DOI: 10.25549/usctheses-c127-643431
title: Development of lentiviral vector mediated gene delivery techniques and stable cell lines for investigating the action of ethanol and other allosteric modulators of glycine and GABAA receptors

description: This thesis has two Aims.  Aim 1 was to develop lentiviral vector mediated gene delivery techniques for expressing glycine receptors (GlyRs) and gamma (γ)-aminobutyric acid (GABA) receptors (GABAARs) in neurons.  Recent studies in our laboratory identified Loop 2 of the extracellular domain of the GlyRs and GABAARs as key targets for the initial actions of ethanol.  These studies also demonstrated that mutations of Loop 2 can produce GlyRs and GABAARs that are supersensitive to ethanol.  This work suggests the exciting possibility that structural modifications of Loop 2 in GlyRs and GABAARs might be used to markedly increase the ethanol sensitivity in target receptor populations in transgenic animals. These mutant receptors could provide the basis for measuring the effects of ethanol on sensitized receptors in which overexpression of high ethanol sensitive mutant receptors in neurons will enable us to see the effects of ethanol on these receptors and the resultant behavioral cascades at very low concentrations that should not elicit responses from endogenous receptors.  However, these earlier studies were carried out in Xenopus oocytes and more recently in a mammalian cell line.  Neither of these models have endogenous GlyRs or GABAARs.  Therefore, we wanted to know whether overexpression of these mutant receptors in the complex neuronal environment will produce the desired changes in receptor sensitivity to ethanol when expressed in neurons. ❧ To address this concern, my project was to develop lentiviral techniques to encode genes of interest as a vector mediated gene transfer system for expressing GlyRs and GABAARs in neurons.  To do this we used HIV-1 based pLVX-IRES-ZsGreen1 vector as the backbone for cloning the GlyR WT, GlyR with mutated Loop 2 (δl2) and mutant gamma (γ) GABAAR (γGABAAR - δl2) mutant gene inserts.  We demonstrated successful transduction using fluorescence microscopy. Initial follow-up studies using patch clamp electrophysiology supported the conclusion that the supersensitive mutant GlyRs were expressed and were functional in neurons.  Hence, these studies provided proof of concept that supersensitive GlyRs can be overexpressed in neurons and will result in supersensitive GlyRs in this preparation. ❧ Aim 2 was to develop stable cell lines expressing α5β3γ2L and α1β2γ2L GABAARs in a mammalian cell line for testing libraries of compounds to determine their selectivity as α5 GABAA receptor inverse agonists within an academic setting.  The α5 subtype selective GABAAR agents represent a favorable approach in the treatment of cognitive disorder such as Alzheimer’s disease, autism and other learning and memory related disorders. ❧ Stably transfected cell lines demonstrate longevity of gene expression without batch variation.  The findings presented under this section demonstrate the step wise procedures that were performed and optimized to generate stable cell lines.  We implemented subsequent sequential transfections methodology, followed by antibiotic selection of transfected clones and propagation of stable clones.  This resulted in successful generation of stable cell lines expressing two (out of three) combination subunit receptors. GFP-Fluorescence microscopy confirmed positive transfections of genes of interest.  Generation of stable cell lines expressing the α5β3γ2L and α1β2γ2L GABAARs are in progress.  Once these are developed, they could be used as a tool for screening the selectivity of currently available GABAAR compounds as well as for the development of novel subtype selective agents that potentiate this receptor subtype.  Hence, these studies lay out the developmental strategy that could also be applied to create stable cell lines for other genes for future experimental purpose.