This service is for BAC-based assembly of gene targeting vectors for use in generating non-conditional (i.e. "global") gene targeting alleles.  This service may be used to make a targeting vector for generation of loxed cassette acceptor alleles, or other such docking sites, in mice.   BAC recombineering allows faster and better construction of targeting vectors, in part by freeing the user from relying on endogenous restriction sites for the design process.  The process usually involves a consultation meeting between the P.I. and Dr. Mortlock to determine the ideal, desired final design of the targeting vector, and the screening process that will eventually be needed to identify a correctly targeted ES clone.  This will determine the specific reagents needed for BAC-based assembly of the targeting vector. 

The first BAC recombination step typically involves inserting a cassette into the gene of interest on a BAC clone, and the second step is a recombination to subclone the BAC genomic sequences and cassette into a plasmid, thus creating the final targeting vector that contains all components needed for gene targeting in ES cells.  The service typically involves two recombineering steps and usually takes 6 - 8 weeks.

Experimental flow for BAC recombineering services: 

1. The principal investigator (PI) schedules a time to meet with Dr. Mortlock (mortlock@chgr.mc.vanderbilt.edu).  Dr. Mortlock is available to design the desired vector or provide consultation, depending on the needs of the PI.  Together they will determine which, if any, reagents must be provided by the PI before vector construction (e.g. specialized expression cassettes) and which reagents will be obtained by Dr. Mortlock on behalf of the PI (e.g. standard, publicly available BAC clones).   The outcome of the meeting will be to create a written plan for vector construction and the final vector design. 
2. Dr. Mortlock creates maps and Genbank files of the vector to be generated.
3. The PI submits the appropriate service form to the TMESCSR including the required documentation, as described on the submission form.
4. The BAC service will be scheduled by the TMESCSR.  Dr. Mortlock will notify the PI of the start date.
5. The PI will be billed the month that service begins.
6. When completed, Dr. Mortlock will provide the PI with the final vector as a glycerol stock of the bacteria stock (whether BAC or plasmid).
7. Intermediate BAC clones (generated as intermediate steps in vector construction) will be stored by the Core as bacterial glycerol stocks. Duplicates of these may be provided to the PI upon request.