Transgenic technologies conventionally rely on the oocyte as a substrate for 
genetic modification. Owing to their accessibility, however, male germ cells, 
including mature sperm, have material advantages for use in transgenesis. Here 
we have exploited lentiviruses to generate transgenic animals via the male 
germline. When pseudotyped lentiviral vectors encoding green fluorescent protein 
(GFP) were incubated with mouse spermatozoa, these sperm were highly successful 
in producing transgenics. Lentivirally transduced mouse spermatozoa were used in 
in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥ 
42% of founders were found to be transgenic for GFP. Inverse PCR strategy for 
integration site analysis demonstrated integration of at least 1 or 2 copies of 
GFP in the transgenics, mapping to different chromosomes. GFP expression was 
detected in a wide range of murine tissues, including testis and the transgene 
was stably transmitted to a third generation of transgenic animals. This 
relatively simple, yet highly efficient, technique for generating transgenic 
animals by transducing spermatozoa with lentiviral vectors in vitro is a 
powerful tool for the study of fertilization/preimplantation development, 
vertical viral gene transmission, gene function and regulation, and epigenetic 
inheritance.