Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals.
Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.
Wyborski
D L
DL
Stratagene, La Jolla, CA 92037, USA. Denise_Wyborski@Stratagene.com
DuCoeur
L C
LC
Short
J M
JM
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D005057:Eukaryotic Cells; D005260:Female; D005333:Fetus / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D008297:Male; D008745:Methylation; D008756:Methylgalactosides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics*; D013862:Thiogalactosides / Q000494:pharmacology; D013997:Time Factors; D014018:Tissue Distribution
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D008756:Methylgalactosides; D011994:Recombinant Proteins; D012097:Repressor Proteins; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase