Name: transient transduction of the strobilated forms of echinococcus granulosus
Uploaded: 2022-09-16T12:30:00
Description: Watch this Scientific Journal Video about Transient Transduction of the Strobilated Forms of Echinococcus granulosus at JoVE.com

Research reported in this publication was supported by Elite Researcher Grant Committee under award number 958680 from the National Institute for Medical Research Development (NIMAD), Tehran, Iran.

This study was approved by the National Institute for Medical Research Development and the Research Ethics Review Committee, No. 958680. Lentiviruses are classified as BSL-2 organisms; hence, all laboratory culture procedures in this protocol were carried out using sterile laboratory practices and conducted under a laminar flow hood according to NIH guidelines. Figure 1 demonstrates a schematic presentation of the study protocol for the different E. granulosus stages.
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<ol>
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Understanding the molecular basis of nematodes and Platyhelminthes biology is crucial to understanding the pathogenicity of zoonotic parasites27. The lack of an effective gene evaluation system is a major obstacle to the direct interpretation of functional genetics of cestode parasites, including Echinococcus species12,27. The present study demonstrates the excellent potential of lentivirus in E. granulosus transient tran...

Cystic echinococcosis (CE) is one of the most important helminth diseases caused by Echinococcus granulosus, a small tapeworm within the family Taeniidae1,2. Extensive studies on immunodiagnostic and vaccine development for E. granulosus have been carried out. However, inadequate knowledge about the molecular basis of parasite biology poses major limitations in the diagnosis, management, and prevention of hydatid disease3,4,5,6.
In recent years, due to the development of genome sequencing and transcriptomic methods, a wide range of molecular studies have been conducted on flatworms by several research groups7,8,9. However, in the world of parasites, advances in gene transfer technology in parasitic flatworms are still limited compared with the highly reproducible transient transduction methods developed for some protozoa10,11,12.
The use of viral delivery systems has emerged as an essential tool for transgene delivery and gene/protein investigations over the last two decades13. Lentivirus infects both dividing and non-dividing cells, thus making it possible to infect postmitotic cells14,15,16. Recent evidence indicates that using a lentivirus-based transduction system in mammalian cells offers the potential to overcome most of the limitations of previous knock-in/knock-down techniques. The design and construction of expression lentiviral vectors with appropriate molecular markers, such as GFP expression, have been described previously16. Therefore, we evaluate lentiviral transient transduction of a GFP reporter gene in the protoscoleces and strobilated worms of E. granulosus.

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	<tbody>
		<tr>
			<td>12-well culture plates</td>
			<td>SPL Life Sciences</td>
			<td>30012</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 culture flask</td>
			<td>SPL Life Sciences</td>
			<td>70325</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>SPL Life Sciences</td>
			<td>30006</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901-500G</td>
			<td>Working concentration: 2.5&#160;mM</td>
		</tr>
		<tr>
			<td>CMRL 1066 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11530037</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>memmert</td>
			<td>ICO150</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technology</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>Dog bile</td>
			<td></td>
			<td></td>
			<td>Isolated from a euthanized dog and sterilized by 0.2 &#956;m syringe filter</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma-Aldrich</td>
			<td>E4009-5G</td>
			<td>prepare 0.1% of Eosin for working exclusion test</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>DNAbiotech</td>
			<td>DB9723-100ml</td>
			<td>Heat inactivation&#160;of&#160;FBS (30 min in 40 &#176;C)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FCS)</td>
			<td>DNAbiotech</td>
			<td>DB9724-100ml</td>
			<td>Heat inactivation&#160;of&#160;FCS (30 min in 40 &#176;C)</td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>BONbiotech</td>
			<td>BN_0012.1.14</td>
			<td>Human embryonic kidney 293T</td>
		</tr>
		<tr>
			<td>HEPES buffered saline (HBS)</td>
			<td>Sigma-Aldrich</td>
			<td>51558-50ML</td>
			<td>2x concentrate</td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P3032-10MU</td>
			<td>Working concentration: 100 IU/mL</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Roche</td>
			<td>10108057001</td>
			<td>Working concentration: 2 mg/mL, pH 2</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>DNAbiotech</td>
			<td>DB0011</td>
			<td>This reagent solve in less than 1 min in D.W</td>
		</tr>
		<tr>
			<td>Polybrene (Transfection reagent)</td>
			<td>Sigma-Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>BioIdea</td>
			<td>BI-1006-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S9137-25G</td>
			<td>Working concentration: 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Third-generation lentiviral plasmid (pCDH513b)</td>
			<td>SBI System Biosciences (BioCat GmbH)</td>
			<td>CD513B-1-SBI</td>
			<td>Transfer vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td>
		</tr>
		<tr>
			<td>Third-generation lentiviral plasmid (pLPI and pLPII)</td>
			<td>Invitrogen (Life Technologies)</td>
			<td>K4975-00</td>
			<td>Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td>
		</tr>
		<tr>
			<td>Third-generation lentiviral plasmid (pMD2G)</td>
			<td>Addgene</td>
			<td>Plasmid 12259</td>
			<td>Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td>
		</tr>
		<tr>
			<td>Tris/EDTA Buffer (TE)</td>
			<td>DNAbiotech</td>
			<td>DB9713-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T9935-50MG</td>
			<td>1x working solutions (pH 7.4&#8211;7.6)</td>
		</tr>
	</tbody>
</table>

