markers (speed congenics) to identify back-cross heterozygous offspring that carry a high proportion of DNA from of the strain needed (e.g., Balb/C). In this way, a congenic strain can be produced in approximately 6 back-crosses. Charles River Laboratories provides this service, but it is expensive.