This work was funded by the Secretar&#237;a Nacional de Ciencia, Tecnolog&#237;a e Innovaci&#243;n (SENACYT), Panam&#225;, grant number NI-177-2016, and Sistema Nacional de Investigaci&#243;n (SNI), Panam&#225;, grant numbers SNI-169-2018, SNI-008-2022, and SNI-060-2022.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64939/64939fig01.jpg" /> 
Figure 1: Summary scheme of the protocol for generating...

<ol>
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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...

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. Species belonging to the L. (Viannia) subgenus are associated with most cases of cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) in Central and South America, particularly in Panama, Colombia, and Costa Rica, with L. panamensis being the main etiological agent of these clinical presentations3,4.
Existing anti-leishmania chemotherapy that includes drugs such as pentavalent antimonials, miltefosine, and amphotericin B is highly toxic and expensive. Furthermore, increased drug resistance during recent decades has been added to the factors that interfere with the effective treatment of patients worldwide5. Substantial differences have been demonstrated across species of the genus Leishmania in relation to drug susceptibility, especially between New and Old World species6,7. For these reasons, it is necessary to direct efforts to the identification and development of new anti-leishmania drugs, paying special attention to species-specific approaches. Studying large libraries of drug candidates with traditional methodologies is quite difficult, given that these methodologies are very laborious for evaluating the activity of compounds against intracellular amastigotes or for performing in vivo experiments8; therefore, it has been necessary to develop new techniques that reduce these disadvantages, including the implementation of reporter genes and development of high-content phenotypic screening assays9.
The use of reporter genes has shown the potential to increase the efficiency of the drug screening process as it facilitates the development of high-throughput and in vivo assays. Recombinant Leishmania parasites expressing several reporter genes have been generated by various research groups. Reporter genes, such as &#946;-galactosidase, &#946;-lactamase, and luciferase, have been introduced in several Leishmania species using episomal vectors, showing limited utility for drug screening in extra- and intra-cellular forms of the parasite10,11,12,13,14,15. These approaches have the limitation of requiring a strong selective pressure in culture to avoid the elimination of the episomal construct, as well as the use of additional reagents to reveal the activity of the reporter gene. Conversely, the green fluorescent protein (GFP) and its variant, the enhanced green fluorescent protein (eGFP), have been used in the generation of a large number of transgenic Leishmania strains for in vitro drug screening assays due to their flexibility and sensitivity, as well as the possibility of automating the screening process using flow cytometry or fluorometry15,16,17,18,19. Despite promising results, cultures of these transgenic strains were highly heterogeneous in their fluorescence levels, since the number of copies of the GFP gene was not the same in all the parasites. Furthermore, maintaining fluorescence required constant selective pressure on the parasites in culture, since the GFP gene was introduced in an episomal construct.
For the reasons stated before, many efforts have focused on developing new methodologies for producing stable recombinant strains. These efforts have mostly relied on the integration of reporter genes into ribosomal loci, taking advantage of the higher transcription rates of ribosomal genes20. Strains of L. infantum and L. amazonensis have been generated having integrated the genes coding for &#946;-galactocidase21, IFP 1.4, iRFP22, and tdTomato23, and they have been evaluated for their usefulness in drug screening assays. Various groups have developed L. donovani strains that express GFP constitutively by integrating its coding gene into the 18S ribosomal RNA locus (ssu locus) through homologous recombination24,25; they showed stable and homogeneous GFP expression in the transfected population, including intracellular amastigotes24,25, and they were successfully implemented in drug screening assays24,25,26. Bolhassani et al.27 developed strains of L. major and L. infantum expressing GFP as an integrated transgene. They used the integration vector pLEXSY, originally designed for the transgenic expression of proteins in a system using L. tarentolae as the host28. The pLEXSY-GFP vector has shown to be very efficient for the generation of different Leishmania strains constitutively expressing GFP24,25,27,29,30. In these parasites, fluorescence is homogeneous and maintained in the intracellular forms, being able to be detected in footpad lesions of infected mice27.
In this work, we describe the methodology used for generating L. panamensis and L. donovani strains expressing the gene encoding for eGFP as an integrated transgene using the pLEXSY system. The strains generated through this process are used in our laboratory for performing drug-screening assays that evaluate the potential anti-leishmania activity of molecules of natural and synthetic origin.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well Microplates</td>
