The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

1. Cell Culture of EMT Induction
Note: EMT can be induced by treatment of TGF&#946; or ectopic expression of transcription factors Twist, Snail, or Zeb1/2 in epithelial cells. Described in this protocol is an inducible EMT system via expression of the Twist-ER fusion protein in immortalized human mammary epithelial cells (HMLE/Twist-ER, a gift of Dr. J Yang, UCSD)9,11,29. Upon 4-hydroxytamoxifen (TAM) treatment, the fusion protein Twist-ER translocates to the nucleus to drive transcription and results in a full EMT transition in 12-14 days9,11,29. Additional EMT inducible systems, such as HMLE/Snail-ER, HMLE/TGF&#946;, and MCF10A/TGF&#946;, can be found in our previous publications11,30.
<ol>
	<li>Maintain HMLE/Twist-ER cells in serum-free mammary epithelial cell growth medium (MEGM) in a 37 &#176;C incubator with 5% CO2. Passage the cells every 2-3&#160;days when they reach 80% confluence. Incubate cells with 0.15% trypsin at 37 &#176;C for 5-10 min, and then inactivate trypsin with 5% calf serum/DMEM. Spin down cells and resuspend them in MEGM. Plate cells in a new tissue culture dish at a ratio of 1 to 4.</li>
	<li>Dissolve TAM in ethanol to make a 200 &#956;M solution. Protect TAM from light and store at -20 &#176;C. Before use, freshly add TAM into MEGM media at a 1:10,000 dilution to make a final concentration of 20 nM TAM.</li>
	<li>For induction of EMT, plate 1.6 x 106 HMLE/Twist-ER cells into a 10 cm tissue culture dish in duplicate. Culture cells with 20 nM TAM-containing MEGM media.</li>
	<li>Plate 1.6 x&#160;106 HMLE/Twist-ER cells into a 10 cm dish in duplicate and treat cells with 0.01% ethanol as a non-TAM-treated control.</li>
	<li>Two days after incubation, take pictures under a light microscope at 10X&#160;magnification to record morphological changes of cells.</li>
	<li>Aspirate the medium from one dish of control or TAM-treated cells. Wash cells with cold PBS, and harvest cells by scrapping half of the plate in RNA lysis buffer for RNA isolation and the other half in RIPA buffer for protein analysis.</li>
	<li>Passage cells from the control or TAM-treated dishes by re-plating 1.6 x&#160;106 cells in a 10 cm dish in duplicate. Continuously treat the cells with 20 nM TAM or vehicle.</li>
	<li>Repeat steps 5 to 7 once every other day until day 14, at which time, TAM-treated Twist-ER cells show a spindle-shaped mesenchymal morphology, whereas vehicle-treated control cells maintain the cobblestone-like epithelial morphology.</li>
</ol>
2. Characterization of EMT
Note: A completion of EMT is indicated by: (1) Cell morphological changes from a cobblestone-like epithelial morphology to a spindle-shaped fibroblastic-like appearance; (2) Loss of E-cadherin localization at cell-cell junctions; and (3) The switched expression of EMT markers, defined by the loss of epithelial markers and gain of mesenchymal markers.
Cell morphological changes are captured by light microscope at 10X&#160;magnification during the time course of EMT induction, described in Section 1. Immuno-fluorescence detection of E-cadherin at cell-junctions is described in this section. The expression of EMT markers is detected by immunoblotting. Common epithelial markers are E-cadherin, &#947;-catenin, and occludin, and mesenchymal markers include fibronectin, N-cadherin and vimentin. General procedures of immunoblotting are described in Section 4.
<ol>
	<li>At day 12 of TAM treatment, seed 1 x 105 cells on a 12 mm glass circular coverslip placed in a well of a 24-well plate.</li>
	<li>48 hr&#160;after seeding, aspirate medium and wash cells with pre-warmed PBS.</li>
	<li>Fix cells with pre-warmed 4% paraformaldehyde for 15 min.</li>
	<li>Wash cells three times with PBS.</li>
	<li>Incubate cells with 0.2% Triton X-100 in PBS for 15 min&#160;at RT.</li>
	<li>Wash cells three times&#160;with PBS.</li>
	<li>Incubate cells in 5% BSA/PBS for 1 hr at RT to block non-specific binding.</li>
	<li>Incubate cells with the primary antibody E-cadherin at a 1:50 dilution in 1% BSA/PBS in a humidified chamber O/N&#160;at 4 &#176;C. NOTE: The range for primary antibody dilution is normally between 1:50 and 1:200, and the optimal dilution for each antibody should be determined experimentally.</li>
	<li>Decant the primary antibody and wash cells three times&#160;with PBS.</li>
	<li>Incubate cells with fluorescent-labeled secondary antibody in a 1:500 dilution for 1 hr at RT. From this step on, protect cells from light.</li>
