Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line.
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.
Ono
Tatsuya
T
Department of Integrated Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan.
Nishijima
Hitoshi
H
Adachi
Noritaka
N
Iiizumi
Susumu
S
Morohoshi
Akane
A
Koyama
Hideki
H
Shibahara
Kei-ichi
K
D015153:Blotting, Western; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D055786:Gene Knockout Techniques; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D011500:Protein Synthesis Inhibitors; D004265:DNA Helicases; D013752:Tetracycline