University of Pennsylvania
DNA Sequencing Facility
Construction of Targeting Vector for Gene Targeting
(In association with the Gene Targeting Service and the Transgenic Mouse Facility at the School of Medicine)
Gene targeting, a methodology for the generation of knock-out and knock-in mice involves construction of a targeting vector (done by this facility), electroporation of embryonic stem (ES) cells with the construct and selection of homologous recombinant ES clones with southern blotting (done by Gene Targeting Service), injection of recombinant cells into blastocysts and generation of chimera (done by Transgenic Mouse Facility ) and finally germ line transmission to develop targeted mouse cell lines.
DNA Sequencing Facility constructs targeting vectors for both conditional knock-out and knock-in mutations using BAC recombineering method of Dr. Neal Copeland.
Ref: Liu et. al. 2003, 13:476-484, Genome Research
- The vector design is to be provided by the investigator. Gene Targeting Service designs vectors for a fee.
- The DNA Sequencing Facility will:
- Order 2 BAC clones if available
- Design and order primers for homology arms (300 - 600 bases) of the retrieval vector and the mini targeting vector(s)
- Make retrieval vector and retrieve genomic target from the BAC DNA by homologous recombination
- Make 1 or 2 mini targeting vectors as required
- In the case of knock-in construct introduce mutation(s) in one of the homology arms
- Introduce loxP or FRT sites into the target using Cre or Flpe recombinases
- Do maxi DNA prep of the final vector
- Verify sequence of the final vector including all exons in the target, two ends of neo gene and LoxP and FRT sites
Turnaround time: 3 - 4 months after receiving the vector designCost:
|Conditional knock-out construct||$4,350|
Note: We also make probes for southern blotting & purify construct DNA for direct microinjection into mouse oocytes.
The DNA Sequencing Facility is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.