Virabind adenovirus miniprep kit

The three rAds, namely rAd-amiRSn, rAd-amiRCD163 and rAd-amiRcon, were generated by transfecting AAV-293 cells (Stratagene, USA) with the rAd vectors according to the instruction manual for AdEasy™ Adenoviral Vector System (Agilent Technologies, USA). The rAds were amplified on AAV-293 cells, purified using ViraBind™ Adenovirus Miniprep Kit (CELL BIOLABS, USA) by following the manufacturer’s protocol, and titrated on AAV-293 cells as fluorescent formation units (FFU)/ml. For transduction of PK-15 cells, the cells were seeded in triplicates at 5 × 10⁴ cells/well on 24-well plates, and grown to 80% confluent growth. After two time wash with PBS, the cells were transduced (37°C for 2 h) with different doses (MOI) of rAds. The transduction of primary PAMs was carried out as previously described [33 (link)]. Briefly, the cells were cultured for 7 days in the growth medium conditioned with 20% of L-929 cell culture medium. The cells were trypsinized, diluted to 10⁵cells/ml with the same medium, mixed with different doses of rAds and centrifuged at 2000 × g for 1 h at 37°C. After wash again with PBS, the cells were grown for additional 48 h in the fresh medium and assayed for GFP-positive cells on BD FACSAia III or by fluorescent microscopy.

To determine whether expression of hDENND1A.V2 converts MA-10 cells to a PCOS phenotype, MA-10 were grown as previously described and were infected with 3 pfu/10⁶ cells of either null adenovirus or adenovirus expressing hDENND1A.V2. The MA-10 cells used in these studies were a generous gift from Dr. Mario Ascoli (University of Iowa). hDENND1A.V2 adenovirus (hDENND1A.V2-pADenoG) was constructed by Applied Biological Materials (Vancouver, BC, Canada), by cloning hDENND1A.V2 from pCMV6-XL4 plasmid encoding the hDENND1A.V2 into pADenoG, from Origene (Rockville, MD, USA). Control empty NULL non-expressing adenovirus (pAdenoG Null) was also obtained from Applied Biological Materials (Vancouver, BC, Canada). Recombinant adenoviruses were propagated and expanded in HEK293T cells, purified using a Virabind Adenovirus Miniprep Kit, Cell Biolabs, Inc (San Diego, CA, USA), and titered by QuickTiter Adenovirus Titer Elisa Kit, Cell Biolabs, Inc (San Diego, CA, USA). Both the DENND1A.V2 or control empty NULL non-expressing adenovirus (pAdenoG Null) were used to infect mouse Leydig MA-10 cells as we previously described [23 (link)].