transient transduction of the strobilated forms of echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Here, we describe a rapid and efficient transient transduction technique in E. granulosus by using third-generation lentiviral vectors. We cultured PSCs in a biphasic culture medium to obtain strobilated worms, as described previously25,26. Protoscoleces develop into strobilated worms after 6 weeks in vitro. Different stages of E. granulosus were observed in the biphasic culture medium, including invaginated PSCs (F...

Watch this Scientific Journal Video about Transient Transduction of the Strobilated Forms of Echinococcus granulosus at JoVE.com

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Transient transaction of the strobilated forms of Echinococcus granulosus. We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors. Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines.There is an urgent need for applied genetic research to understand mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of functional genetics of cestode parasites, including Echinococcus species. Advances in gene transferring in parasitic flatworms are still limited in comparison with protozoan parasite.The use of viral delivery system has emerged as an essential tools for transgene delivery and gene/protein investigation over the last two decades. So we evaluated the lentiviral transient transduction of GFP reporter gene to the protoscoleces and strobilated worms of Echinococcus granulosus. Figure 1 demonstrates a schematic presentation of the study protocol for Echinococcus granulosus stages.One, collecting hydatid cysts. Collect liver hydatid cysts from naturally infected sheep routinely slaughtered at an abattoir. Completely sterilize the cyst surface using sterile gauze and 70%alcohol before aspirating the protoscoleces.Aspirate 20 to 50 mL of hydatid fluid out into sterile 50 mL conical tubes. After draining, blot out the cyst with a sharp blade, transfer the germinal layer into a sterile 50 mL conical tube, and shake it for a short period to increase the collection of protoscoleces. Wash the protoscoleces with PBS buffer three to five times.Observe the protoscoleces in 0.1%eosin for five minutes to determine the protoscoleces'viability under a light microscope. Use protoscoleces with at least 95%viability for culture. Treat the protoscoleces precipitate with two mL of pepsin for 15 to 20 minutes.To isolate individual protoscoleces, resuspend the protoscoleces sediment in PBS and pass it through two layers of sterile gauze into a new sterile 50 mL conical tube. Two, biphasic cultivation of protoscoleces of Echinococcus granulosus to obtain adult worms. Add 10 mL of the liquid phase medium to each 25 centimeter squared culture flask.Add approximately 5, 000 protoscoleces to the culture flask and transfer it to the CO2 incubator. After 24 to 48 hours, transfer the protoscoleces to a new flask with the solid phase and add one mL of the same fresh liquid phase medium per flask. Transfer the flasks into the incubator, 37 degrees Celsius, 5%CO2.Every five to seven days, replace the medium with fresh liquid phase medium. Three, monophasic cultivation of protoscoleces of a Echinococcus granulosus. Ensure that monophasic cultivation is done 24 to 48 hours before transduction.Culture approximately 5, 000 protoscoleces in a 25 centimeter squared culture flask with RPMI and 10%FBS. Transfer the flasks into the CO2 incubator until use. Four, cell culture for virus production and preparation.Transfer 500, 000 HEK cells to a 25 centimeter squared flask containing 5 mL of DMEM with 10%FBS, 100 U/mL penicillin, and 100 micrograms/mL streptomycin. Incubate the HEK2 cells at 37 degrees Celsius in 5%CO2 and passage them in the complete culture medium every 48 hours. Finally use low-passage HEK cells for transduction.Five, production and preparation of the virus with the third-generation lentiviral vectors. Day one, after trypsinizing the HEK cells in a 6-well culture plates, seed 70, 000 cells per well with fresh antibiotic-free DMEM containing 10%FBS. Use cells at 50 to 60%confluency for transduction by the calcium phosphate method.Day two, replace the cell culture medium two to three hours before the experiment with fresh antibiotic-free DMEM containing 2%FBS. In a 1.5 mL tube, mix third-generation lentiviral plasmids, including 7 micrograms/microliter pCDH513b transfer vector, 4 micrograms/microliter PLPII, 4 micrograms/microliter PLPI, and 2 micrograms/microliter PMD2G helper vectors. Add HEPES buffer to the mixture to reach the volume of 422 microliters.Add 16 microliters of 1%TE buffer to the mixture. Add 62 microliters of 2.5 mL calcium chloride to the tubes and mix well. Add 500 microliters of 2x HBS buffer to the mixture, drop by drop within one