The BAC mutagenesis service can be used to generated BAC transgene vectors with engineered point mutations or deletions where precise engineering is critical, i.e. without any remaining foreign DNA left behind along with the desired modification.  This involves inserting a bacterial selection cassette capable of both positive and negative selection into the desired location in the BAC.  Positive selection is used to select for the insertion event and negative selection is used in a second recombination step to switch the selection marker with the final mutation of interest.  This procedure typically involves two recombineering steps and takes approximately 6 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.

Please note that a set of guidelines for BAC recombineering of transgenes and targeting vectors can be found at vanderbiltresearch.org in the TMESCSR protocol collection under "BAC recombineering and transgenesis."