Studies on lentiviral-mediated transgenesis

Transgenic animals are essential research tools, whether to address basic biological questions or to develop preclinical models of human diseases. Their generation through the injection of naked plasmid DNA into the male pronucleus of a fertilized oocyte has been the standard practice for almost 3 decades. However, this approach is invasive, largely limited to the mouse and results in low rates of transgene integration. Although the inefficiency of pronuclear injection can be overcome in small animals by high-throughput screening for transgene integration, this becomes economically more challenging in larger animals, such as sheep, pigs or cows, because of the extreme costs associated with this procedure ($60'000-$300'000). Thus, new strategies to enhance the production and the variety of transgenic animals would be an important asset. One such approach has emerged through the use of lentiviral vectors, a gene delivery system that can efficiently transfer and stably integrate its cargo into a wide variety of cell types from numerous species, and has been successfully applied to generate transgenic mice, rats, pigs and cows. The increasing use of lentivector-mediated transgenesis warrants a full analysis of its modalities. As a step towards this goal, the present study aimed at characterizing the genotypic features of transgenic mice generated by lentiviral infection of zygotes. First, as an indirect method to measure the kinetics of lentiviral integration in early embryos, we examined the rates of transmission of individual proviruses from G0 transgenic mice to their G1 progeny using a PCR-based technique specific for each integrant. The mean rate of transmission of 44% for individual proviruses suggests that vector integration was most often established between the post-S phase of the one-cell stage and pre-S phase of the two-cell stage. We then moved on to define proviral integration site selection in lentivector-generated transgenic mice. Using LAM-PCR-mediated amplification and sequencing of the host genome-provirus junctions, we found that the frequency of proviral integration inside a gene was 1.75- and 1.88-fold higher in transgenic mice and 3T3 fibroblasts, respectively, than expected from the distribution of annotated genes in the mouse genome (Pmice .03e 09; P3T3 5.55e 16). Moreover, integration was not influenced by the transcriptional orientation of the target gene and tended to favour the middle of transcribed regions. We also observed a subtle difference in the integration patterns of lentiviral vectors in 3T3 fibroblasts and transgenic mice, with the latter exhibiting a slightly higher frequency of integration into repeats. Although this difference was statistically not significant, it might reflect the elevated transcriptional activity of repetitive elements in preimplantation embryos. Since proviruses favour intragenic localization, we analyzed G2 mice homozygous for specific lentiviral integrants using PCR-based techniques and o