Efficient generation of transgenic mice by lentivirus‐mediated modification of spermatozoa

Abstract

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.

Lentiviral vector transduction of spermatozoa as a tool for the study of early development

“…3B). Similar results were also obtained on transduction of murine spermatozoa using lentiviruses displaying murine mbSCF [24].…”

Regulation and roles of Ca2+ stores in human sperm

“…2009 , Chandrashekran et al . 2014 a , b ). Regulation of sperm function is therefore dependent primarily on post-translational processes.…”

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

“…Compared to the first-and second-generation vectors, the third generation is safer and yields higher titers (Dull et al, 1998). Major applications of the third generation have been in generating transgenic animals, studying gene functions, modeling neurological diseases and developing therapeutic approaches and hepatocyte transplantation Chandrashekran et al, 2014;Li et al, 2012;Nguyen et al, 2009).…”