			<td>Corning</td>
			<td>CLS3340</td>
			<td>Flat bottom clear, black polystyrene, sterile, lid</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A4718</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A8351</td>
			<td>BioXtra, suitable for cell culture</td>
		</tr>
		<tr>
			<td>BglII restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R0144S</td>
			<td>2,000 units. 10,000 units/mL</td>
		</tr>
		<tr>
			<td>Cell Culture Flasks</td>
			<td>Corning</td>
			<td>CLS430168</td>
			<td>Surface area 25 cm2, canted neck, cap (plug seal)</td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>Bio-Rad</td>
			<td>17001401</td>
			<td></td>
		</tr>
		<tr>
			<td>CyFlow Space</td>
			<td>Sysmex</td>
			<td>Not available</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td>powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>G2526</td>
			<td>&#160;The rate of migration varies with gel composition. Dilute 1:3 to 1:6 with sample before loading.</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporation System</td>
			<td>Bio-Rad</td>
			<td>1652660</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Gene Pulser/MicroPulser Electroporation Cuvettes</td>
			<td>Bio-Rad</td>
			<td>1652086</td>
			<td>Pkg of 50, 0.2 cm&#8211;gap sterile electroporation cuvette, for use with the Gene Pulser and MicroPulser Systems, for mammalian and other eukaryotic cells</td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>50 mg/mL in deionized water, liquid, 0.1 &#956;m filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>GoTaq Long PCR Master Mix</td>
			<td>Promega</td>
			<td>M4021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IXplore Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>KpnI-HF restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R3142S</td>
			<td>4,000 units. 20,000 units/mL</td>
		</tr>
		<tr>
			<td>LB Broth with agar</td>
			<td>Sigma-Aldrich</td>
			<td>L3147</td>
			<td>Highly-referenced nutrient-rich microbial growth powder medium with Agar, suitable for regular&#160;E.coli&#160;culture.</td>
		</tr>
		<tr>
			<td>LB Broth&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2542</td>
			<td>Liquid microbial growth medium</td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT Horizontal Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>1640300</td>
			<td>Mini horizontal electrophoresis system, includes 8- and 15-well combs, 7 cm x 10 cm UV-transparent tray</td>
		</tr>
		<tr>
			<td>pEGFP-N1-1x</td>
			<td>Addgene</td>
			<td>172281</td>
			<td>Expressing eGFP mRNA fused with 1 tandem repeat of a 50-base sequence</td>
		</tr>
		<tr>
			<td>pLEXSYcon2.1 expression kit</td>
			<td>Jena Bioscience</td>
			<td>EGE-1310sat</td>
			<td>Contains integrative constitutive expression vector pLEXSY-sat2.1. Antibiotic selection of transfectants with Nourseothricin (NTC, clonNAT). Contains all primers for diagnostic PCRs and sequencing.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Millipore</td>
			<td>529552</td>
			<td>Molecular Biology Grade - CAS 7447-40-7 - Calbiochem</td>
		</tr>
		<tr>
			<td>PureYield Plasmid Miniprep System</td>
			<td>Promega</td>
			<td>A1222</td>
			<td>Up to 15 &#956;g of Transfection-Ready Plasmid from 3 mL cultures.</td>
		</tr>
		<tr>
			<td>Schneider&#8242;s Insect Medium</td>
			<td>Sigma-Aldrich</td>
			<td>S0146</td>
			<td>Medium used in our laboratory for culturing Leishmania.</td>
		</tr>
		<tr>
			<td>SOC Medium</td>
			<td>Sigma-Aldrich</td>
			<td>S1797</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>for molecular biology, DNase, RNase, and protease, none detected, &#8805;99% (titration)</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>RDD038</td>
			<td>BioReagent, suitable for cell culture, suitable for insect cell culture, &#8805;99.0%, free-flowing, Redi-Dri</td>
		</tr>
		<tr>
			<td>SwaI restriction enzyme</td>
			<td>New England BioLabs</td>
			<td>R0604S</td>
			<td>2,000 units. 10,000 units/mL</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Corning</td>
			<td>CLS431212</td>
			<td>regenerated cellulose membrane, diam. 4&#160;mm, pore size 0.2&#160;&#956;m</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td>Thermal cycler system, includes 96-well thermal cycler, power cord, tube support ring</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>M1801</td>
			<td>Joins two DNA strands with cohesive or blunt ends</td>
		</tr>
		<tr>
			<td>Tris-Borate-EDTA buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T4415</td>
			<td>BioReagent, suitable for electrophoresis, 10&#215; concentrate</td>
		</tr>
		<tr>
			<td>Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9285</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold Ultracompetent Cells</td>
			<td>Agilent</td>
			<td>200317</td>
			<td>XL10-Gold Kanr Ultracompetent Cells, 10 x 0.1 mL. Features the kanamycin-resistance gene on the F&#39; episome, for extremely demanding cloning in chloramphenicol-resistant vectors. Efficiency: &#62; 5 x 10 9 transformants/&#181;g pUC18 DNA.</td>
		</tr>
	</tbody>
</table>

