Development of Leishmania Species Strains with Constitutive Expression of eGFP

Summary

Here, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene for eGFP as a stable integrated transgene using the pLEXSY system. Transfected parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity in both species were selected for further use in drug screening assays.

Abstract

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people worldwide. Infection with L. donovani can result in fatal visceral disease. In Panama, Colombia, and Costa Rica, L. panamensis is responsible for most of the reported cases of cutaneous and mucocutaneous leishmaniasis. Studying a large number of drug candidates with the methodologies available to date is quite difficult, given that they are very laborious for evaluating the activity of compounds against intracellular forms of the parasite or for performing in vivo assays. In this work, we describe the generation of L. panamensis and L. donovani strains with constitutive expression of the gene that encodes for an enhanced green fluorescent protein (eGFP) integrated into the locus that encodes for 18S rRNA (ssu). The gene encoding eGFP was obtained from a commercial vector and amplified by polymerase chain reaction (PCR) to enrich it and add restriction sites for the BglII and KpnI enzymes. The eGFP amplicon was isolated by agarose gel purification, digested with the enzymes BglII and KpnI, and ligated into the Leishmania expression vector pLEXSY-sat2.1 previously digested with the same set of enzymes. The expression vector with the cloned gene was propagated in E. coli, purified, and the presence of the insert was verified by colony PCR. The purified plasmid was linearized and used to transfect L. donovani and L. panamensis parasites. The integration of the gene was verified by PCR. The expression of the eGFP gene was evaluated by flow cytometry. Fluorescent parasites were cloned by limiting dilution, and clones with the highest fluorescence intensity were selected using flow cytometry.

Introduction

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with a wide range of clinical manifestations. This disease is prevalent in 98 countries, and its annual incidence is estimated at 0.9 to 1.6 million cases¹. Leishmania species that are pathogenic to humans are divided into two subgenera, namely L. (Leishmania) and L. (Viannia). Infection with some species belonging to the L. (Leishmania) subgenus, such as L. donovani and L. infantum, may result in visceral leishmaniasis (VL), which is fatal if left untreated²....

Protocol

To keep the samples sterile, all steps involving parasite culture should be performed inside a biosafety level 2 (BSL-2) hood or according to local health and safety regulations. A graphical summary of this protocol can be found in Figure 1.

Figure 1: Summary scheme of the pro....

Results

After building the pLEXSY-eGFP construct and transforming competent E. coli cells, colonies containing the construct with the eGFP insert will generate an approximately 859 bp product after running the colony PCR described in section 1.2 (Figure 3A). Total digestion of the purified plasmid from positive colonies using SwaI should give two characteristic fragments in gel electrophoresis, a 2.9 kbp fragment that is the portion of the PLEXSY vector containing all the necessary.......

Discussion

The advantages and disadvantages of various reporter genes have been studied in several protozoan parasites. Among them, GFP and eGFP are intrinsically fluorescent and allow easy quantification and imaging. The fluorescent activity of these proteins can be detected with minimal manipulation using fluorescence microscopy, fluorimetry, or flow cytometry. Few studies have been carried out for generating GFP-expressing L. (Viannia) strains, despite the demonstrated robustness of GFP and their derivatives as.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 Well Microplates	Corning	CLS3340	Flat bottom clear, black polystyrene, sterile, lid
Agarose	Sigma-Aldrich	A4718
Ampicillin sodium salt	Sigma-Aldrich	A8351	BioXtra, suitable for cell culture
BglII restriction enzyme	New England BioLabs	R0144S	2,000 units. 10,000 units/mL
Cell Culture Flasks	Corning	CLS430168	Surface area 25 cm2, canted neck, cap (plug seal)
ChemiDoc Imaging System	Bio-Rad	17001401
CyFlow Space	Sysmex	Not available
D-(+)-Glucose	Sigma-Aldrich	G7021	powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Fetal Bovine Serum	Sigma-Aldrich	F7524
Gel Loading Buffer	Sigma-Aldrich	G2526	The rate of migration varies with gel composition. Dilute 1:3 to 1:6 with sample before loading.
Gene Pulser Xcell Electroporation System	Bio-Rad	1652660	The system is composed of a main unit, two accessory modules, the capacitance extender (CE module) and the pulse controller (PC module), and a ShockPod cuvette chamber.
Gene Pulser/MicroPulser Electroporation Cuvettes	Bio-Rad	1652086	Pkg of 50, 0.2 cm–gap sterile electroporation cuvette, for use with the Gene Pulser and MicroPulser Systems, for mammalian and other eukaryotic cells
Gentamicin solution	Sigma-Aldrich	G1397	50 mg/mL in deionized water, liquid, 0.1 μm filtered, BioReagent, suitable for cell culture
GoTaq Long PCR Master Mix	Promega	M4021
HEPES solution	Sigma-Aldrich	H0887	1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture
Inverted microscope	Olympus	IXplore Standard
KpnI-HF restriction enzyme	New England BioLabs	R3142S	4,000 units. 20,000 units/mL
LB Broth with agar	Sigma-Aldrich	L3147	Highly-referenced nutrient-rich microbial growth powder medium with Agar, suitable for regular E.coli culture.
LB Broth	Sigma-Aldrich	L2542	Liquid microbial growth medium
Mini-Sub Cell GT Horizontal Electrophoresis System	Bio-Rad	1640300	Mini horizontal electrophoresis system, includes 8- and 15-well combs, 7 cm x 10 cm UV-transparent tray
pEGFP-N1-1x	Addgene	172281	Expressing eGFP mRNA fused with 1 tandem repeat of a 50-base sequence
pLEXSYcon2.1 expression kit	Jena Bioscience	EGE-1310sat	Contains integrative constitutive expression vector pLEXSY-sat2.1. Antibiotic selection of transfectants with Nourseothricin (NTC, clonNAT). Contains all primers for diagnostic PCRs and sequencing.
Potassium Chloride	Millipore	529552	Molecular Biology Grade - CAS 7447-40-7 - Calbiochem
PureYield Plasmid Miniprep System	Promega	A1222	Up to 15 μg of Transfection-Ready Plasmid from 3 mL cultures.
Schneider′s Insect Medium	Sigma-Aldrich	S0146	Medium used in our laboratory for culturing Leishmania.
SOC Medium	Sigma-Aldrich	S1797
Sodium chloride	Sigma-Aldrich	S3014	for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sodium phosphate dibasic	Sigma-Aldrich	RDD038	BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.0%, free-flowing, Redi-Dri
SwaI restriction enzyme	New England BioLabs	R0604S	2,000 units. 10,000 units/mL
Syringe filters	Corning	CLS431212	regenerated cellulose membrane, diam. 4 mm, pore size 0.2 μm
T100 Thermal Cycler	Bio-Rad	1861096	Thermal cycler system, includes 96-well thermal cycler, power cord, tube support ring
T4 DNA Ligase	Promega	M1801	Joins two DNA strands with cohesive or blunt ends
Tris-Borate-EDTA buffer	Sigma-Aldrich	T4415	BioReagent, suitable for electrophoresis, 10× concentrate
Wizard Genomic DNA Purification Kit	Promega	A1120
Wizard SV Gel and PCR Clean-Up System	Promega	A9285
XL10-Gold Ultracompetent Cells	Agilent	200317	XL10-Gold Kanr Ultracompetent Cells, 10 x 0.1 mL. Features the kanamycin-resistance gene on the F' episome, for extremely demanding cloning in chloramphenicol-resistant vectors. Efficiency: > 5 x 10 9 transformants/µg pUC18 DNA.