Production of Disease Resistance Livestock by RNA Interference

In addition to targeting CAEV we have also expanded our objectives to include targeting key gene sequences required for replication of the virus which causes Foot and Mouth disease and the endogenous prion protein gene (PrP). A mouse model has also been added to our work plan. Transgenic mice will be produced which express a gene coding for a shRNA which confers resistance to roto virus.

Genetic engineering farm animals to confer resistance to viral infections represents one of the most promising approaches for reducing the economic losses due to infectious disease of livestock. Very recent research involving the phenomenon of RNA interference indicates that technologies which take advantage of this evolutionary conserved process can be developed to generate transgenic animals resistant to a wide variety of different infectious diseases. In brief, when small hairpin RNAs (shRNAs) composed of gene sequences homologus to key gene sequences encoding the infectious agent are introduced into cells and/or embryos, these inhibit replication of the infectious agent by way of RNA interference. We propose to produce transgenic goats which encode shRNAs that target caprine arthritis encephalitis virus (CAEV), foot and mouth disease virus (FMDV), and prion protein (PrP). Once these transgenic animals have been produced they will be challenged/tested to determine if they are resistant to disease. We expect the results of this research to result in transgenic goats that are resistant to CAEV, FMD, and prion disease (scrapie).

The general approach to achieve our objectives is as follows: 1) Nucleotide sequences of the CAEV viral genome previously demonstrated essential for viral replication will be identified. shRNAs targeted at these key sequences will be delivered into established caprine cell lines via oligonucleotide delivery methods. The cells will then be challenged with CAEV and assayed to determine the effect of the shRNAs on viral replication. 2) Once we have identified candidate sequences that confer viral resistance in cell culture, gene constructs that code for these shRNAs will be generated and transfected into the genome of a caprine cell line. This will be achieved using a vector system already proven effective in our lab, and result in the production of a stable, transgenic cell line resistant to infection by CAEV. To confirm this, transgenic cells will be infected with CAEV and assayed to measure their response to viral infection. 3) Transgenic cell lines demonstrated to confer resistance to CAEV propagation will then be utilized for somatic cell nuclear transfer to produce cloned transgenic goats. 4) Transgenic goats will be challenged with CAEV and appropriate assays and control experiments performed to determine their response to CAEV infection. A similar approach will be utilized to target other genes of interest i.e. FMDV, PrP, and roto virus. In addition, we have recently adapted/developed an alternative technology involving the utilization of a lentiviral vector system to produce transgenics which may allow us to bypass the cloning step. In this case, recombinant lentiviral vectors containing genes encoding the shRNAs of interest will be injected into the perivitelline space of 1-cell goat (or mouse) embryos. Following injection of the recombinant lentiviral vectors, the embryos will be transferred into recipient females (surrogates) for the production of transgenic offspring expressing the shRNAs of interest.

The long term goal of this research project is to exploit the phenomenonon of RNA interference (RNAi) to develop technology for the production of genetically engineered livestock which are resistant to disease. The general approach is to construct recombinant lentiviral vectors capable of delivering genes encoding short hairpin RNAs (shRNAs) into the genome of livestock cells and embryos. Expression of these shRNAs in transgenic livestock should result in their phenotype being resistant to disease. The three diseases we are currently focused on include Scrapie (Prion disease) Foot and Mouth Disease (FMD) and CAEV, in goats. Most work this past year has focused on developing lentiviral vectors that can consistantly deliver genes encoding shRNAs into goat cell lines and embryos. In addition, given the tools we already had available to begin this work, most of our effort has focused on producing animals resistant to scrapie by targeting the prion protein (PrP). Based on previous studies conducted in mice, silencing this gene should result in genetically engineered animals resistant to scrapie. Thus far we have successfully utilized a lentiviral vector system to produce both goat cell lines and embryos that express green flourescent protein (GFP is used as a marker gene)and shRNAs targeting PrP. The vector system has not proven as consistent when injecting embryos. As such, much of our effort has involved the production of transgenic cell lines then using these (green cells are selected) for cloning to produce cloned transgenic goats which express GFP and the hairpin targeting PrP. We have now transferred cloned transgenic embryos into a number of recipient females (11), but to date have not obtained any pregnancies. We are currently in the process of reevaluating the cell line we are using to determine if there is a problem with the cell line in terms of cloning i.e. performing control experiments. In addition, we are continuing to explore construction of new and improved lentiviral vectors so we can infect embryos directly and avoid the need for cloning. Additional work has involved the design and initial construction of shRNAs that target CAEV.

The ability to produce genetically engineered livestock resistant to disease will have a dramatic impact on animal agriculture. Current approaches for prevention and treatment of livestock diseases are extremely expensive and represent billions of dollars in economic losses to the livestock industry each year. In addtion, livestock genetially engineered to be resistant to disease should prove an extremely beneficial deterrant to potential threats of bioterrorism where our livestock herd are the target.