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The long-term goal of the proposed project is to exploit the phenomenon of RNA interference (RNAi) to produce livestock in which specific endogenous genes have been targeted for silencing. The goal of this project is to develop and test novel methods for genetically engineering cattle in which genes encoding either Prion Protein (PrP) or myostatin (GDF8) have been targeted for silencing by RNAi.<P>
The specific objectives of the proposed research are as follows: <ol>
<li> Develop reliable effective recombinant lentiviral vectors containing constructs coding for shRNAs targeting either PrP or GDF8 for silencing, <li> Develop efficient techniques for delivery of lentiviral vectors into early stage bovine embryos, resulting in infection, integration and expression of shRNAs targeting PrP or GDF8 for silencing, <li> Evaluate transgene integration and expression in bovine embryos produced in vitro prior to transfer to synchronized recipient cows for establishment of pregnancy, <li> Collect and analyze transgenic fetal tissues for quantitative assessment of shRNA integration, expression and effectiveness, using Southern blotting, quantitative Real Time PCR and Western blotting, respectively. </ol> Given our previous experiences and preliminary data we expect to produce transgenic bovine fetuses in which genes encoding myostatin and PrP have been effectively targeted for silencing by RNAi. More important, we anticipate this work to result in new, efficient, and powerful methods which can be used to study functional genomics in a wide variety of different livestock species.
NON-TECHNICAL SUMMARY: The ability to inactivate (knock out) specific genes in mice has provided an extremely powerful tool to study gene function in this species. Unfortunately the technology utilized to inactivate genes in mice is not readily adaptable to other species including livestock. RNA interference (RNAi) is a naturally occurring phenomenon that results in gene silencing. The purpose of this research is to develop new RNAi-based technologies that can be utilized to effectively and efficiently silence the expression of specific genes in livestock species.
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APPROACH: The approach of the proposed research is as follows: <ol> <li> Nucleotide sequences essential for the expression, translation, and function of genes encoding PrP and myostatin will be identified. <li> Recombinant lentiviral vectors carrying genes encoding green fluorescent protein (GFP) and short hairpin RNAs (shRNAs) targeting these key gene sequences will be produced. <li> The viral vectors will then be injected into the perivitelline space of in vitro matured bovine ova followed by in vitro fertilization and embryo culture. <li> Following 7 days of culture, blastocysts expressing GFP (indicative that they are transgenic and also expressing a shRNA targeting a specific gene) will be transferred into synchronized recipient cows to establish pregnancy. <li> Other embryos expressing only GFP or GFP and a nonsense shRNA will be produced and transferred to synchronized recipient cows to serve as controls. <li> Bovine fetuses will be collected from pregnant cows at 90 days of gestation. <li> Tissue samples will be collected from the fetuses for analysis and quantification of gene and protein expression. <li> Phenotypic measurements/observations of the resulting fetuses will also be documented and recorded.</ol> Given our previous experiences and preliminary data we expect to produce transgenic bovine fetuses in which genes encoding myostatin and PrP have been effectively targeted for silencing by RNAi. More important, we anticipate this work to result in new, efficient, and powerful methods which can be used to study functional genomics in a wide variety of different livestock species.