To maximize use of the reagents, requests for adenovirus constructions are tabulated and posted on our PPG server so that all members of the program are informed about their status and availability. The posting includes sufficient information on the expressed proteins to allow all PPG investigators to determine which ones could be useful in their own experiments. This important communication tool keeps members of the PPG continuously up to date on the activities of this core.
The facility is currently transitioning over nearly completely to the commercial method for producing recombinant adenovirus, AdEasy, since it uses clonal selection of the recombinant virus in bacteria and does not require plaque purification in the host 293 cell line. The core uses the pAdlox method only in the rare cases where the AdEasy method cannot be used (Ad Easy requires the use of a rare cutting restriction enzyme, Pac I, and its restriction base sequence cannot be present in the cDNA to be cloned).
The facility is also looking into new methods for expressing large proteins such as the cytoskeletal spectrins and the sarcoplasmic reticulum-associated ryanodine receptor using a "gutless" form of the virus and a helper virus. In addition, the constructions used for RNA interference (RNAi), is an important tool used to "knock-down" expression of candidate proteins. The constructions used for RNAi can be incorporated into adenovirus for delivery into cardiomyocytes and will be suited for addressing specific aims of all three projects.
The Molecular Genetics Core will also assist gentoypyiing mice with target deletions in line line that are developed by the PPG members and are acquired in conjunction with the PPG.