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.
School of Biological and Molecular Sciences, Oxford Brookes University, UK.
L A
LA
Smith
M
M
Amar
R J
RJ
Harvey
M G
MG
Darlison
F G
FG
Earley
D J
DJ
Beadle
L A
LA
King
I
I
Bermudez
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