Name: methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1
Uploaded: 2016-05-27T14:00:00
Description: Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

This work was supported by Merit Review Awards to Douglas D. Ross and to Arif Hussain from the Department of Veterans&#39; Affairs.

1. In Silico Prediction of Alternative First Exons of Bcrp1 Using BLAST Analysis of Mouse EST Database
Note: This protocol describes how to search the mouse expressed sequence tag (EST) database for ESTs with sequence similarity to exon 2 of Bcrp1 (which contains the translational start site) and then how to align the matching EST sequences to genomic sequences to ascertain their location in the mouse Bcrp1 gene relative to the 5&#39; end of Bcrp1 exon 2.
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	<li>Obtain the sequen...

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+resistance+protein+(Bcrp)+and+the+testis--an+unexpected+turn+of+events.">Breast cancer resistance protein (Bcrp) and the testis--an unexpected turn of events.</a> Asian J Androl. 15 (4), 455-460 (2013).</li><li>Xie, Y., 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=Bcrp1+transcription+in+mouse+testis+is+controlled+by+a+promoter+upstream+of+a+novel+first+exon+(E1U)+regulated+by+steroidogenic+factor-1.">Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1.</a> Biochim Biophys Acta. 1829 (12), 1288-1299 (2013).</li><li>Maliepaard, 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=Subcellular+localization+and+distribution+of+the+breast+cancer+resistance+protein+transporter+in+normal+human+tissues.">Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.</a> Cancer Res. 61 (8), 3458-3464 (2001).</li><li>Fetsch, P. 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A., Van De Ven, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+gene+expression+by+alternative+promoters.">Regulation of gene expression by alternative promoters.</a> FASEB J. 10 (4), 453-460 (1996).</li><li>Zong, Y., Zhou, S., Fatima, S., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mouse+Abcg2+mRNA+during+hematopoiesis+is+regulated+by+alternative+use+of+multiple+leader+exons+and+promoters.">Expression of mouse Abcg2 mRNA during hematopoiesis is regulated by alternative use of multiple leader exons and promoters.</a> J Biol Chem. 281 (40), 29625-29632 (2006).</li><li>Natarajan, K., 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=Identification+and+characterization+of+the+major+alternative+promoter+regulating+Bcrp1/Abcg2+expression+in+the+mouse+intestine.">Identification and characterization of the major alternative promoter regulating Bcrp1/Abcg2 expression in the mouse intestine.</a> Biochim Biophys Acta. 1809 (7), 295-305 (2011).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wizard&#174; SV Gel and PCR Clean-Up System - INSTRUCTIONS FOR USE OF PRODUCT. , (2009).</li><li>New_England_Biolabs. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quick Ligation Kit Protocol - M2200S. , (2014).</li><li>Roche. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> XtremeGENE HP DNA Transfection Reagent Protocol - Manual version 1.0). , (2010).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quick Protocol for the use of the Dual-Luciferase Reporter Assay. , (2009).</li><li>Carninci, P., 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=The+transcriptional+landscape+of+the+mammalian+genome.">The transcriptional landscape of the mammalian genome.</a> Science. 309 (5740), 1559-1563 (2005).</li><li>VanBuren, V., 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=Assembly,+verification,+and+initial+annotation+of+the+NIA+mouse+7.4K+cDNA+clone+set.">Assembly, verification, and initial annotation of the NIA mouse 7.4K cDNA clone set.</a> Genome Res. 12 (12), 1999-2003 (2002).</li><li>Bonaldo, M. 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The majority of the methods and representative results presented are described in previous work entitled &#34;Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1,&#34; which was published in 20135. In addition to the representative results depicted here, the previous paper provided estimates of alternative first exon utilization using 5&#39;-RACE PCR and RT-PCR methodology. Furthermore, in-silico identificati...

