Guidelines for Submitting Constructs for the Molecular Biology Core (Core D)

To Create Recombinant Adenoviruses

1. In order for the Core to create viruses, a pShuttle vector harboring the gene of interest is expected to be submitted
1. In general, creation of a recombinant adenovirus requires 3 major steps.  First, construction of pShuttle vectors containing the gene of interest for homologous recombination in bacteria.  Secondly, generation of recombinant adeoviral plasmids by homologous recombination in E.coli.(BJ5183).  And finally, production of adenoviruses in HEK293 cells.
2. A gene of interest is inserted into pShuttle CMV vectors or pShuttle vectors at the multiple cloning sites (MCS, see the map of vectors for detail) by following conventional molecular cloning procedures.  Please submit your plasmid at a known DNA concentration from 0.5 to 1 ug/ul.
3. Molecular Biology Core provides the pShuttle CMV or pShuttle vectors as well as the foreword and reverse primers used for pShuttle vectors sequencing and PCR.
4. Make sure that your DNA insert does not contain either Pac1 or Pme1 restriction site because these two sites exist in pShuttle vectors and are needed for viral construction. If your insert DNA does contain these sites, it will be necessary to perform site-directed mutagenesis before proceeding to the cloning steps.

1. Evidence of the quality of the plasmid submitted
1. To minimize invalid effort in the subsequent steps of viral creation, the Core expects you to submit the following evidence to show that your pShuttle contains a valid gene of interest:

• PCR and/or restriction enzyme digestion results that prove that the gene of interest is correctly inserted into the MCS of the pShuttle.
• It is highly recommended to verify your insert in the pShuttle by sequencing at least the junction between vector and insert.
• A western blot must be submitted along with your pShuttle plasmid to show that the pShuttle harboring your gene of interest expresses the protein expected by transfecting a cell line (e.g. 293 cells) with your pShuttle plasmid.  Molecular weight markers and negative controls must be included on the same western.

For more information contact Dr. Qiangrong Liang at qliang@usd.edu

                                           Or Tricia Krueger (RA) at takruege@usd.edu