New Approaches for Exploring Functional Genomics in Livestock

NON-TECHNICAL SUMMARY: In recent years, genetic engineering of animals has begun to play a major role in society and will undoubtedly continue to do so, mainly to develop new products that could benefit agriculture, animal and human health. Thus far, the great majority of GM animals have involved mice being produced for utilization in biomedical research. However, considering the benefits, the application of the technology to produce GM animals representing species other than mice is imminent once adequate research and improvement has been achieved. This past year, the first product approved for treating human disease, an anticlotting drug produced in the milk of transgenic goats (Antithrombin III) was introduced into the European market by Genzyme Biotherapeutics LLC, and is expected to be available in the US within the coming year. The market for this single product alone has been estimated at more than $200 million per year. Several other proteins/pharmaceuticals produced in the milk of transgenic livestock are currently in clinical trials. It is anticipated that other products including nutriceuticals and improved food e.g. low fat milk, lean meat, fortified milk and meat, and many others will someday be produced from GM animals. GM livestock will someday be produced that are resistant to parasites and numerous diseases. These will help reduce the need for pesticides and thwart many concerns involving bioterrorism. The development and application of GM animals will someday represent billions of dollars in the economy including the creation of hundreds of thousands of new jobs. At this point, the potential benefits of GM animals for society are limited only to man's imagination. Although the potential benefits of genetic engineering livestock are enormous, methods utilized to produce GM livestock are inefficient and extremely expensive. The long term goal of the proposed research is to develop improved methods for producing GM livestock. More specifically, we will investigate the utilization of non-relicative lentiviral vectors for producing transgenic livestock. This approach is commonly employed for human gene therapy and shows great promise as an efficient tool for genetic engineering livestock.

<P>

APPROACH: 1) Nucleotide sequences essential for the expression, translation, and function of genes encoding FMDV, DNMT1, GDF8 and PrP will be identified. 2) Recombinant lentiviruses carrying genes encoding green fluorescent protein (GFP, or other molecular markers e.g. DS Red) and short hairpin RNAs (shRNAs) targeting these key gene sequences will be produced. 3) The lentiviruses will then be injected into the perivitelline space of in vitro matured bovine ova followed by in vitro fertilization and embryo culture. 4) Following 7 days of culture, blastocysts expressing GFP (indicative that they are transgenic and expressing the appropriate shRNA will be transferred into synchronized recipient cows for the production of transgenic fetuses. 5) Other embryos expressing GFP and a non-relevant hairpin, in addition to non-transgenic in vitro produced embryos will be produced and transferred to synchronized recipient cows to serve as controls. 6) Fetuses will be collected at 90 days of gestation and tissue samples obtained for analysis of gene and protein expression to compare treatment vs control groups. 7) Morphological measurements/observations will also be documented and recorded to address other pertinent questions e.g. Do the fetuses exhibit green fluorescence Do those in which myostatin was targeted for silencing express signs of increased muscle development 8) Once we have confirmed our ability to produce transgenic fetuses, additional transgenic embryos will be produced and transferred into synchronized recipient cows. Any pregnancies that are obtained will be allowed to go to term for the production of transgenic calves.