Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.
BACKGROUND
We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines.


METHODS
Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes.


RESULTS
The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells.


CONCLUSIONS
New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.
Microbiology and Tumor Biology Center, Karolinska Institute, 17177 Stockholm, Sweden. alepro@ki.se
Alexei I
AI
Protopopov
Jinfeng
J
Li
Gösta
G
Winberg
Rinat Z
RZ
Gizatullin
Vladimir I
VI
Kashuba
George
G
Klein
Eugene R
ER
Zabarovsky
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins; D002460:Cell Line; D002893:Chromosomes, Human, Pair 3; D005786:Gene Expression Regulation / Q000187:drug effects*; D016147:Genes, Tumor Suppressor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D016513:Mice, SCID; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology; D013752:Tetracycline / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C098416:MAP-kinase-activated kinase 3; D017346:Protein Serine-Threonine Kinases; D000070957:MutL Protein Homolog 1; D013752:Tetracycline