Targeted Silencing of RPE65 in the Retinal Pigment Epithelial Cell Line ARPE–19 Using Lentivirus–Mediated siRNA Delivery

Abstract

Abstract: : Purpose: The human RPE cell line, ARPE–19, has proven to be an advantageous cell model to study retinal pigment epithelium physiology because it retains many RPE–specific morphologic and biochemical markers. RPE65 protein, preferentially expressed in RPE cells, is required to maintain physiological functions of the RPE and retina. However, the role of RPE65 in RPE cells remain poorly understood. We generated siRNA–expressing recombinant lentiviruses to knockdown RPE65 expression in ARPE–19 cells and to evaluate its effect on the modulation of NF–ΚB and JNK signaling pathways. Methods: Two siRNA hairpin sequences targeting the human RPE65 gene were first subcloned into the pSUPER plasmid (Oligoengine) under the H1–RNA promoter (pH1). pH1–siRNA silencing cassette was then introduced into a lentiviral vector expressing GFP as a marker gene. Empty lentiviral construct, only expressing GFP, was used as control. Constructs were confirmed by sequencing and lentivirus stocks were prepared. ARPE–19 cells were transduced with virus particles containing control– and RPE65 siRNA– constructs at an MOI of 10. The decrease in RPE65 level was assessed by real–time RT–PCR. Modulation of the transcription factor NF–ΚB was assessed by western blot and immunofluorescence and JNK activity by measuring phosphorylated GST–c–Jun substrate in a kinase assay. Results: Efficient and stable expression of the integrated target sequences was observed in ARPE–19 cells, with 90 to 100 % of the transduced cells expressing GFP. Quantitative RT–PCR experiments showed siRNA–mediated reduction of RPE65 gene expression by 70 %. NF–ΚB protein level was not changed between control– and siRNA–expressing ARPE–19 cells, as observed in western blot experiments. As well, immunofluorescence studies did not show any changes in NF–ΚB activity as reflected by the absence of nuclear translocation of the transcription factor. Downregulation of RPE65 in retinal epithelial cells did not mediate the activation of c–Jun terminal kinase. Conclusions: Lentiviral–mediated expression of target siRNA leads to efficient knockdown of human RPE65. Downregulation of RPE65 does not activate intracellular stress responses dependent on either NF–ΚB or JNK pathways. ARPE–19 cells stably expressing RPE65 siRNA represent a valuable cellular model for detailed studies of RPE65–dependent retinal epithelium functions

Keywords: retinal pigment epithelium • gene modifiers • signal transduction