Name: stable knockdown of genes encoding extracellular matrix proteins in the c2c12 myoblast cell line using small-hairpin (sh)rna
Uploaded: 2020-02-12T13:30:00
Description: Watch this Scientific Journal Video about Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA at JoVE.com

D.H. is supported by the National Institutes of Health (National Institute for Arthritis and Musculoskeletal and Skin Diseases, NIAMS, grant number AR070748) and seed funding from the Leni &#38; Peter W. May Department of Orthopedics, Icahn School of Medicine at Mt. Sinai.

1. Preparing the shRNA Plasmid DNA from Escherichia coli
<ol>
	<li>Generation of clonal bacterial colonies carrying the shRNA plasmids
	<ol>
		<li>Obtain glycerol stocks of E. coli carrying target-specific shRNA plasmids and a control plasmid from commercial sources (Table of Materials). 
		NOTE: Three different shRNA plasmids were used, targeting different regions of the murine Adamtsl2 mRNA. One shRNA was selected to target the 3&#39;-untranslated region (3&#39;UTR) of...

<ol>
	<li>Tanzer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+of+extracellular+matrix.">Current concepts of extracellular matrix.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 11 (3), 326-331 (2006).</li><li>Hubmacher, D., Apte, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+the+extracellular+matrix:+novel+insights.">The biology of the extracellular matrix: novel insights.</a> Current Opinion in Rheumatology. 25 (1), 65-70 (2013).</li><li>Sakai, L. Y., Keene, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix:+The+driving+force+of+mammalian+diseases.">Extracellular matrix: The driving force of mammalian diseases.</a> Matrix Biology. 71-72, 1-9 (2018).</li><li>Le Goff, C., 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=ADAMTSL2+mutations+in+geleophysic+dysplasia+demonstrate+a+role+for+ADAMTS-like+proteins+in+TGF-beta+bioavailability+regulation.">ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.</a> Nature Genetics. 40 (9), 1119-1123 (2008).</li><li>Dubail, J., Apte, S. 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H., 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=ADAMTS-like+2+(ADAMTSL2)+is+a+secreted+glycoprotein+that+is+widely+expressed+during+mouse+embryogenesis+and+is+regulated+during+skeletal+myogenesis.">ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.</a> Matrix Biology. 26 (6), 431-441 (2007).</li><li>Khodabukus, A., Baar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+serum+origin+on+tissue+engineered+skeletal+muscle+function.">The effect of serum origin on tissue engineered skeletal muscle function.</a> Journal of Cellular Biochemistry. 115 (12), 2198-2207 (2014).</li><li>Bajaj, 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=Patterning+the+differentiation+of+C2C12+skeletal+myoblasts.">Patterning the differentiation of C2C12 skeletal myoblasts.</a> Integrative Biology. 3 (9), 897-909 (2011).</li><li>Mashinchian, O., Pisconti, A., Le Moal, E., Bentzinger, C. 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S., 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=Conditions+that+promote+primary+human+skeletal+myoblast+culture+and+muscle+differentiation+in+vitro.">Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro.</a> American Journal of Physiology-Cell Physiology. 306 (4), 385-395 (2014).</li><li>Conejo, R., Valverde, A. M., Benito, M., Lorenzo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+produces+myogenesis+in+C2C12+myoblasts+by+induction+of+NF-kappaB+and+downregulation+of+AP-1+activities.">Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities.</a> Journal of Cellular Physiology. 186 (1), 82-94 (2001).</li><li>Dodds, E., Dunckley, M. G., Naujoks, K., Michaelis, U., Dickson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofection+of+cultured+mouse+muscle+cells:+a+direct+comparison+of+Lipofectamine+and+DOSPER.">Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.</a> Gene Therapy. 5 (4), 542-551 (1998).</li><li>Balc&#305;, B., Din&#231;er, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfection+of+mouse-derived+C2C12+myoblasts+using+a+matrigel+basement+membrane+matrix.">Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix.</a> Biotechnology Journal. 4 (7), 1042-1045 (2009).</li><li>Xia, D., 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=Overexpression+of+chemokine-like+factor+2+promotes+the+proliferation+and+survival+of+C2C12+skeletal+muscle+cells.">Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells.</a> Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1591 (1), 163-173 (2002).</li><li>Tapia, O., Gerace, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Nuclear+Lamina+Proteins+in+Myoblast+Differentiation+by+Functional+Complementation.">Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.</a> Methods in Molecular Biology. 1411, 177-194 (2016).</li><li>Yamano, S., Dai, J., Moursi, A. 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We describe here a protocol for the stable knockdown of ECM proteins in C2C12 myoblasts and for phenotypic analysis of the differentiation of C2C12 myoblasts into myotubes. Several factors determine the outcome of the experiment and need to be considered carefully. Maintaining C2C12 cells in the proliferating phase is a critical step to keep the C2C12 cells in the myoblast precursor state. Retaining the capability of C2C12 cells to consistently differentiate into myotubes depends on i) the passage number of the cells, ii...

