Leishmania chagasi: A tetracycline-inducible cell line driven by T7 RNA polymerase

Chaoqun Yao; Jiwen Luo; Chia-Hung Christine Hsiao; John E Donelson; Mary E Wilson

doi:10.1016/j.exppara.2007.01.001

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Leishmania chagasi: A tetracycline-inducible cell line driven by T7 RNA polymerase

Journal article

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Leishmania chagasi: A tetracycline-inducible cell line driven by T7 RNA polymerase

Chaoqun Yao, Jiwen Luo, Chia-Hung Christine Hsiao, John E Donelson and Mary E Wilson

Experimental parasitology, Vol.116(3), pp.205-213

2007

DOI: 10.1016/j.exppara.2007.01.001

PMCID: PMC2231517

PMID: 17320870

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https://www.ncbi.nlm.nih.gov/pmc/articles/2231517View

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Abstract

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 α- 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.

Operator

α- TUB

Tetracycline induction

RNA interference

base pair

TETR

hygromycin phosphotransferase

RNAP

HPT

tetracycline repressor

β-galactosidase

NPT

neomycin phosphotransferase

P T7

LUC

enhanced green fluorescence protein

polymerase chain reaction

major surface protease

RNAi

eGFP

puromycin N-acetyl transferase

α-tubulin

RNA polymerase

MSP

βGAL

T7t

T7 RNAP terminator

PAC

Trypanosomatid

Leishmania

intercoding region

luciferase

tetracycline operator

Repressor

O tet

T7RNAP promoter

PCR

Details

Title: Subtitle: Leishmania chagasi: A tetracycline-inducible cell line driven by T7 RNA polymerase
Creators: Chaoqun Yao - Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
Jiwen Luo - Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
Chia-Hung Christine Hsiao - Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA
John E Donelson - Program in Molecular Biology, University of Iowa, Iowa City, IA 52242, USA
Mary E Wilson - Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Experimental parasitology, Vol.116(3), pp.205-213
DOI: 10.1016/j.exppara.2007.01.001
PMID: 17320870
PMCID: PMC2231517
NLM abbreviation: Exp Parasitol
ISSN: 0014-4894
eISSN: 1090-2449
Publisher: Elsevier Inc
Language: English
Date published: 2007
Academic Unit: Microbiology and Immunology; International Programs; Epidemiology; Biochemistry and Molecular Biology; Internal Medicine
Record Identifier: 9984001207902771

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