Quantificazione differenziale dell’uptake di diversi costrutti episomali in spermatozoi suini.

In the last twenty years many acknowledgements were conceived about Sperm Mediated Gene Transfer (1), but it is whether to define differential construct uptake on sperm membrane, in order to operate efficiently with different exogenous DNAs. The aim of this study was to develop a simple and reliable method for differential uptake quantification during the co-incubation phase between exogenous DNA and spermatozoa. We recurred to construct specific-fluorophores (Cyanine 3, 3.5 and 5) and spectrophotometric readings at every time point. Experimental data were then validated through Real Time PCR: both the assays showed clearly that there are no preferences in construct uptake by sperm membrane, and moreover, that most of the exogenous DNA (>70% for each construct) was seized from the solution by the spermatozoa.