Localized replacement of GABAA receptor γ2 subunits with bicistronic viral vectors
-
1
Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary
Classical genetic methods used in neurobiology act slowly compared to the time scale at which neurons and neuronal circuits operate. However mutations affecting only the drug sensitivity of ion channels can be used for selective, reversible and fast manipulations. Here we applied lentivirus-mediated functional replacement of floxed, benzodiazepine insensitive point mutant GABAAR γ2 subunits (flox-γ2BIS) with wild type, benzodiazepine sensitive γ2 subunits (wt-γ2BS) to locally rescue the benzodiazepine sensitivity of GABAA receptors in targeted mouse CNS neurons. We compared three different strategies to co-express Cre-recombinase (Cre) and the wt-γ2BS subunit ensuring the reliable replacement of wt-γ2BS with the γ2BIS in all affected cells. Injecting a mixture of independent Cre and wt-γ2BS expressing lentiviruses in neocortex resulted in only partially overlapping expression of the Cre and the AU1-tagged wt-γ2BS as revealed by double immunofluorescent reactions. The bicistronic lentiviral construct containing IRES was expressed in very few cells and although co-expressed with Cre, the AU1-tagged wt-γ2BS showed cytoplasmic localization. More cortical cells expressed the bicistronic 2A peptide-containing constructs. Importantly, Cre was perfectly co-expressed with AU1-tagged wt-γ2BS targeted to plasma membrane of the infected cells. Our results demonstrate that the short 2A sequence based strategy is a reliable tool for achieving lentivirus-mediated GABAAR subunit replacement.
Keywords:
Molecular and cellular neurobiology,
Neuroscience
Conference:
13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011.
Presentation Type:
Abstract
Topic:
Molecular and cellular neurobiology
Citation:
Sümegi
M,
Lorincz
A and
Nusser
Z
(2011). Localized replacement of GABAA receptor γ2 subunits with bicistronic viral vectors.
Front. Neurosci.
Conference Abstract:
13th Conference of the Hungarian Neuroscience Society (MITT).
doi: 10.3389/conf.fnins.2011.84.00036
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
03 Mar 2011;
Published Online:
23 Mar 2011.
*
Correspondence:
Dr. M. Sümegi, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, sumegi@koki.hu