More than half of human genes are regulated by alternative promoters1. Each alternative promoter can contain regulatory elements that may be different from those in other alternative promoters. The promoter(s) utilized in one tissue may differ from those used in another tissue. For example, it is possible that activation of a given signaling pathway may trigger the alternative promoter for a gene utilized in one tissue, yet have no effect on or repress a separate alternative promoter for the same gene that is utilized in another tissue.
Expression of the Bcrp1 gene is governed by alternative promoters. Bcrp1 is the murine orthologue of the human Breast Cancer Resistance Protein (BCRP) gene. BCRP is an ATP-binding cassette (ABC) transporter, formally designated ABCG22,3. As an apical plasma membrane protein, BCRP/Bcrp1 functions to efflux a wide variety of natural and xenobiotic substrates3,4. In humans and mice, BCRP/Bcrp1 is highly expressed in pharmacologically relevant organs such as liver (bile canaliculi), intestine, and kidney, as well as organs with blood-tissue barriers such as placenta, brain and testis2,5-12. Expression of BCRP/Bcrp1 in hematopoietic and other stem cells, including cancer stem cells, may confer resistance of these cells to xenobiotics and cancer chemotherapeutic drugs3.
In early work to understand the regulation of BCRP expression in normal and neoplastic cells, 5&#39; rapid amplification of cDNA ends (5&#39;-RACE) analysis of BCRP mRNA was performed to determine its exact transcriptional start site13. Not only were multiple transcriptional start sites found; also encountered were three alternative forms of the first exon, which in BCRP is untranslated. These alternative first exons&#160;&#8212; designated E1a, E1b, E1c &#8212; were expressed differently in a variety of human tissues. Two additional first exon variants were discovered in a BLAST search of the human EST database using the second exon of BCRP13. Four matches revealed a first exon &#62;70 kb upstream from exon 2 which were designated E1u; four other matches revealed BCRP mRNA that lacked a first exon entirely, which were designated E1-13. The presence of alternative leader exons is considered to be a manifestation of alternative promoter usage14.
In mice, four alternative first exons of Bcrp1 are described that may correspond to alternative promoters that govern Bcrp1 transcription in different mouse tissues; these exons/promoters are designated E1U, E1A, E1B and E1C, and are located approximately 70, 58, 15, and 5 kb upstream from Bcrp1 exon 25,15. The E1A mRNA isoform was found to be predominant in murine hematopoietic stem cells, heart, lung, spleen, and brain, whereas the E1B isoform was expressed in mouse intestine, fetal liver cells, and erythroid precursor cells in bone marrow5,15. The promoter upstream from E1B was shown to be the major alternative promoter governing Bcrp1 transcription in mouse intestine, regulated at least in part, by phospho-cyclic-AMP response element binding protein (p-CREB) and a CREB response element unique to that alternative Bcrp1 promoter16. The E1C mRNA isoform is predominantly expressed in adult murine liver and kidney5. The E1U isoform is undetectable in most tissues tested except for murine testis, where it is the predominant isoform expressed5. Bcrp1 expressed in rat testes is found in both somatic (endothelial tight junctions, peritubular myoid cells, and Sertoli cells) and germ cells (in the seminiferous endothelium, where it may protect late-stage spermatids4). The region upstream from E1U contains a functional response element for steroidogenic factor-1 (SF-1)5. Bcrp1 mRNA and protein are markedly reduced in the testes of Sertoli cell-specific SF-1 knockout mice, suggesting that Bcrp1 expression in murine Sertoli cells is controlled by SF-15.
The protocols presented detail methods to detect alternative first exons of Bcrp1 in-silico, and to establish luciferase-based reporter assays for putative promoters upstream from the alternative first exons identified.

