Ribozyme-mediated reduction of the GABA(A) receptor (alpha)1 subunit

Citation
Jr. Subramaniam et al., Ribozyme-mediated reduction of the GABA(A) receptor (alpha)1 subunit, MOL BRAIN R, 92(1-2), 2001, pp. 149-156
Citations number
45
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Neurosciences & Behavoir
Journal title
MOLECULAR BRAIN RESEARCH
ISSN journal
0169-328X → ACNP
Volume
92
Issue
1-2
Year of publication
2001
Pages
149 - 156
Database
ISI
SICI code
0169-328X(20010815)92:1-2<149:RROTGR>2.0.ZU;2-U
Abstract
As an approach to understanding the role of the alpha1 subunit of the GABA( A) receptor, ribozymes were designed to reduce expression of this subunit p rotein by hydrolysis of cel subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all k nown murine GABA(A) receptor subunits at the amino acid and nucleotide sequ ence level. Two ribozymes were designed and synthesized: one against the ex tracellular domain and the other against the cytoplasmic domain. These ribo zymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro trans cribed mRNA. The stable cell line, 122, expressing recombinant human GABA(A ) alpha1, beta2 and gamma 2S subunits of receptor was stably transfected wi th the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55-60% reduction of alpha1 mRNA in clones of cells transf ected with either the single ribozyme (Cy) or with both ribozymes (EC). The alpha1 protein level was reduced 75% in a stable Cy clone and more than 90 % in a stable EC clone when compared with al expression in 122 cells and th e vector transfected (Zeo) cells. Electrophysiological analysis revealed th at the GABA(A) receptor properties were very similar in 122 cells and in st able clones in which the subunit protein expression had been greatly reduce d. No significant difference was detected in the potentiation of the recept or response by either bretazenil or zolpidem. These data demonstrate the ef ficacy of the ribozyme approach in dramatically reducing GABA(A) subunit pr otein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice. (C ) 2001 Elsevier Science BY All rights reserved.