Development of siRNA strategy to knockdown the regulatory contractile proteins in lymphatic muscle

Abstract

The aim of this study was to develop a fast and efficient method to screen short interfering RNA (siRNA) sequences and incorporate them into recombinant adenoviruses to specifically knockdown regulatory myosin light chain 20, smooth muscle α‐tropomyosin (TM) and cardiac troponin C (TnC) in lymphatic muscle. siRNA sequences targeting each specific gene were either custom‐designed or purchased directly from Dharmacon. We used lymphatic muscle cells (LMCs) derived from rat mesenteric or thoracic duct lymphatics to test the efficiency of the siRNA sequences to knockdown a specific gene. After 48‐72 hrs, quantitative Western blot analyses were performed on the siRNA transfected or control LMCs to verify the percentage of knockdown of the targeted genes. Results demonstrated that myosin light chain kinase and troponin C were knocked down more than 90% in LMCs using the specific siRNAs. siRNA targeted to α‐TM SM isoform decreased the level of α‐TM SM up to 50–60% in the LMCs. We optimized the required siRNA concentrations and the transfection time for each specific gene. The steps required for design of a hairpin siRNA construct containing the target siRNA sequences and the generation of adenoviruses will be discussed. Our data demonstrate a strategy to screen siRNA sequences targeted to specific genes, which may play an important role in regulating lymphatic contractility. AHA 09POST2280005 SC; NIH RO1HL80526 MM.