The production of a stably transformed insect cell line expressing an invertebrate GABAA receptor beta-subunit.
We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a beta-subunit of the Lymnaea stagnalis GABAA receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1. LGbeta1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.
Smith
L A
LA
School of Biological and Molecular Sciences, Oxford Brookes University, UK.
Amar
M
M
Harvey
R J
RJ
Darlison
M G
MG
Earley
F G
FG
Beadle
D J
DJ
King
L A
LA
Bermudez
I
I
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008195:Lymnaea / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014170:Transformation, Genetic
D018076:DNA, Complementary; D011963:Receptors, GABA-A