This service is for BAC-based assembly of gene targeting vectors for use in generating conditional alleles 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.  

For construction of conditional targeting vectors, typically the goal is to create a targeting vector with a single loxP site flanking a critical exon, and a “floxed” ES cell-selectable marker (e.g. Neo) on the other side.  This takes three total BAC recombineering steps.  Typically, a genomic fragment is retrieved from the BAC into a plasmid in the first step.  The second and third steps are used to insert the "orphan" loxP, and a loxP+FRT-flanked selection cassette such as neo, thus creating the final targeting vector that contains all components needed for gene targeting in ES cells.   This service typically involves three recombineering steps and takes 8 - 12 weeks.  For more complex targeting vectors, additional steps may be required at additional cost.

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."