	<li>Decant the secondary antibody and wash three times&#160;with PBS.</li>
	<li>Stain cells with 0.1-1 g/ml DAPI or Hoechst for 10 min.</li>
	<li>Rinse cells with PBS three times.</li>
	<li>Mount coverslips with a drop of mounting medium, and seal coverslips with nail polish.</li>
	<li>Observe cells and take images under a 63X&#160;fluorescent microscope.</li>
</ol>
Staining with additional antibodies can be performed to confirm the EMT phenotype. For example, N-cadherin and &#945;-smooth muscle actin can be stained for a mesenchymal phenotype29,31,32. Reorganization of cytoskeletal structure can be monitored by staining cells with phalloidin for F-actin11. In addition, assays for cell motility and cell-death resistance can be performed to examine the properties of EMT11.
3. Detection of Splice Isoforms Using qRT-PCR Assays
<ol>
	<li>Primer Design 
	NOTE: Primer pairs should be carefully designed to amplify distinct splice isoforms. Exon skipping, the most commonly observed alternative splicing event, is shown here as an example to illustrate the strategy of primer design. Figure 1A depicts a four-exon pre-mRNA, with the third exon as a variable exon. Inclusion of exon 3 results in the production of a longer splice isoform, whereas skipping of exon 3 generates a shorter splice isoform. Primers need to be designed to distinguish the exon-3-included isoform and exon-3-skipped isoform, and to detect the total transcript of this mRNA.
	<ol>
		<li>For detection of exon inclusion, carefully design a forward primer inside the variable exon 3, and a reverse primer inside the nearby constitutive exon 4, allowing for the specific amplification of the variable exon-included isoform. Make sure not to design both forward and reverse primers within the same variable exon in order to avoid PCR products amplified from genomic DNA or pre-mRNA.</li>
		<li>To specifically amplify exon skipping events, design one of the primers spanning the junction of the constitutive exon 2 and exon 4 that would otherwise be destroyed when variable exon 3 is included. Design the other primer inside the constitutive exon 4. As such, PCR products are generated only when the variable exon 3 is excluded and the constitutive exon 2 and exon 4 are subsequently joined together.</li>
		<li>To detect the total transcripts of the gene, design the primers within two constitutive exons 1 and 2. Thus, PCR products are amplified from all isoforms.</li>
		<li>Make sure the melting temperature (Tm) for all primers is approximately 55 &#176;C, the length of each primer is approximately 18-22 nucleotides, and the size of PCR products is between 80 and 150 base pairs.</li>
	</ol>
	</li>
	<li>RNA extraction: Extract RNA using a total RNA isolation kit (see Table of Materials).
	<ol>
		<li>Collect cells in 350 &#956;l RNA lysis buffer.</li>
		<li>Add 350 &#956;l of 70% ethanol to the lysate and mix thoroughly.</li>
		<li>Transfer the sample to the RNA purification column.</li>
		<li>Centrifuge at 10,000 x&#160;g for 1 min&#160;and discard flow-through.</li>
		<li>Wash the column once with 500 &#956;l of RNA wash buffer I and twice with RNA wash buffer II.</li>
		<li>Remove residual RNA wash buffer II from the column by centrifugation at maximum speed for 2 min.</li>
		<li>Transfer the column into a new 1.5 ml tube, add 50 &#956;l nuclease-free water at the center of the column matrix and elute RNA by centrifugation at the maximum speed.</li>
	</ol>
	</li>
	<li>Synthesis of First-strand cDNA
	<ol>
		<li>Determine the concentration and quality of RNA by UV absorption measurement. NOTE: Good quality RNA should have a 260 nm / 280 nm absorbance ratio of approximately 2.0.</li>
		<li>Set up reverse transcriptase (RT) reactions that contain 250 ng total RNA, 0.05 &#956;g random hexamer primers, 50 pmol MgCl2, 10 pmol dNTP, 2 &#956;l 5&#215; GoScript buffer, and 1 &#956;l RT enzyme in a final volume of 20 &#956;l.</li>
		<li>Perform RT reactions at 42 &#176;C for 1 hr, followed by incubation at 70 &#176;C for 5 min&#160;to inactivate the RT enzyme.</li>
	</ol>
	</li>
	<li>Real-time PCR
	<ol>
		<li>Set up 20 &#956;l reactions that consist of 10 &#956;l SYBR green master mix, 0.1-1.0 &#956;l cDNA template, and 5 pmol forward and reverse primers.</li>
		<li>Run real-time PCR with 40 cycles of the following steps: 95 &#176;C denaturation for 10 sec, 58 &#176;C annealing for 10 sec, and 60 &#176;C extension for 30 sec. Perform PCR reactions in triplicate.</li>
