Development of lentiviral vector mediated gene transfer techniques as tools to overexpress/knockdown P2X4 receptors in vitro

The current study focused on the development of recombinant lentiviral (rLV) mediated gene transfer techniques that would aid in testing the hypothesis that the purinergic P2X4 receptors (P2X4Rs) play an important role in behavioral effects of alcohol. Ethanol inhibits ATP-induced currents in P2X4Rs recombinantly expressed in oocytes and HEK293 cells. Building evidence suggests that P2X4Rs play a role in alcohol consumption. P2X4Rs are widely expressed in neurons and glial cells, however little is known about the contribution of neuronal and/or glial P2X4Rs in alcohol-induced behaviors. In order to study the action of ethanol on P2X4Rs in these cells, it is necessary to develop tools to over-express/knockdown the expression of the receptors. The recombinant rLV gene delivery techniques were developed to express P2X4Rs in neuronal and HEK293 cells (Aim 1); to generate HEK293 cell line stably expressing P2X4Rs (Aim 2) and to inhibit P2X4Rs in microglial cells (Aim 3).;To address this issue, Lentivirus encoding P2X4R-GFP fusion construct was produced and was used to transduce neuronal and HEK293 cells. Fluorescence microscopy and western immunoblotting results confirmed the successful transduction of the P2X4Rs in the cells. The lentivirus encoding the P2X4R-GFP was then used to generate a HEK293 cell line stably expressing the P2X4Rs. Fluorescence microscopy and patch clamp technology was used to observe the stable expression of the P2X4Rs in HEK293 cells over a period of 47 days. A rLV containing shRNA oligonucleotides targeting the P2X4Rs was generated. Microglial BV-2 cells were transduced with the recombinant lentivirus and the knockdown of P2X4Rs was tested using fluorescence imaging and western immunoblotting. rLV infection of BV-2 cells with two different shRNA oligonucleotides resulted in more than 50% reduction of P2X4R protein expression per shRNA tested. Infection of BV2 cells with the combination of the two rLV shRNA oligonucleotides almost completely inhibited the expression of P2X4Rs (upto 85 %). The results suggest that the recombinant rLV vector can be successfully used for efficient gene delivery of P2X4Rs in neuronal and HEK293 cells as well as for knocking down P2X4Rs in microglial cells. This work sets the stage for the use of rLV technology to investigate the physiological and cellular roles of neuronal and microglial P2X4Rs in ethanol-induced behaviors.