Gene Targeting in ES Cells
To see background, more information is available here.
Resources and recommendations
Currently we perform blastocyst injections of ES clones that were either produced by ourselves or our customers. Furthermore, we are actively using gene trapped and gene targeted ES cell clones produced by the International Knockout and Gene Trap Consortiums (see Useful Link and References). The Consortiums have genetically engineered ES cell clones on the 129 and C57BL/6 backgrounds. At this time our specific attention is focused on the ES clones with a C57BL/6N genetic background. To produce new mutant mouse line we recommend the purchase 2-3 different clones, with the price being approximately $500- $700 per one ES cells clone. The generation of a new mouse line will cost $4000-$6000 including the cost of ES clone, delivery of ES cells and the generation of chimeric mice via blastocyst injection.
It is known that ES cell lines accumulate chromosomal abnormalities during culture that reduce or completely abolish the chance of generating of germline chimeras (Robertson, 1987; Longo et al., 1997).Therefore we, in collaboration with the Cytogenetics Core, are able to improve the quality of your mutant ES clones by a selection of euploid subclones. 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).
Responsible parties and timeline
|1||Variable||Analysis of ES cell clones for targeted replacement of desired locus||Customer/Company|
|2||Week 1||Grow up 2 independent clones of ES cells confirmed to be targeted. Microinject each clone into 80-100 blastocysts in order to produce chimeric offspring||TMM|
|3||Week 4||Offspring born||TMM|
|4||Week 8||Transfer chimeras to customer||Customer|
|5||Week 10||Mate chimeras to identify germline transmitting mice||Customer|
|6||Week 15||Genotyping of pups||Customer|
- 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.
- Fill out, sign and submit the Project Request Form to TMM.
- If the ES cells were not produced by TMM please provide the following materials:
- ES cells and information about the origin of these ES cells.
- Information about the health status of ES cells.
- Protocol for handling of the ES cells.
Service fees include
- Purchase of virgin C57BL/6 or C57B6-A females (donors of embryos).
- Expansion of positive ES cell clone for injection into blastocysts.
- Injection of cells of one ES cell clone into 80-100 C57Bl/6 or C57Bl/6-albino blastocysts.
- Transfer of microinjected embryos into pseudopregnant recipients.
- Generation of 15-20 pups.
- 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
- Animal housing charges of litters after weaning.
- Breeding of chimeras for germline transmission. Health testing of the produced mice.
- Shipping charges.
- Customers will be charged for the time while mice remaining within the Transgenic Core’s mouse room after weaning.
According to 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.