development of leishmania species strains with constitutive expression of egfp

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.

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...

Watch this Scientific Journal Video about Development of Leishmania Species Strains with Constitutive Expression of eGFP at JoVE.com

Development of Leishmania Species Strains with Constitutive Expression of eGFP

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.

Development of Leishmania Species Strains with Constitutive Expression of eGFP

Protozoan parasites of the genus Leishmania cause leishmaniasis, a disease with variable clinical manifestations that affects millions of people ...

This protocol is key for generating strains that can be used in high-throughput screening assays to evaluate the potential and traditional activity of molecules Demonstrating the procedure will be Juana Quintero and Laura Pineda, research assistants from my laboratory. To begin, amplify the target gene using a high fidelity polymerase to preserve the coding sequence. Add the primers to the reaction mix in a final concentration of 0.2 micromolar and run a PCR cycling protocol as described in the manuscript.Purify the amplified PCR fragment using a conventional PCR product purification kit. To trim the ends of the eGFP PCR product, set up a reaction with Kpn I enzyme according to the manufacturer's instructions and incubate at 37 degrees Celsius for one hour. Then add 100 millimolar of sodium chloride and 10 units of Bgl II to the reaction and incubate for 15 minutes.After digestion, purify the reaction as shown earlier. Next, digest pLEXSY expression vector with Bgl II and Kpl I following the sequential digestion as demonstrated earlier. Ligate 100 nanograms of digested pLEXSY vector and 28 nanograms of digested eGFP in a 20 microliter reaction containing ligase buffer and three units of T4 DNA ligase.Incubate overnight at four degrees Celsius. At the end of the incubation, transform competent Escherichia coli cells with the ligation product using standard transformation protocols. Plate the transformed cells in LB ampicillin agar and incubate for 24 hours at 30 degrees Celsius to select recombinant clones.After screening the colonies, purify plasmid DNA from a positive clone for subsequent transfection using a commercial plasmid isolation kit. To produce linearized fragments of the plasmid, take at least 10 micrograms of the purified pLEXSY eGFP plasmid and digest it with SWA1 for three to four hours at 25 degrees Celsius. At the end of the incubation, heat inactivate the digestion product at 65 degrees Celsius for 20 minutes.Then run the digestion product in agarose gel electrophoresis to separate the resulting fragments. Purify the expression cassette using a commercial agarose gel extraction kit according to the manufacturer's instructions. Grow the parasite cultures until there are enough log phase or early stationary phase promastigotes.Pool the contents of multiple culture flasks if required. Centrifuge the parasite culture at 2, 000 G for three minutes at room temperature. Resuspend the pellet in electroporation buffer at four degrees Celsius to get a concentration of one times 10 to the eighth parasites per milliliter and keep it on ice for 10 minutes.Simultaneously, pre-chill tubes with 2 to 10 micrograms of linearized pLEXSY eGFP construct in 50 microliters of water or 10 millimolar tris buffer with pH 8.0 and electroporation cuvettes of two millimeter diameter. Next, add 350 microliters of pre-chilled parasites to the tube with the linearized plasmid and transfer the entire 400 microliters to the electroporation cuvette on ice. Electroporate the parasite cells with plasmid and in parallel, parasite cells without plasmid DNA as a negative control.Put the cuvette on ice for 10 minutes. Transfer the