Construction of Targeting Vector for Gene Targeting
Description of Service
Gene targeting, a procedure for the generation of knock out mice involves construction of a targeting vector, electroporation of embryonic stem (ES) cells, selection of homologous recombinant ES clones, injection of recombinant cells into blastocysts, generation of chimera and finally germ line transmission to develop targeted mouse cell lines. DNA Sequencing facility constructs the targeting vector following the steps mentioned below-
- Design cloning strategy together with the user
- Generate PCR products for left arm, right arm and middle arm (for conditional vectors
- Clone PCR products
- Sub-clone into relevant vectors
- Screen clones at each step by restriction digestion and/or sequencing
- Make Probes for Southern Blotting
- Also purify construct DNA for direct microinjection into mouse oocytes