Journal:

Article Title: Helper-Free Foamy Virus Vectors

doi: 10.1089/hum.1998.9.17-2517

Figure Lengend Snippet: Time course of vector production by transient transfection of 293T cells. Three independent transfections were performed with plasmids pCGPES and pCGPMAPΔBel, and at 24, 36, 48, 56, 72, and 120 hr 100 μ l of vector-containing medium was removed from each well and frozen at −80°C until assayed. Samples were then thawed together at room temperature and titered on FAB cells. Controls of cells transfected with vector plasmid alone were negative (<1 AP FFU/ml). Values shown are titers (AP FFU/ml), with each symbol type representing a different transfection.

Article Snippet: The cell lines used included human embryonic kidney 293 cells (ATCC CRL-1573), simian virus 40 (SV40) T antigen-transformed 293T cells ( DuBridge et al ., 1987 ), NIH 3T3 TK − mouse fibroblasts ( Wei et al ., 1981 ), human fibrosarcoma HT-1080 cells ( Rasheed et al ., 1974 ), SV40-transformed African green monkey kidney COS-1 cells (ATCC CRL-1650), baby hamster kidney BHK-21 cells ( Macpherson and Stoker, 1962 ), normal human foreskin fibroblasts ( Palmer et al ., 1987 ), and FAB hamster HFV β -galactosidase ( β -Gal) indicator cells ( Yu and Linial, 1993 ).

Techniques: Plasmid Preparation, Transfection

Journal:

Article Title: Helper-Free Foamy Virus Vectors

doi: 10.1089/hum.1998.9.17-2517

Figure Lengend Snippet: Comparison of vector harvest methods. 293T cells were transfected with both the helper construct pCGPES and vector construct pCGPMAPΔBel, and 72 hr later supernatants (vector-containing medium) or suspensions of cells and supernatants were harvested as follows and titered on FAB cells. Harvest conditions included supernatant filtered without freezing (SUP); filtered supernatant that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T); filtered supernatant with glycerol added to 40% (v/v) that was frozen at −80°C for 30 min, then thawed at room temperature (SUP 1×F/T GLYC); supernatant combined with a suspension of the transfected cells that was frozen at −80°C for 30 min, thawed at room temperature, then filtered (SUSP 1×F/T); or supernatant combined with a suspension of the transfected cells that was frozen in an ethanol–dry ice bath and thawed at 37°C three times, then filtered (SUSP 3×F/T). Controls of untransfected cells or cells transfected with vector plasmid pCGPMAPΔBel alone produced no AP FFU (<1/ml). Supernatant from a control well transfected with the same transfection mixture used for all treatments was tested by FAB assay and did not contain Bel-dependent RCR (<1 BFFU/ml). Mean titer values (AP FFU/ml) with standard errors from three independent measurements are plotted. Values in parentheses indicate the fold reduction as compared with unfrozen supernatants.

Article Snippet: The cell lines used included human embryonic kidney 293 cells (ATCC CRL-1573), simian virus 40 (SV40) T antigen-transformed 293T cells ( DuBridge et al ., 1987 ), NIH 3T3 TK − mouse fibroblasts ( Wei et al ., 1981 ), human fibrosarcoma HT-1080 cells ( Rasheed et al ., 1974 ), SV40-transformed African green monkey kidney COS-1 cells (ATCC CRL-1650), baby hamster kidney BHK-21 cells ( Macpherson and Stoker, 1962 ), normal human foreskin fibroblasts ( Palmer et al ., 1987 ), and FAB hamster HFV β -galactosidase ( β -Gal) indicator cells ( Yu and Linial, 1993 ).

Techniques: Plasmid Preparation, Transfection, Construct, Produced