Transgenic mice have become a staple in the study of basic biology and disease, including Alzheimer's. Even so, the method predominantly used today to create these animals remains technically difficult, costly, and impractical in other animal species. A different approach published today in Sciencexpress, tomorrow in print, appears to overcome these limitations and may complement the current method, according to Carlos Lois, David Baltimore, and colleagues at the California Institute of Technology in Pasadena.
Currently, scientists inject tiny amounts of transgene directly into the pronucleus of the zygote, requiring micromanipulation expertise. Efforts to use certain retroviruses as vectors for transgenes have been stumped because retroviruses often become epigenetically silenced, i.e., not expressed, during early development (Jahner et al., 1982). Lois et al. tried lentiviruses, which are being used in some experimental gene therapy regimens, and found that they are indeed expressed in transgenic founder animals, as well as their progeny. The authors first created a lentivirus-based vector with the gene for green fluorescent protein (GFP) as the readout under the control of a ubiquitous promoter. After injecting picoliters of high-titer virus solution into the space between the zona pellucida and the embryo cell itself, the virus integrated into about 80 percent of the subsequently born founder pups; another 80 percent of those displayed green fluorescent paws, tails, and faces. All major tissues, including brain, expressed GFP. Progeny of the transgenic mice expressed GFP uniformly.
The authors also tried a simpler delivery method: they stripped the zona pellucida off the single-cell embryos and bathed them in virus solution. These "naked" embryos had a lower rate of implantation than the intact ones, but on the plus side, the method enabled the scientists to better control the number of integrated virus copies by adjusting the virus titer in the bath solution. It also obviates the need for tricky micromanipulation and might therefore make transgenic technology accessible to more laboratories, the authors state.
This method worked with two tissue-specific vector constructs: One using the myogenin promoter led to GFP expression specifically in skeletal muscle, and another one using the T cell-specific lck promoter caused GFP expression exclusively in the thymus.
Finally, the authors made transgenic rats. In one initial experiment, they found the virus in 13 of 22 founder pups, a rate far more efficient than previously possible.
This method is no panacea, the authors caution. It does make creating transgenic animals easier and, potentially, allows for the efficient generation of transgenic birds or even primates. (Unlike mice, APP-, PS1- or tau-transgenic primates, could perhaps be studied with modern PET and MRI imaging machines.) However, achieving sufficient viral titers and accommodating large transgenes can be difficult. Besides, lentiviruses cause chronic diseases in humans, raising the question whether it is safe in the long run to have numerous lentiviral constructs circulating among primate populations.—Gabrielle Strobel
- Jähner D, Stuhlmann H, Stewart CL, Harbers K, Löhler J, Simon I, Jaenisch R. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature. 1982 Aug 12;298(5875):623-8. PubMed.
- Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002 Feb 1;295(5556):868-72. PubMed.