Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish.
Effective transgenesis methods have been successfully employed in many organisms including zebrafish. However, accurate spatiotemporal control of transgene expression is still difficult to achieve. Here we describe a system for chemical-inducible gene expression and demonstrate its feasibility for generating transgenic driver lines in zebrafish. The key element of this system is a hybrid transcription factor engineered by fusion of the DNA-binding domain of the bacterial LexA repressor, a truncated ligand-binding domain of the human progesterone receptor, and the activation domain of the human NF-kappaB/p65 protein. This hybrid transcription factor (LexPR transactivator) binds to the synthetic steroid, mifepristone (RU-486), and functions in a ligand-dependent manner to induce expression of the gene(s) placed under the control of a synthetic operator-promoter sequence that harbors LexA binding sites. Transgene expression is strictly controlled and can be induced at any stage of the life cycle through administration of mifepristone in the water. To demonstrate the utility of this system, we generated stable transgenic lines which allow inducible tissue-specific expression of activated K-ras(V12). Combined with the Ac/Ds-mediated transgenesis, the LexPR expression system has many potential applications in the fields of genetics and biotechnology.
Emelyanov
Alexander
A
Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore.
Parinov
Serguei
S
D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015735:Mifepristone / Q000494:pharmacology*; D009030:Mosaicism / Q000187:drug effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D015735:Mifepristone; D016283:Proto-Oncogene Proteins p21(ras); D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C485184:enhanced green fluorescent protein