to two minutes using a Pasteur pipette.Mix gently until a semi-opaque solution is obtained and incubate the mixture for 20 minutes at room temperature. At the end, add the mixture to each plate slowly and distribute it with slow circular motions. Incubate the plates at 37 degrees Celsius in a 5%CO2 incubator and replace the medium after four to six hours with antibiotic free DMEM containing 10%FBS.Day three, confirm successful transient transduction and cell viability using an inverted fluorescence microscope. Day four, harvest viruses from the culture medium by collecting each well's supernatant and store them at negative 70 degrees Celsius until use. Six, transient transduction of different stages of Echinococcus granulosus with the virus.Day one, use a 12-well culture plate for gene transfer. Culture 5, 000 to 50, 000 HEK cells in triplicates with a complete DMEM medium as the internal reference. Culture 150 fresh protoscoleces in triplicates in RPMI and 10%FBS.Culture 30 strobilated worms in triplicate into DMEM and 10%heat-inactivated FBS. Prepare a mixture of 1 million viruses with four micrograms/mL transfection reagent to transduce the HEK cells and different stages of the worm. Add the mixture to each plate slowly and distribute it with slow circular motions.Incubate the plates for four to six hours in a CO2 incubator, 37 degrees Celsius, 5%CO2. Replace the medium for each sample with the same complete culture medium to minimize the toxic effects of the transfection reagents. Day two, repeat two previous steps to increase the efficiency of transient transduction for the HEK cells, protoscoleces, and strobilated worms.Incubate all the plates for 24 to 48 hours in a CO2 incubator with the same culture conditions based on the type of cell media. Day three, make sure the transduction is successful by using fluorescence microscopy. Representative results.Here we describe a rapid and efficient transient transduction technique in Echinococcus granulosus by using third-generation lentiviral vectors. We cultured protoscoleces in biphasic culture media to obtain strobilated worms according to our previous studies. Protoscoleces are developed into the strobilated worms after a six-week in vitro culture.Different stages of Echinococcus granulosus were observed in the biphasic culture medium, including invaginated protoscoleces, Figure 2A, evaginated protoscoleces, Figure 2B, and strobilated worms with first and third proglottid formation, Figure 2C through E.Protoscoleces and the strobilated worms obtained from monophasic and biphasic cultivations, respectively, were transfected by GFP-expressing lentiviruses, Figure 3. HEK cells clearly expressed GFP as transient transduction process control. The adult worms expressed GFP most distinctly in the tegumental layer.However, protoscoleces have demonstrated a somewhat lower level of GFP expression after 48 hours. Some cyst fluid residues and or germinal layer debris around the protoscoleces caused some fluorescence background in the transfected samples. The intensity of fluorescence in HEK cells and strobilated worms increased after 24 and 48 hours.Understanding the molecular basis of nematodes and Platyhelminthes biology is one of the most important frontiers in the pathogenicity of zoonotic parasites. The lack of an effective gene evaluation system is a major obstacle to direct interpretation of functional genetics of cestode parasites, including Echinococcus species. The major goal of this work was to illustrate the process of lentiviral vector transduction which will be critical in future Echinococcosis research.The results show that strobilated worms are more responsive to transient transduction of the gene than the protoscoleces, which may be the result of extensive tegumental structure in the strobilated forms. However, due to the nature of lentiviral transduction, the fluorescence intensity in the multicellular protoscoleces and strobilated worms is typically much lower than monolayer HEK cells. There is an urgent need for applied genetic research at the genomic and transcriptomic levels for Echinococcus as well as a wide variety of parasitic and free-living flatworm model systems.Therefore, developing the gene transfer process to tapeworms paves the way for potential use of the new techniques such as virus-based gene mapping or CRISPR-Cas9-mediated gene editing. This work present lentiviral transduction in a tapeworm model and demonstrate promising outcomes with potential implication for flatworm biology. Yet more in-depth studies are required to improve the methods and develop the applicability of these techniques for future experiments.

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Research

JoVE Journal

Genetics

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...