Establishment of stable melanoma cell line expressing a novel gene, jpk, using a tetracycline-controlled gene expression system.
Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.
Kim
Byung-Gyu
BG
Department of Anatomy and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Sodaemoongu Shinchondong 134, Seoul 120-752, Korea.
Cheng
Meang Sub
MS
Park
Hyoung Woo
HW
Kim
Myoung Hee
MH
D000818:Animals; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D008546:Melanoma, Experimental; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011691:Puromycin; D012313:RNA; D013752:Tetracycline