6 LENTIVIRAL TRANSGENESIS IN MICE AND NONHUMAN PRIMATES

Abstract

Transgenic technology is a powerful tool for investigating gene function and regulation in species ranging from fly to higher primates. The role of transgenic animal modeling has become more prominent in biomedical research; therefore, a highly efficient method for producing transgenic animals is critical for the advancement of animal modeling of genetic disorders, especially in species with limited access such as nonhuman primates. Lentiviral transgenesis is one of most efficient methods in generating transgenic animals, and has been applied in different species including mice, rats, pigs, and cattle. Here we evaluated lentiviral transgenesis by an in-depth investigation on the effect of gene construct and the method of viral delivery in mice; thus the prospect of creating transgenic nonhuman primates can be assessed. Lentiviruses carrying 25 different gene constructs in the same viral backbone were created and microinjected into the cytoplasm or the perivitelline space (PVS) of mouse zygotes; these zygotes were then compared to those subjected to the traditional pronuclear injection (PI) method. Embryo development was not affected by PVS, whereas intracytoplasmic injection produced a mild effect on embryo development, which was dependent on the manipulation skill. We found that intracytoplasmic injection of lentivirus had the highest transgenic rate (weaned pups) of approximately 54.22% (199/367), whereas the transgenic rate using PVS injection was 40.74% (22/54). However, the transgenic rate of PI was only 9.09% (4/44), which was significantly lower than the other two methods. Germline transmission was confirmed in over 90% of the transgenic lines produced by lentiviral gene transfer. In addition to the effect due to gene delivery method, variations in gene transfer efficiency were also observed when lentiviruses with different constructs were used. Our interest was to translate the lentiviral gene transfer technique into nonhuman primates for the development of a model for human disease. We evaluated the in vitro developmental rate of Rhesus macaques embryos that were microinjected into the PVS with lentiviruses carrying mutant genes leading to neurodegenerative diseases. The blastocyst rate of the lentivirus injection group (26%; 62/238) was not different from that of the control (25%; 13/52), which was without lentivirus injection. This indicates the feasibility of applying the lentiviral gene transfer technique to nonhuman primates. We carried out embryo transfers to surrogate female monkeys; however, the confirmation of pregnancy and the success of a developing nonhuman primate model of human disease were not available at the time of this writing. Here we demonstrate that lentiviral transgensis by cytoplasmic injection or PVS injection is a promising method to generate transgenic animals at high efficiency, and is superior to the traditional methods. Thus the production of a nonhuman primate model of human genetic diseases is foreseeable, and will have a significant impact on transgenic animal modeling as well as the advancement of biomedicine.

This work was supported by the NCRR/NIH.