Description (including epitope tag if present):
The initial construct consisted of human cDNA for NFATc1 fused to GFP and cloned into the multiple cloning site of pAC-CMV-pLpA. The resulting plasmid was cotransfected into HEK293 cells with plasmid pJM17 which contains the Ad5 genome. Homologous recombination between the two plasmids resulted in the replacement of the Ad5 early region 1 with the mouse cDNA for NFATc1-GFP, generating a replication deficient recombinant virus.
Source:
http://www.med.umich.edu/vcore/plasmids/pACCMVpLpA%28-%29loxP-SSP.htm- The adenoviral vector is currently commercially available through Seven Hills Bioreagents:
http://www.sevenhillsbioreagents.com/inc/sdetail/612
How used:
Produces a strong GFP signal 18-36 hour post-transfection in isolated acinar cells (see "Imaging the dynamic subcellular localization of flourescently-tagged transcription factors in isolated acinar cells: NFATc1-GFP as a proof-of-princle" in the methods section of Pancreapedia). We were also able to establish transfection efficiency sufficient for imaging in acinar and non-acinar derived secondary cell culture including AR42J, 266-6, NIH-3T3, NIH-3T3 CCK-AR, CHO and HEK293.
Publications describing the vector and usage:
- Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo.Gurda GT, Guo L, Lee SH, Molkentin JD, Williams JA. Mol Biol Cell. 2008 Jan;19(1):198-206.