Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by the E. coli lac repressor.
Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that the Escherichia coli ldc operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two synthetic lac operators. Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-beta-D-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of the E. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.
Izquierdo
R E
RE
Department of Medicine, Albany Medical College, New York 12208, USA.
Breese
K
K
Jain
S
S
Carestio
D
D
Jung
L
L
Figge
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D013006:Growth Hormone / Q000235:genetics*; D006801:Humans; D007763:Lac Operon; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D010957:Plasmids; D012097:Repressor Proteins / Q000494:pharmacology*; D014162:Transfection / Q000379:methods
D012097:Repressor Proteins; D013006:Growth Hormone; D015500:Chloramphenicol O-Acetyltransferase