Analysis of endogenous S1P and LPA receptor expression in CHO-K1 cells.
The CHO-K1 cell line is commonly used for studies of recombinantly expressed proteins, including proteins of the G protein-coupled receptor (GPCR) family. This laboratory has used CHO-K1 cells for the functional characterization of Edg family GPCRs. However, parental CHO-K1 cells respond to lysophospholipids in in-vitro functional assays, which suggests expression of endogenous Edg family GPCRs. To determine the repertoire of Edg family receptor expression in this cell line, alignments of human and rodent sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) receptor sequences were used to design semi-redundant oligonucleotide pairs. A portion of each receptor gene coding sequence was amplified from Chinese hamster genomic DNA and the resultant gene fragments sequenced. Species-specific oligonucleotide pairs were designed using this novel sequence information and used to detect expression of S1P(1,2,4) and LPA(1) transcripts in CHO-K1 cells by RT-PCR.
Holdsworth
Gill
G
Biology Division, Celltech R and D Ltd., 216 Bath Road, Slough, Berkshire SL1 4EN, UK. Gill.Holdsworth@Celltechgroup.com
Slocombe
Patrick
P
Hutchinson
Gillian
G
Milligan
Graeme
G
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D049368:Receptors, Lysophosphatidic Acid / Q000235:genetics*; D049349:Receptors, Lysosphingolipid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D049349:Receptors, Lysosphingolipid