		<li>Check dissociation curves and make sure that a single peak is observed for each primer set.</li>
		<li>Obtain the quantification cycle (Cq) values by calculating the second derivative values of amplification curves.</li>
		<li>Calculate the relative quantity (RQ) of target mRNAs in induced samples as compared to the uninduced sample using the &#8216;delta delta Cq (&#916;&#916;Cq)&#8217; method as shown by the following formula. Use housekeeping genes, such as GAPDH or TBP, as a reference.</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> 
To more accurately calculate relative values of splice isoforms, the use of multiple reference genes should be considered. The results can be analyzed using a modified absolute value quantification method described by Pfaffl et al33,34.
4. Examination of Splice Isoforms at the Protein Level
<ol>
	<li>Collect cells in 100 &#956;l ice-cold RIPA buffer.</li>
	<li>Set lysates on ice for approximately 10 min.</li>
	<li>Centrifuge at 14,000 x g for 10 min&#160;at 4 &#176;C, and collect the supernatant.</li>
	<li>Measure protein concentration by Bradford assays.</li>
	<li>Dilute protein samples in SDS loading buffer and load 20-40 &#956;g of proteins into 10% SDS-PAGE gels.</li>
	<li>Run SDS-PAGE gels at 20 mA for 1.5 hr.</li>
	<li>Transfer proteins to PVDF membranes in a wet transfer system at 4 &#176;C for 3 hr&#160;at 85 V. Adjust the transfer time according to the molecular weight of target proteins.</li>
	<li>Block membrane in 5% non-fat milk for 30 min&#160;to 1 hr at RT.</li>
	<li>Incubate membrane with a primary antibody at 4 &#176;C O/N. The range for primary antibody dilution is normally between 1:200 and 1:5,000, determine the optimal dilution for each antibody experimentally. The dilution of antibodies used in this protocol can be found in &#8220;Table of specific reagents and equipment&#8221;.
	<ol>
		<li>To detect splice isoforms, use specific antibodies that recognize a particular isoform. Alternatively, use an antibody that recognizes the epitope in the constitutive exon coding region and detect splice isoforms simultaneously based on their different protein sizes.</li>
		<li>At the same time, monitor EMT by immunoblotting for EMT markers. Use E-cadherin, &#947;-catenin, and occludin as epithelial markers, and fibronectin, N-cadherin and vimentin as mesenchymal markers.</li>
	</ol>
	</li>
	<li>Wash membrane three times&#160;with TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at a 5-min&#160;interval.</li>
	<li>Incubate membrane with an HRP-conjugated secondary antibody in a 1:10,000 dilution for 1 hr at RT.</li>
	<li>Wash membrane three times&#160;with TBS-T at a 5-min&#160;interval.</li>
	<li>Visualize protein with a chemiluminescence detection system and expose the membrane to an autoradiography film.</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+post-transcriptional+regulatory+switch+in+polypyrimidine+tract-binding+proteins+reprograms+alternative+splicing+in+developing+neurons.">A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons.</a> Genes Dev. 21, 1636-1652 (2007).</li><li>Salomonis, N., 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=Alternative+splicing+regulates+mouse+embryonic+stem+cell+pluripotency+and+differentiation.">Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.</a> Proc Natl Acad Sci USA. 107, 10514-10519 (2010).</li><li>Calarco, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vertebrate+nervous+system+alternative+splicing+and+development+by+an+SR-related+protein.">Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.</a> Cell. 138, 898-910 (2009).</li><li>Grabowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+takes+shape+during+neuronal+development.">Alternative splicing takes shape during neuronal development.</a> Curr Opin Genet Dev. 21, 388-394 (2011).</li><li>Seol, D. W., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caspase-9+variant+missing+the+catalytic+site+is+an+endogenous+inhibitor+of+apoptosis.">A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis.</a> J Biol Chem. 274, 2072-2076 (1999).</li><li>Srinivasula, S. 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=Identification+of+an+endogenous+dominant-negative+short+isoform+of+caspase-9+that+can+regulate+apoptosis.">Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis.</a> Cancer Res. 59, 999-1002 (1999).</li><li>Akgul, C., Moulding, D. A., Edwards, S. 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The authors declare that they have no competing financial interests.

