Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line

• Published in: Journal of biotechnology 2009-04, Vol.141 (1), p.1-7
• Main Authors: Ono, Tatsuya, Nishijima, Hitoshi, Adachi, Noritaka, Iiizumi, Susumu, Morohoshi, Akane, Koyama, Hideki, Shibahara, Kei-ichi
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Blotting, Western; Cell Line; Conditional gene knockout; DNA Helicases - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - drug effects; Gene knockout; Gene Knockout Techniques; Gene targeting; Humans; Promoter Regions, Genetic - genetics; Protein Synthesis Inhibitors - pharmacology; Tetracycline - pharmacology; Tetracycline-inducible gene expression system; Transcription, Genetic - drug effects
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2008.12.010

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled us to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a ‘tetracycline-inducible conditional gene knockout’ for the proliferation-associated SNF2-like gene ( PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function.