BAC-based vectors are particularly useful substrates for engineering transgenes involving vertebrate genomic DNA.   “Recombineering” refers to any of several similar methods that use homologous recombination in E.coli cells in order to engineer desired modifications within BAC clones.  The most widely used applications of this technology are to quickly build (1) targeting constructs that can be used for directing homologous recombination (“knockout”) manipulations in mouse ES cells; (2) reporter or “driver” constructs to express desired gene products in transgenic animals or cells; (3) deletion, insertion or point mutation engineering in BACs for sophisticated gene expression/function studies.   

For many typical projects we can offer a complete BAC engineering service, from assistance in vector design and engineering strategy to building and validating the construct.  In addition to BAC recombineering services we offer BAC DNA prep services (column-based purification), and consultation for BAC-based project design/execution.  

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."