To see background, more information is available here.

Resources and recommendations

CRISPR technology offers the potential to directly target genes in single-cell eggs without the need for embryonic stem (ES) cell application, significantly facilitating gene targeting. However, in certain knock-in projects requiring the use of several kilobases (kb) of donor DNA, the efficiency of homology recombination (HR) is notably low. To enhance HR efficiency in such cases, the combination of CRISPR and ES cells proves to be invaluable. Consequently, we continue to utilize ES cells for gene targeting purposes. Several ES cell lines on C57Bl/6NJ, 129/Sv, and 129xC57Bl/6 the background is available in TMM for gene targeting. To prevent differentiation of ES cells, they are cultured on the special media contained LIF. The cells have high capacity to colonize germline of chimeric mice and were successfully used to generate many novel knock out mouse lines.

Mouse Embryonic Fibroblasts (MEF)

To support ES cells growth we have wild type MEF on CD-1 and C57Bl/6 background. To perform ES clones selection after transfection we have multiple-drug resistant MEFs prepared from the Tg(DR-4) mouse strain (Tucker et al., 1997)   from Jackson Lab that displayed resistance to concentrations of the drugs G418, 6-thioguanine, puromycin and hygromycin well above those used normally for the selection of drug-resistant ES cells.

Targeting

Design of CRISPR target and sgDNA, is performed by TMM. Customer designs and provides 50 µg of DNA comprising the targeting construct, linearized and precipitated in EtOH.To transfect ES cells the sgRNA, Cas9 and targeting DNA construct  will be electroporated with a batch of 2x10⁷ cells from the requested ES cell line. Following drug-selection, ~ 100 ES cell colonies will be isolated and replica plates generated, which will be forwarded to the PI's laboratory for genotyping. The master plates will be stored at - 190° C freezer for further analysis and the production of chimeric mice. We are also capable of eliminating differentiated ES cells and improving the germline transmission capacity of your mutant ES clones by culturing and subcloning under special conditions (for example, culture with RESGRO or ESGRO-2i Medium, Millipore).

Currently, we conduct blastocyst injections using embryonic stem (ES) clones, which are either generated in-house or provided by our customers. Additionally, we actively incorporate gene-trapped and gene-targeted ES cell clones obtained from the International Knockout and Gene Trap Consortiums, as indicated in the Useful Links and References section. These Consortiums offer genetically engineered ES cell clones with backgrounds on both 129 and C57BL/6 strains. Our present focus is specifically on ES clones with a C57BL/6NJ genetic background.

For the creation of a new mutant mouse line, we recommend the purchase of 2-3 different clones, with each ES cell clone priced approximately between $800 and $1200. The overall cost for generating a new mouse line is estimated to be in the range of $5000 to $7000. This cost includes the purchase of ES clones, delivery of ES cells, and the generation of chimeric mice through blastocyst injection.

Service ordering

1. Before initiating a transgenic mouse project, please receive approval of the Animal Care and Use Committee (IACUC) and the Institute Biosafety Committee (IBC) for your animal use and recombinant DNA protocols.
2. Fill out, sign and submit the Project Request Form to TMM.
3. If the ES cells were not produced by TMM please provide the following materials:
   1. ES cells and information about the origin of these ES cells.
   2. Information about the health status of ES cells.
   3. Protocol for handling of the ES cells.

Service fees include

1. Purchase of virgin C57BL/6 or C57B6-A females (donors of embryos).
2. Expansion of positive ES cell clone for injection into blastocysts.
3. Injection of cells of one ES cell clone into 80-100 C57Bl/6 or C57Bl/6-albino blastocysts.
4. Transfer of microinjected embryos into pseudopregnant recipients.
5. Generation of 15-20 pups.
6. Animal housing charges of litters before weaning (3 weeks).

If we use ES clones generated by us, we guarantee the generation of 4-5 chimeras with 50% agouti on the fur. However, we don’t guarantee germline transmission. 

Service fees do not include

1. Animal housing charges of litters after weaning.
2. Breeding of chimeras for germline transmission. Health testing of the produced mice.
3. Shipping charges.
4. Customers will be charged for the time while mice remaining within the Transgenic Core’s mouse room after weaning.

Additional services

According to the customer’s request, TMM can perform a germline transmission assay. For this purpose the animals will be housed in our colony beyond the 3 weeks of weaning age. Furthermore, Transgenic Core can generate aggregation chimeras. Moreover, TMM can tag, collect tail tissue samples, extract DNA, and genotype the offspring by PCR. These additional services will be charged as extra fees including the additional costs for the cage(s) per diem.

Service fees

See fees for Blastocyst injection/Chimeras.