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			<td>5&#39;-RACE PCR kit</td>
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			<td>Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors.</td>
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			<td>For plasmid miniprep - isolation and purification of plasmid DNA from bacterial colonies</td>
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			<td>part of the TOPO TA cloning kit</td>
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			<td>pGL3-basic luciferase containing vector</td>
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			<td>pRL-TK Renilla luciferase expressing vector</td>
			<td>Promega</td>
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			<td>Bacterial artificial chromosome</td>
			<td>BACPAC Resources Center, Children&#8217;s Hospital Oakland Research Institute, Oakland, CA</td>
			<td>RP23-285A12 and RP24-314E24</td>
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			<td>Wizard&#174; SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
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			<td>Useful for purifying PCR products following digestion with restriction endonucleases</td>
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			<td>SOFTWARE:</td>
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			<td></td>
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			<td>Ensembl software</td>
			<td>Ensembl project</td>
			<td>http://Ensembl.org&#160;</td>
			<td>The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online.</td>
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			<td>Blast software</td>
			<td>NCBI</td>
			<td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= 
			Web&#38;PAGE_TYPE=BlastHome</td>
			<td></td>
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			<td>Primer 3 software</td>
			<td>Simgene</td>
			<td>http://simgene.com/Primer3</td>
			<td>Useful for designing primers for 5&#39;-RACE PCR</td>
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		<tr>
			<td>NCBI Nucleotide database</td>
			<td>NCBI</td>
			<td>http://www.ncbi.nlm.nih.gov/nucleotide/</td>
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			<td>Name</td>
			<td>Company&#160;</td>
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			<td>CELL LINES:</td>
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			<td>TM4 murine Sertoli cells</td>
			<td>ATCC, Manassas, Virginia</td>
			<td>CRL-1715&#8482;</td>
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			<td>Luminometer</td>
			<td>Turner Biosystems</td>
			<td>TD-20/20</td>
			<td>This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates.</td>
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			<td>NanoDrop spectrophotometers</td>
			<td>Thermo Scientific, Inc.</td>
			<td>NanoDrop 2000</td>
			<td>Spectrophotometer can use very small quantities of sample</td>
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			<td>DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software</td>
			<td>BeckmanCoulter Inc, Fullerton, CA</td>
			<td>DU 800</td>
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</table>

methods to discover alternative promoter usage and transcriptional regulation of murine bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

Identification of Bcrp1 alternative promoter utilization in mouse testis by analysis of leader exons
When the EST database at NCBI was probed (April 2015) using the steps outlined in Protocol 1, the ESTs found that were contiguous with the 5&#39; end of exon 2 in Bcrp1 mRNA are shown in Table 1, along with their position in genomic DNA relative...

Watch this Scientific Journal Video about Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 at JoVE.com

