The long term goal of the proposed research is to produce transgenic animals which can be used as models for biomedical research by employing RNA interference to knock-down expression of specific genes. The ability to genetically engineer animals has proven to be an invaluable tool for biomedical research. Transgenic mice have been extremely useful for investigating human health issues including cancer, diabetes, cardiovascular disease, AIDS, kidney disease, and immunology, just to name a few. Other animal models are also important for research involving human health and welfare, as mice are not completely representative of human genetics and physiology. Valuable research leading to new information useful for developing treatments for (or the prevention of) human diseases can be derived from comparative studies involving the use of different animal species for biomedical research. Transgenic animals representing species other than mice would be extremely useful; unfortunately, the ability to produce transgenic animals other than mice has proven difficult and in many cases cost-prohibitive. In addition, the utilization of embryonic stem cells (ES cells) for gene targeting by homologous recombination to create models for human development and disease has thus far proven unsuccessful in species other than mice. In recent times, several exciting breakthroughs promise efficient methods to allow the production of transgenic animals representing a wide variety of different species. The utilization of lentiviral vectors to produce transgenic mice has proven to be extremely effective. We have recently demonstrated that lentiviral vectors can also be efficiently used to produce transgenic bovine embryos. In addition, techniques involving RNA interference (RNAi) have now been developed which allow silencing of specific genes by introducing short hairpin RNAs (shRNAs) into the genome, thus bypassing the need for homologous recombination. Finally, the utilization of recombinant lentiviral vectors in combination with techniques involving RNAi has now been successfully utilized to create transgenic mice in which specific genes were successfully targeted for silencing. These techniques should be applicable to a wide variety of mammalian species.
The specific aim of this proposal is to develop and utilize a lentiviral vector system to produce transgenic goats encoding shRNAs targeting myostatin (GDF 8). Single-cell embryos will be produced, and then infected with a recombinant lentiviral vector carrying gene constructs coding for the shRNAs targeting myostatin. Alternatively, a recombinant lentiviral vector will be used to deliver shRNAs targeting myostatin into caprine fibroblasts followed by nuclear transfer to produce cloned transgenic embryos in which myostatin has been targeted for silencing. Embryos will be transferred into synchronized recipient females for the production of transgenic offspring. The resulting offspring will be analyzed to determine if they are transgenic and exhibit the appropriate phenotype. We expect to produce transgenic goats in which expression of myostatin has been effectively silenced by RNAi. We anticipate these animals will exhibit increased muscle growth and mass when compared to controls. Success of this project will lead to techniques which can be applied to a wide variety of different animal species, greatly expanding their usefulness as models for research involving human health and disease.
|Tessanne, K; Golding, M C; Long, C R et al. (2012) Production of transgenic calves expressing an shRNA targeting myostatin. Mol Reprod Dev 79:176-85|
|Long, Charles R; Tessanne, Kimberly J; Golding, Michael C (2010) Applications of RNA interference-based gene silencing in animal agriculture. Reprod Fertil Dev 22:47-58|
|Golding, Michael C; Long, Charles R; Carmell, Michelle A et al. (2006) Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci U S A 103:5285-90|