Cloning and characterization of a retroviral plasmid, pCC1, for combination suicide gene therapy

• Published in: BioTechniques Vol. 28; no. 3; pp. 572 - 576
• Main Authors: Kuiper, M, Sanches, R, Gäken, J A, Bignon, Y J
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis Group 01-03-2000
• Subjects: Animals; Cloning, Molecular; Ganciclovir - pharmacology; Genetic Therapy; Interleukin-2 - biosynthesis; Plasmids; Rats; Retroviridae - genetics; Simplexvirus - enzymology; Thymidine Kinase - genetics
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/00283cr03

Introduction of the herpes simplex virus thymidine kinase (HSV-TK) gene into mammalian cells confers specific sensitivity to killing by the anti-herpes drug ganciclovir (GCV). This gene has therefore been used in a number of cancer gene therapy protocols as a therapeutic gene. However, the therapeutic efficacy of HSV-TK/GCV in cancer gene therapy experiments can be augmented by additional therapeutic genes. We have cloned a retroviral plasmid, pCC1, containing a fusion gene of HSV-TK and Sh-ble driven by an internal simian virus 40 early promoter. This gene encodes a fusion protein that confers GCV sensitivity and Zeocin resistance when introduced into mammalian cells. A multiple cloning site (MCS) allows the introduction of a second therapeutic gene under the transcriptional control of the Moloney murine leukemia virus 5' long terminal repeat. We have generated packaging cell lines electroporated with pCC1 or pCC1 rtIL-2 S (rat interleukin-2 gene cloned in the sense direction in the MCS), the supernatants of which transfer GCV sensitivity only, or both GCV sensitivity and rtIL-2 production, respectively to rat ovarian cancer cells. This plasmid may be useful for the study of combination suicide gene therapy strategies.