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

The overall goal of this set of protocols is to detect alternative first exons in the mRNA of a gene such as Bcrp1 and to establish luciferase-based reporter assays for the putative promoters upstream of the alternative identified first exons. The in-silico methods used can help answer key questions in the transcription and regulation field such as how mRNA expression is controlled in different tissues. The main advantage of this technique is that it uses publicly available genetic data to identify alterative first exons in the mRNA of gene of interest, which implies alternative promoter usage.This method that prepares report constructs and conducts reporter assays can help answer key questions in the gene regulatory field such as the functional activity of a putative alternative promoters. To begin this procedure, obtain the sequence for mouse Bcrp1 exon 2 by inputting the mRNA reference sequence ID into the search window of the Ensembl genome browser and click Go.Next, select a full length sequence that contains 16 exons. Click the Download sequence option.Then, choose the FASTA format and click Preview. In the next screen, copy the preview sequence of exon 2 into the clipboard. Navigate to the blast homepage on the NCBI website.Select Mouse genome. Paste the exon 2 sequence from the clipboard into the query box. After that, select ESTs from the Database drop down.Optimize it for Highly similar sequences and then choose BLAST. When the BLAST run is complete, the results page will appear. Under the Descriptions subheading of the results page, select All, then Download, followed by FASTA in the drop down, and then choose Continue.A txt file will appear. Open and copy the entire file into the clipboard. To identify the location of the EST sequence that is five prime to exon 2 in the Bcrp1 gene, on the BLAST homepage under the Specialized BLAST subheading, select Align two sequences using BLAST.Paste the text file from the clipboard into the query box and enter 372099104 in the Subject Sequence box. Optimize it for Highly similar sequences under Program Selection and run BLAST. Once the results window appears, view the alignments graphically by clicking Graphics in the Alignments window.Use the right and left arrows and zoom to focus on Bcrp1/ABCG2 and the sequence alignments. Then, save the sequence alignments by selecting All in the Descriptions box. Under the Download drop down, select Hit Table and then click Continue.As the position of the five prime end of exon 2 corresponds to the nucleotide 58, 655, 638 in the chromosome six contig, designate this as plus one and then calculate the position of the partial sequences of each EST five prime to 58, 655, 638. A critical step in this procedure is the prediction of multiple first exons. The alternative promoters are hence identified here.Using the sequence of E1U obtained from the EST database, designate the first five prime nucleotide of E1U as plus one. Obtain a back clone of mouse chromosome six that contains the Bcrp1/ABCG2 gene sequence and the sequence at least 100 kilobases upstream of the Bcrp1/ABCG2 gene. Then, identify a suitable reporter vector, such as the luciferase reporter plasmid pGL3-Basic.Determine the multiple cloning sites in the reporter vector. KpnI, SacI, MluI, NheI, SmaI, XhoI, BglII, and HindIII are the restriction endonucleae sites in the multiple cloning site of the pGL3-Basic vector in the five prime to three prime orientation. Next, select the two kilobase region upstream of the E1U exon and paste the sequence in the DNA5 followed by running the program.The details of the single and multiple digestion sites will be displayed. After that, compare the list of digestion sites on the E1U region with the MCS on the pGL3-Basic vector in an excel sheet. Subsequently, prepare forward and reverse primers for PCR that contain restriction endonucleae sites using commercial gene primer synthesis services or primer selection software.Select a forward primer located about two kilobases upstream of the E1U sequence and a reverse primer within the E1U sequence. Then, amplify the E1U genomic region using these primers with PCR 01 to one microgram of Bak and PCR Master Mix containing high fidelity taq polymerase. Afterward, verify the length of the amplified PCR product by comparing it against a one kilobase DNA ladder using 0.8%TAE agarose gel electrophoresis.Afterward, digest the obtained PCR product and the pGL3-Basic vector using restriction endonucleases specific for restriction digestion sites introduced into the forward and reverse primers. Then, verify the digestion of the PCR product using agarose gel electrophoresis. Cut the digested vector DNA and the two kilobase PCR band from the agarose gel.Next, extract the digested vector DNA and the two kilobase PCR band from the gel slices using a commercial SV gel and PCR clean-up systems kit. Measure the concentration of the purified PCR product and the purified vector using UV spectrometry. Subsequently, prepare the E1U reporter construct by ligating the purified restriction enzyme digested PCR product and the pGL3-Basic firefly luciferase reporter vector using the T4 DNA ligase quick ligation kit thereby producing the E1U reporter construct named pGL3 E1U.In this step, culture the TM4 cells in 24-well plates at an initial density of 200, 000 cells per well. Six hours after placing the cells in culture, transfect the cells with 0.2 micrograms of MP pGL3-Basic vector or 0.2 micrograms of pGL3 vector along with four nanograms of pRL-TK as an internal control. Thirty hours following transfection, remove the growth media from the transfected cells.Wash the cells with PBS, then remove the wash solution. After that, measure the firefly and Renilla luciferase activity using a commercial kit. Express the activity for each tube as the ratio of the firefly luciferase activity divided by the internal control.This step confirms the functional activity of the testis specific promoter. Here is the reporter assay for Bcrp1 promoters E1U, E1A, E1B, and E1C in bulbs these are totally cell-derived TM4 cells. Data are expressed as the luminescence of firefly luciferase normalized to that of Renilla luciferase.The asterisk indicates a significant difference from the MP pGL3 vector control. This graph shows the effects of mutation of the SF-1 response element in the Bcrp1 E1U reporter construct on luciferase activity. Bcrp1 reporter constructs with a putative SF-1 binding region were transfected into TM4 cells with and without the enforced expression of SF-1.In the figure, a denotes a significant difference from the empty vector pGL3 control;b signifies a statistically significant difference compared to the E1U promoter construct in the vector transfected cells. While attempting this procedure, it's important to remember to select an appropriate cell line for the reporter assay. The cell line should express the gene of interest under the control of the promoter in the reporter construct.Following this procedure, other methods, such as quantitative RT-PCR can be performed in order to facilitate evaluation of alternative promoter usage in multiple tissue samples. After watching this video, you should have a good understanding of how to predict alternative promoter usage for a gene of interest and to determine the functionality of that promoter.

methods-to-discover-alternative-promoter-usage-transcriptional

Research

JoVE Journal

Biology

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...