electroporated parasites to five milliliters of the appropriate culture medium and incubate at 26 degrees Celsius for 20 hours. Approximately 20 hours post-electroporation, observe the cultures under a microscope and add the appropriate selective antibiotic depending on the pLEXSY plasmid used.Follow the cultures microscopically until there is a clear difference between parasites electroporated with and without plasmid DNA. To verify parasite fluorescence through flow cytometry, centrifuge one milliliter of the stationary phase culture at 2, 000 G for three minutes at room temperature. After washing the cells twice in PBS, resuspend the final pellet in one milliliter of PBS.Run samples and collect 20, 000 events at a speed of 0.5 microliters per second. Set gain values for the forward scatter, side scatter, and fluorescence one channels as 225.0, 200.0, and 520.0 respectively. Create a gate G1 containing the parasite population.Filter the FL1 channel through that gate to determine the autofluorescence of promastigotes and set a range gate G2 for the FL1 channel. Run the transfected parasites to verify if there is fluorescence. Record the percentage of parasites in G1 that are fluorescent and the mean of fluorescence intensity in G2.Purify genomic DNA from two to five milliliters of a stationary phase parasite culture by conventional phenol chloroform extraction or with a commercial kit.Perform diagnostic PCR for pLEXSY-sat2.1 using a cycling protocol as described in the manuscript. After confirmation of fluorescence through flow cytometry, prepare a dilution of recombinant parasites at a concentration of five promastigotes per milliliter. Add 100 microliters of this dilution into each well of a 96-well plate and leave the plate undisturbed for at least 12 hours.Follow the plate microscopically at a minimum magnification of 100X until wells with parasite growth are detected. When a well is full of parasites, use the content to inoculate a five milliliter culture. Once the clone seeded cultures reach the stationary phase, verify fluorescence using flow cytometry as described earlier.Select clones with 98%to 99%of fluorescent parasites and the highest mean fluorescence intensity for in vitro and in vivo assays. Colony PCR of the E.coli cells transformed with pLEXSY eGFP construct generated an 859 base pair product. Total digestion of the plasmid using SWA1 resulted in two fragments, a 2.9 kilobase pair fragment containing all the necessary elements for replication and selection in E.coli and a seven to eight kilobase pair fragment representing the linearized expression cassette that is used for transfection.Flow cytometry analysis revealed that 82%of the transfected L.panamensis parasites expressed eGFP. After polyclonal selection, 98.44%of L.donovani parasites analyzed by flow cytometry were fluorescent in comparison with 82.0%of L.panamensis. The successful integration of pLEXSY eGFP into the SSU locus resulted in a 2.3 kilobase pair product in the diagnostic PCR.Clones with the highest percentage of fluorescent parasites and mean fluorescence intensity was selected for further use in drug screening assays. This method allows the production of recombinant Leishmania parasites with the

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556 Views.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP). This protocol is key for generating strains that can be used in high-throughput screening assays to evaluate the potential and traditional activity of molecules Demonstrating the procedure will be Juana Quintero and Laura Pineda, research assistants from my laboratory. To begin, amplify the target gene using a high fidelity polymerase to preserve the coding sequence. Add the primers to the reaction mix in a final concentration of 0.2 micromolar and run a PCR cycling protocol as described in t...