Name: sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

This work was supported by a grant from the National Sciences and Engineering Research Council of Canada (NSERC) (#298527) (BB). CT was supported by an institutional FARE scholarship. AV was supported by a NSERC Graham Bell Ph.D. scholarship. BB held a Canada Research Chair in Human Retrovirology (Tier 2). Thanks to Beatrix Beisner for help in revising the text.

The UQAM ethics committee has approved these protocols, which are in accordance with the guidelines of the ethics committee of St-Luc Hospital of the Centre Hospitalier Universitaire de Montr&#233;al (Montr&#233;al, Canada). Participants signed an informed consent form.
1. Medium Preparation and Isolation of Primary Villous Cytotrophoblasts
<ol>
	<li>Prepare culture medium for human primary villous cytotrophoblasts by supplementing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with 25 mM HEPES, 1...

<ol>
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Studies on human placental function and development have been greatly improved by protocols aimed at optimizing isolation of various placental cell populations. One of the best studied placental cell population remains the villous cytotrophoblasts, the study of which has greatly benefited from optimized protocols permitting efficient and reliable isolation. This has further allowed a number of experiments, such as transfection and promoter studies. Using a previously described protocol 3, pure populations of p...

Human placental dysfunction is associated with the development of several pregnancy-associated diseases like preeclampsia and intrauterine growth restriction 1. An important cell constituent of the placenta is the trophoblasts, which can be classified as either extravillous or villous cytotrophoblasts. Upon fusion, villous cytotrophoblasts further differentiate into the syncytiotrophoblast layer, a multinuclear cell structure with an important role in feto-maternal exchange and hormone production 2. Human primary villous cytotrophoblasts and their differentiated counterparts represent important biological samples and allow researchers to study a number of placenta-related processes, such as cell fusion, in culture. Furthermore, substantial efforts are ongoing to identify markers that will facilitate appropriate management and improve preventive therapies specific to pregnancy-related diseases. Laboratories routinely isolate primary human villous cytotrophoblasts from fresh placentas, using a standard isolation procedure based on trypsin digestion of placenta villi 3. As cultured cytotrophoblasts lose their capacity to proliferate and quickly differentiate in a syncytiotrophoblast-like layer upon culture 4, very efficient transfection methods and optimized analysis approaches are needed. Previous studies have determined optimal conditions of transfection of these primary cytotrophoblasts 5. Herein, a different method of siRNA transfection, which has been previously tested in this cell type 6, is presented. In comparison to a lipofection-based approach, this microporation method improves transfection, as assessed by the extent of silencing of specific genes.
Promoter and gene expression studies also provide a better understanding of placental function. Although more difficult to use owing to the short time frame for which primary villous cytotrophoblasts can be cultured, promoter analyses using standard protocols can nonetheless be addressed, as previously published 7. Electrophoretic Mobility Shift Assay (EMSA) is one of these commonly used in vitro methods, allowing for fast and easy monitoring of DNA-protein interactions. Nuclear extracts from these primary trophoblasts were used to test a region of the Syncytin-2 promoter for specific interactions. Results revealed that bound factors could be detected at different time points of culture and in a specific and reproducible manner.
Data presented in this protocol confirm that our transfection approach and the EMSA protocol can be used for isolated primary villous cytotrophoblasts and will be of great use to study the diverse functions of villous cytotrophoblasts in normal or pathological conditions.

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			<td>Cell Signaling</td>
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			<td>Montr&#233;al Biotech</td>
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sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

Fresh placentas from term pregnancies were used to isolate human primary villous cytotrophoblasts to conduct the set of experiments presented in the Protocol section. Following their isolation, we first analyzed the purity of cytotrophoblasts through the use of the cytokeratin-7 marker (Figure 1). Cell preparations were thus stained using a monoclonal anti-cytokeratin-7 antibody. Figure 1 represents results from a typical experiment following purification...

Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

The overall goal of this protocol is to demonstrate optimal conditions for the transfection of freshly isolated primary villous cytotrophoblasts and to present a method for the identification of DNA protein interaction at promoter sites. This method can help answer key questions in the field of placenta research, such as those related to genes involves in cytotrophoblast differentiation and the regulatory mechanisms involved in their expression. The main advantage of this technique is that they're easy to perform and very efficient.Furthermore, the time needed to conduct this experiment is short and can include a lot of different samples. Demonstrating these procedures will be Chirine Toufaily a former Phd student from my lab, as well as Yong Xiao, a research associate. Here, primary villous cytotrophoblasts are isolated from fresh placentas according to standard protocols.The first step of this protocol is to analyze the purity of the preparation by FACS analysis. First, spin one times ten to the sixth of the freshly isolated cytotrophoblasts in micro centrifuge tubes at 300 times G for five minutes. Following the centrifugation, discard the supernatant.Add one milliliter of pre-warmed PBS and repeat the centrifugation. After removing the PBS by aspiration add one milliliter of cold methanol to the cells and incubate at minus 20 degrees Celsius for 20 minutes. Next, after pelleting the cells by centrifugation, remove the methanol by aspiration.Then add one milliliter of room-temperature PBS to the pellet to rehydrate the cells. After spinning the cells and removing the PBS, add PBS containing FBS with FC receptor blocking reagent and incubate for 45 minutes at room-temperature to eliminate non-specific binding. Once the incubation time has elapsed, wash the cells twice with 500 microliters of room-temperature PBS centrifuging at 300 times G for five minutes each time.After removing the last PBS wash, resuspend the cells in 100 microliters of room-temperature PBS. Then incubate the cells with a mouse monoclonal FITC conjugated anti-human cytokeratin 7 antibody clone LP5K, or a control isotype-matched non-specific antibody for 4 minutes at room-temperature in the dark. After washing the cells in PBS, analyze florescents by flow cytometry.Use cell preparations that demonstrate a minimum of 96%CK7 positive cells by flow cytometry for further experiments. To transfect human primary villous cytotrophoblasts using a microporation device first prepare six well plates containing two milliliters of supplemented DMEM medium and place them in a 37 degree Celsius tissue culture incubator. Next, transfer aliquots of 1.5 times 10 to the 6th isolated primary cytotrophoblasts to micro centrifuge tubes.Following a centrifugation to pellet the cells wash with one milliliter of Dulbecco's PBS. After removing the PBS, and gently resuspending the washed cells in 100 microliters of resuspension buffer add 300 nanograms of Syncytin-2 siRNA or controlled scrambled siRNA. And then aspirate the cell siRNA mixture using a 100 microliter transfection pipette.Now insert the pipette into the transfection station and subject the cells to a single 1300 volt pulse of 30 milliseconds duration. Then immediately transfer the cells to the previously prepared six well plates and gently rock the plate for 30 seconds to ensure an even distribution of the cells. It's very important no to make air bubble while resuspending the cells and to transfer them immediately to the medium.Otherwise this will affect that viability of cells and the transfection efficiency. Incubate the plate at 37 degrees Celsius in a humidified carbon dioxide incubator for 24 to 48 hours. First, wash the cultured cytotrophoblasts twice with one milliliter of pre-warmed DPBS.After aspirating the DPBS add 500 microliters of 0.25%Trypsin-EDTA and incubate for one to three minutes at 37 degrees Celsius in a carbon dioxide incubator. Next, stop the Trypsin treatment by adding 500 microliters of serum containing growth medium and transfer the cells from each well into a 1.5 milliliter micro centrifuge tube. Pellet cells by centrifugation at 300 times G for two minutes at room temperature.After washing and pelleting the cells once more, carefully remove all supernatant without disrupting the pellet and place the tubes on ice until protein extraction. Prepare the phosphorylation reaction in a micro centrifuge tube by adding 15 nanograms of a forward oligonucleotide along with 1XT4 Polynucleotide kinase buffer, one unit of T4 Polynucleotide kinase, and 20 microcuries of gamma-32P labeled ATP to nuclease free water, for a final volume of 20 microliters. Incubate the reaction at 37 degrees Celsius for 30 minutes.Then, stop the reaction by adding 5 microliters of 0.5 molar EDTA. Bring the volume up to 50 microliters with nuclease free water. Then proceed to the removal of unincorporated nucleotides and the hybridization of the complimentary strand according to the instructions in the written portion of the protocol.Setup the DNA binding reaction by first adding 10 micrograms of nuclear extract 1X gel shift binding buffer to nuclease free water for a final volume of 10 microliters. For the competition reaction add one microliter of cold non-specific or specific double-stranded oligonucleotide. Next, add one microliter of the radioactive oligonucleotide probe to each reaction.Incubate the reaction at room-temperature for 20 minutes. Then add one microliter of 10X gel loading buffer per reaction. Load each sample onto a non-denaturing polyacrylamide gel and run the gel for 1.5 to 2 hours at 150 volts.After migration, wrap the gel and the glass in plastic wrap. Then position the wrapped gel in an exposure cassette and place a phosphorus screen over it. Leave the cassette at 4 degrees Celsius for 24 hours.The next day remove the screen from the exposure cassette and insert it into a phosphorimaging device for scanning. Microporation of a scrambled control or a Syncytin-2 specific siRNA in villous cytotrophoblasts is compared to a lipid based transfection approach by western blot detection of Syncytin-2 and the control protein GAPDH. Syncytin-2 protein levels were normalized in terms of band intensity with corresponding GAPDH signals.Microporation conclusively leads to a more significant knockdown of Syncytin-2 expression. This image shows an EMSA analysis of nuclear extracts villous cytotrophoblasts incubated with a probe corresponding to a promoter region of Syncytin-2 with or without an excess of specific, non-specific a cold oligonucleotide for 24 or 48 hours. The Syncytin-2 promoter drived probe showed the presence of two DNA protein complexes.Addition of cold wildtype oligonucleotide competed for the formation of the complex while no competition was observed with a cold, unrelated, oligonucleotide. While attempting this procedure, it's very important to carefully handle cytotrophoblasts after transfection and to transfer them immediately to the medium. Following the EMSA procedure, a supershift assay can be performed in order to identify transcription factor that bound to DNA.After watching this video you should have a good understanding on how to handle and carefully transfect a cytotrophoblast as well as how to perform an EMSA assay with these kind of cells.

sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

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 ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

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 ...