The Escherichia coli LexA repressor-operator system works in mammalian cells.
We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells. The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40. This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein. We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene. Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter. Necessary 3' signals were from the HSV tk gene. Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor. We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences.
Smith
G M
GM
Leicester University/ICI Joint Laboratory, School of Biological Sciences, University of Leicester, UK.
Mileham
K A
KA
Cooke
S E
SE
Woolston
S J
SJ
George
H K
HK
Charles
A D
AD
Brammar
W J
WJ
D000818:Animals; D001426:Bacterial Proteins / Q000502:physiology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D005822:Genetic Vectors; D008322:Mammals / Q000235:genetics; D051379:Mice; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology*; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein