Human gene therapy vol:19 issue:10 pages:1196-1196
European Society of Gene & Cell Therapy location:Brugge, Belgium date:13-16 November 2008
The ability to introduce and express exogenous genes of interest or to knock down the expression of specific genes in animals has become the method of choice to develop novel (gene) therapeutic strategies and to model human diseases like Parkinson’s disease. One approach to generate transgenic animals is to use HIV1 - derived lentiviral vectors (LV) as gene delivery vehicles because they are able to stably integrate into the genome. This technique is especially useful to create non-murine transgenic animals. To evaluate the feasibility and efficiency of different LV-based methods, we generated transgenic mice and rats carrying the green fluorescent protein (eGFP) gene driven by a ubiquitously expressing CMV promoter.
In this study we compared two techniques to create transgenic rodent animals using lentiviral vectors. First, transgenic mice were generated by transduction of preimplantation embryos at morula stage with high titer eGFP lentiviral vectors. 24 hours after transduction, eGFP positive blastocysts were transferred into the uteri of pseudopregnant females. Secondly, transgenic rats were produced via injection of lentiviral vectors into the perivitelline space of one-cell stage oocytes. The next day two-cell stage embryos were transferred into the oviduct of pseudopregnant females. Positive offspring was determined via fluorescence microscopy and via PCR on genomic DNA.
Results and Conclusion
Both techniques, morula transduction as well as subzonal LV injection of oocytes resulted in the generation of eGFP transgenic mice and rats, respectively. Depending on the vector titer, 15 to 50% of the offspring expressed the eGFP transgene as determined by genomic PCR. 75% of these positive founders had stable germline transmission. Although the efficiency is comparable for both methods, the litter size and the number of successful transfers was at least four-fold higher using the subzonal injection of LV. In addition, the latter technique can be used to generate higher transgenic animals (rats, monkey), which is an advantage over morula transduction.