AbstractBackgroundLentiviral vectors allow stable gene transfer into nonreplicating cells and are increasingly used in clinical gene therapy approaches. Vectors derived from different origins can show distinct target cell transduction properties. Therefore, the construction of modern vector systems of different viral origin remains desirable. The generation of safe and efficient lentivirus‐derived transfer vectors by gradual enhancing cloning steps is a time‐consuming process that depends on the presence of suitable restriction sites. Multiple‐step cloning protocols also enhance the risk of acquisition of mutations or other genetic instabilities.MethodsWe constructed novel HIV‐2 and SIVsmmPBj‐derived transfer vectors by amplification of three essential segments of the viral genome [5′‐long terminal repeat (LTR), rev responsive element, ΔU3‐3′‐LTR] on the template of the lentiviral full‐length genome by a highly flexible three‐step fusion polymerase chain reaction approach. Further necessary vector elements, as well as a multiple cloning site, were included into the resulting vector by extension of the primer sequences. The respective vesicular stomatitis virus G pseudotyped lentiviral vector particles were generated and analysed.ResultsTwo novel transfer vectors of different lentiviral origin were successfully generated. Titers for the corresponding SIVsmmPBj‐ and HIV‐2‐derived vectors reached up to 9.9 × 107 transforming units (TU)/ml and 1.2 × 108 TU/ml, respectively. The specific capacity to transduce primary human monocytes was maintained in both newly‐generated vector systems.ConclusionsWe anticipate that this novel and fast way of generating any lentiviral transfer vector will improve the generation of such vectors. The HIV‐2‐ and SIVsmmPBj‐derived vectors described will prove valuable for future gene therapy strategies. Copyright © 2010 John Wiley & Sons, Ltd.