Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21

✍ Scribed by Alexei I. Protopopov; Jinfeng Li; Gösta Winberg; Rinat Z. Gizatullin; Vladimir I. Kashuba; George Klein; Eugene R. Zabarovsky

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 310 KB
• Volume: 4
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.283

✦ Synopsis

Abstract

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.

Method

Two retroviral vectors pLNCtTA‐hCMV and pLNCtTA‐EF1α 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. Copyright © 2002 John Wiley & Sons, Ltd.