Figure 1

Estimating the proportion of successfully transduced cells. a. Significant transfer of plasmid occurred in the transfection – transduction process. Cos-1 cells were transfected with HVP without any helper plasmid using the calcium/BBS method. The supernatant was used to mock transduce Jurkat cells. LTR could be detected by PCR using primers NI2F and NI2R (see Methods) for at least four weeks after mock transduction. Control reactions were performed. DNA from untransduced Jurkat cells (J) and transduced, puromycin selected cells (S) was used as negative and positive extraction controls respectively. Reactions were also performed on HVP plasmid (P) or water (W) as positive and negative controls respectively. b. The method of transfection affects the extent to which passive transfer of plasmid occurs. The experiment was conducted as in (a), transfection was performed by the DEAE dextran method instead of the calcium/BBS method. LTR could be detected by PCR one week after mock transduction, but was no longer evident in the second week. Control reactions were performed, labelled as in (a). c. The ampicillin resistance gene is a target that can be used to detect passively transferred plasmid. A simplified, linearised diagram of the plasmid HVP is shown. The ampicillin resistance gene can be amplified by PCR (highlighted in red) using primers AmpF and Amp R (see Methods) to detect passively transferred plasmid, as it is distinct from the region coding for the viral vector, from which transcribed RNA could be packaged. d. Plasmid persists in transduced cells for at least three weeks. DNA was extracted from transduced cells. PCR for the ampicillin resistance gene was performed. A faint band can just be appreciated at three weeks post transduction.