EMBRYONIC STEM CELL ELECTROPORATION
In vivo inactivation of a gene is possible by the gene targeting technique. The appropriate gene targeting vector is inserted into the embryonic stem cells (ES) and after homologous recombination events the endogenous target gene is inactivated. Injection of ES recombinant cells into blastocysts give rise to chimeric mice that should be properly crossed to test germ-line transmission of the mutation.
The Users are instructed and helped in the correct planning of the gene targeting vector by CFCM staff.
If the user already has a vector, CFCM requires discussing it before electroporation, to verify for minimal requirements.
Characteristics of the targeting vector:
To ensure success of the homologous recombination some fundamental parameters should be respected during targeting vector construction: the genomic regions to be included in the vector MUST be isolated from an Sv129 mouse genomic library. These regions must be isogenics to the DNA of the ES cells used by the Service (TBV2, Sv129 strain).
The vector should be constructed in order to eliminate most of the coding region of the targeting gene. In that way you are sure to create a non-functional protein. The length of arms can vary from 2 to 5 kb, unbalanced arms are better than equal, and in general long arms have more possibilities to recombine than short arms.
The gene targeting construct can be programmed for conventional or conditional knock-out. It has to contain double selection: the gene that confers Neomycin resistance under the control of TK or PGK promoter as positive selection and the gene coding for Thymidine kinase under the control of TK or PGK promoter as a negative selection. Some vectors are distributed by the Facility.
Two replicas per each 96-wells plate will be stored at –80°C refrigerator for one month and a half. Then recombinant clones should be expanded: cells frozen on 96-wells plate do not survive longer storing.
This is the reason why the screening for homologous recombination events must be done timely by the user (three weeks).
The service will start electroporation of the targeting vector only in the case the user has developed a proven strategy to detect homologous recombination events.
Two replica plates containing ES colonies grown onto gelatin will be given to the User to extract DNA and perform Southern blot or PCR screening (Mini-Southern protocol).
Guarantees: if positive and negative selections are present in the vector and the size is under 15 kb, the picking of 144 clones is guaranteed. In case a higher number of resistant clones are present, they will be picked until a maximum of 384 clones.
CFCM does not guarantee the presence of recombinant clones.
Some DNA sites appear impervious to homologous recombination, whereas sites a few kilobases away may be much more open to integration. But even at the same site, the frequency of homologous versus non-homologous recombination events can vary by a factor of ten from one day to the next.
What we need:
- 400 micrograms of Qiagen quality gene targeting vector DNA.
- A map of the construct and a map of the targeting strategy.
- The restriction enzyme for linearization should be clearly indicated.
- A picture of the linearized vector.
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