Name: development of leishmania species strains with constitutive expression of egfp
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Description: Watch this Scientific Journal Video about Development of Leishmania Species Strains with Constitutive Expression of eGFP at JoVE.com

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...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+green+fluorescent+protein+reporter+system+in+Leishmania+spp.+for+the+in+vitro+and+in+vivo+screening+of+antileishmanial+drugs.">Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.</a> Acta Tropica. 122 (1), 36-45 (2012).</li><li>Bastos, M. S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achievement+of+constitutive+fluorescent+pLEXSY-egfp+Leishmania+braziliensis+and+its+application+as+an+alternative+method+for+drug+screening+in+vitro.">Achievement of constitutive fluorescent pLEXSY-egfp Leishmania braziliensis and its application as an alternative method for drug screening in vitro.</a> Mem&#243;rias do Instituto Oswaldo Cruz. 112 (2), 155-159 (2017).</li><li>Vincze, T., Posfai, J., Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NEBcutter:+A+program+to+cleave+DNA+with+restriction+enzymes.">NEBcutter: A program to cleave DNA with restriction enzymes.</a> Nucleic Acids Research. 31 (13), 3688-3691 (2003).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon-based+approach+for+live-cell+imaging+of+RNA+transcripts+with+minimal+target+engineering+at+the+single-molecule+level.">A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level.</a> Scientific Reports. 7 (1), 1550 (2017).</li><li>Green, M., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. 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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

development-leishmania-species-strains-with-constitutive-expression

Research

JoVE Journal

Genetics

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...