Abstract
Transfection technology is an important tool in the investigation of gene function and the modulation of gene expression, thereby contributing to the advancement of basic cellular research, drug discovery, and target validation. Creation of the mutant cells through gene disruption and exogenous protein expression with noticeable phenotype like reporter genes are among other key applications. In this chapter, protocols for generating recombinant Leishmania expressing EGFP or EGFP-Luciferase and their applications are given in detail.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zahedifard F, Gholami E, Taheri T, Taslimi Y, Doustdari F et al (2014) Enhanced protective efficacy of nonpathogenic recombinant Leishmania tarentolae expressing cysteine proteinases combined with a sand fly salivary antigen. PLoS Negl Tropical Dis 8:e2751
Saljoughian N, Taheri T, Zahedifard F, Taslimi Y, Doustdari F et al (2013) Development of novel prime-boost strategies based on a tri-gene fusion recombinant L. tarentolae vaccine against experimental murine visceral leishmaniasis. PLoS Negl Trop Dis 7:e2174
Doroud D, Zahedifard F, Vatanara A, Najafabadi AR, Taslimi Y et al (2011) Delivery of a cocktail DNA vaccine encoding cysteine proteinases type I, II and III with solid lipid nanoparticles potentiate protective immunity against Leishmania major infection. J Control Release 153:154–162
Roy G, Dumas C, Sereno D, Wu Y, Singh AK et al (2000) Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol Biochem Parasitol 110:195–206
LeBowitz JH, Coburn CM, McMahon-Pratt D, Beverley SM (1990) Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci U S A 87:9736–9740
Ha DS, Schwarz JK, Turco SJ, Beverley S (1996) Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 77:57–64
Misslitz A, Mottram JC, Overath P, Aebischer T (2000) Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol 107:251–261
Lye LF, Kang SO, Nosanchuk JD, Casadevall A, Beverley SM (2011) Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major. Mol Biochem Parasitol 175:58–67
Robinson KA, Beverley SM (2003) Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania. Mol Biochem Parasitol 128:217–228
Varela MRE, Lorena Muñoz D, Robledo SM, Kolli BK, Dutta S et al (2009) Leishmania (Viannia) panamensis: an in vitro assay using the expression of GFP for screening of antileishmanial drug. Exp Parasitol 122:134–139
Bolhassani A, Taheri T, Taslimi Y, Zamanilui S, Zahedifard F et al (2011) Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Exp Parasitol 127:637–645
Taheri T, Saberi Nik H, Seyed N, Doustdari F, Etemadzadeh M-H et al (2015) Generation of stable L. major +EGFP-LUC and simultaneous comparison between EGFP and luciferase sensitivity. Exp Parasitol 150:44–55
Beverley SM, Clayton CE (1993) Transfection of Leishmania and Trypanosoma brucei by electroporation. Methods Mol Biol 21:333–348
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Laboratory Press, Cold Spring Harbor, NY
Myler PJ, Fasel N (2007) Leishmania after the genome. Caister Academic Press, Norfolk, UK, p 306
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Taheri, T., Seyed, N., Rafati, S. (2016). DNA Integration in Leishmania Genome: An Application for Vaccine Development and Drug Screening. In: Thomas, S. (eds) Vaccine Design. Methods in Molecular Biology, vol 1403. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3387-7_34
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3387-7_34
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3385-3
Online ISBN: 978-1-4939-3387-7
eBook Packages: Springer Protocols