Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase.
Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.
Yao
Chaoqun
C
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA. chaoqun-yao@uiowa.edu
Luo
Jiwen
J
Hsiao
Chia-Hung Christine
CH
Donelson
John E
JE
Wilson
Mary E
ME
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D016054:DNA, Protozoan / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000502:physiology; D018314:Leishmania infantum / Q000166:cytology* / Q000187:drug effects / Q000235:genetics; D008647:Mesocricetus; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000502:physiology; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D014404:Tubulin / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
D016054:DNA, Protozoan; D012097:Repressor Proteins; D014404:Tubulin; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D013752:Tetracycline