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Abstract

Prion Protein (PrP) Targeted for Silencing by RNAi

Amanda Kowalski*, Mike Peoples, Sarah Canterberry, Kim Tessanne, Charles Long

Reproductive Sciences Laboratory, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX 77843

 

Abstract

Objective: The specific aim of this project is to test the feasibility of utilizing a recombinant lentiviral vector system to produce transgenic bovine fetuses encoding short hairpin RNAs (shRNAs) targeting the prion protein (PrP). 

Sample population: Bovine fetal fibroblasts.

Procedure: We are specifically using short hairpin RNA (shRNA) technology as a method of RNAi.  To test our hypothesis, four shRNA sequences specific to caprine and bovine PrP were found after analysis with the computer algorithm specific for shRNAs on the RNAi Central (Hannon lab) website.  We inserted the shRNA into a lentiviral dsRed expression vector and transformed into bacterial cells to amplify.  Next, we will test the relative knockdown of PrP via a dual vector expression system.  The coding region of bovine PrP was inserted downstream of the Luciferase reporter protein in the PGL3 vector.  The PGL3 vector will be co-transfected with a vector containing a PrP specific shRNA.  Relative luciferase activity will be measured and the shRNA that has statistically greater than 90% knockdown (ie >90 decreased relative luciferase activity) will be used in the near future to create transgenic PrP knockdown bovine fetuses.

Results: 293T cells were successfully transfected with the correct sequences of DNA via plasmid.  Fibroblast fluorescence from infection is pending.

Conclusion: It appears through this experiment that RNAi can be used to knock down PrP from our careful analysis of the transfection potential of several DNA sequences; however, conclusive evidence is pending.