The procedure described here enables the detection of alternative splicing in an inducible EMT model. As such, dynamic alterations of splice isoform expression can be captured throughout the time course of EMT. This method has advantages over the use of different epithelial- or mesenchymal-representing cell lines for comparison of alternative splicing because distinct genetic backgrounds from un-related cell lines could unduly influence alternative splicing. However, the successful induction of EMT must be carefully conf...

The epithelial-mesenchymal transition (EMT) is a developmental program that drives organ morphogenesis and tissue remodeling during embryogenesis. When abnormally activated, EMT promotes tumor metastasis and organ fibrosis1,2. Compelling studies have described the importance of transcriptional regulation during the process of EMT, defined by several transcription factors, such as Twist, Snail, and ZEB, which repress the expression of the adherens junction protein E-cadherin, resulting in loss of a cobble-stone like epithelial morphology and gain of a spindle-shaped mesenchymal phenotype3-8. Recent studies through genome-wide analysis of RNAs revealed that there exists a group of genes whose splicing patterns are associated with either epithelial or mesenchymal phenotypes9,10. Work from our lab functionally connected alternative splicing and EMT. By studying the cell surface adhesion molecule CD44, we demonstrated that CD44 alternative splicing is tightly regulated during EMT, and more importantly, that CD44 splice isoform switching causally contributes to EMT11.
Alternative splicing represents a widespread and conserved model of gene regulation, as up to 95% of human multi-exon genes are alternatively spliced12-14. By generating multiple protein products from a single gene, alternative splicing constitutes an essential mechanism for protein diversity, adding another layer of complexity to the human genome. As such, dysregulation of alternative splicing could potentially lead to profound biological effects causing human diseases. Indeed, aberrant alternative splicing in diseases has been documented for over a decade15-25, including recent findings that mutations in genes encoding the spliceosome machinery are commonly found in myelodysplastic syndromes26-28. Therefore, developing reliable methods for the detection of alternatively spliced isoforms is of great importance in the study of diverse biological processes including EMT.
Here we provide a protocol to detect changes in alternative splicing using an inducible EMT model. The methods for designing PCR primers and detecting splice isoforms are suitable not only for the study of alternative splicing during EMT, but also for the study of alternative splicing in other biological processes. Investigating alternative splicing during EMT is imperative in order to better understand the mechanisms of EMT and tumor metastasis, thus facilitating the development of effective strategies to treat cancer metastasis.

<table border="0" cellpadding="0" cellspacing="0" width="1167">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

The procedures described above provide a robust method to detect alternative splicing during EMT. Representative results of CD44 splice isoform switching during Twist-induced EMT are given below as an example.
Twist-induced EMT in HMLE/Twist-ER cells was characterized by a transition from a cobble-stone like epithelial phenotype to an elongated fibroblastic phenotype (Figure 2A), the absence of epithelial markers E-cadherin, &#947;-catenin and occludin and the upregulation of ...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

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12.8K Aufrufe.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Serie: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

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.

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 ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...