Extracellular matrix (ECM) proteins provide structural support for all tissues, mediate cell-cell communication, and determine cell fate. The formation and dynamic remodeling of ECM is thus critical to maintain tissue and organ homeostasis1,2. Pathological variants in several genes coding for ECM proteins give rise to musculoskeletal disorders with phenotypes ranging from muscular dystrophies to pseudomouscular build3,4. For example, pathogenic variants in ADAMTSL2 cause the extremely rare musculoskeletal disorder geleophysic dysplasia, which presents with pseudomuscular build, i.e., an apparent increase in skeletal muscle mass5. Together with gene expression data in mouse and humans, this suggests a role for ADAMTSL2 in skeletal muscle development or homeostasis6,7.
The protocol that we describe here was developed to study the mechanism by which ADAMTSL2 modulates skeletal muscle development and/or homeostasis in a cell culture setting. We stably knocked down ADAMTSL2 in the murine C2C12 myoblast cell line. C2C12 myoblasts and their differentiation into myotubes is a well-described and widely used cell culture model for skeletal muscle differentiation and skeletal muscle bioengineering8,9. C2C12 cells go through distinct differentiation steps after serum withdrawal, resulting in the formation of multinucleated myotubes after 3&#8722;10 days in culture. These differentiation steps can be reliably monitored by measuring mRNA levels of distinct marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC). One advantage of generating stable gene knockdowns in C2C12 cells is that genes that are expressed at later stages of C2C12 differentiation can be targeted more efficiently, compared to transient knockdown achieved by small-interfering (si) RNA, which typically lasts for 5&#8722;7 days after transfection, and is influenced by the transfection efficiency. A second advantage of the protocol as described here is the relatively fast generation of batches of C2C12 knockdown cells using puromycin selection. Alternatives, such as CRISPR/Cas9-mediated gene knockout or the isolation of primary skeletal muscle cell precursors from human or target-gene deficient mice are technically more challenging or require the availability of patient muscle biopsies or target-gene deficient mice, respectively. However, similar to other cell culture based approaches, there are limitations in the use of C2C12 cells as model for skeletal muscle cell differentiation, such as the two-dimensional (2D) nature of the cell culture set-up and the lack of the in vivo microenvironment that is critical to maintain undifferentiated skeletal muscle precursor cells10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Fisher Chemical</td>
			<td>191784</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Bioreagents</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1760-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter Countesse II</td>
			<td>Invitrogen</td>
			<td>A27977</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Thermo Scientific</td>
			<td>P4205987</td>
			<td></td>
		</tr>
		<tr>
			<td>C2C12 cells</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Chemical</td>
			<td>183172</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Bioreagents</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (Amplification Grade)</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>EMD Millipore</td>
			<td>MAB374</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>VWR Life Sciences</td>
			<td>19C2656013</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse secondary antibody (IRDYE 800CW)</td>
			<td>Li-Cor</td>
			<td>C90130-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti mouse secondary antibody (Rhodamine-red)</td>
			<td>Jackson Immune Research</td>
			<td>133389</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Chemical</td>
			<td>A144S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (Shaker)</td>
			<td>Denville Scientific Corporation</td>
			<td>1704N205BC105</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Amresco, VWR Life Sciences</td>
			<td>2707C122</td>
			<td></td>
		</tr>
		<tr>
			<td>Midiprep plasmid extraction kit</td>
			<td>Qiagen</td>
			<td>12643</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin 4 (myosin heavy chain)</td>
			<td>Invitrogen</td>
			<td>14-6503-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Invitrogen</td>
			<td>2086310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Life Sciences</td>
			<td>241</td>
			<td></td>
		</tr>
		<tr>
			<td>non-ionic surfactant/detergent</td>
			<td>VWR Life Sciences</td>
			<td>18D1856500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MP</td>
			<td>199983</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Bioreagents</td>
			<td>BP399-4</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin antibiotics</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petridishes</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene tubes</td>
			<td>Fisherbrand</td>
			<td>149569C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>33576300</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Fisher Scientific</td>
			<td>BP2956100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR (Real Time)</td>
			<td>Applied Biosystems</td>
			<td>4359284</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcription Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Thermo Scientific</td>
			<td>TK274910</td>
			<td></td>
		</tr>
		<tr>
			<td>sh control plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>07201820MN</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 3086 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092578</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 972 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092579</td>
			<td></td>
		</tr>
		<tr>
			<td>sh 1977 plasmid</td>
			<td>Sigma-Aldrich</td>
			<td>TRCN0000092582</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer (Nanodrop)</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One C</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Reagent Master Mix</td>
			<td>Applied Biosystems</td>
			<td>743566</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid</td>
			<td>Acros Organics</td>
			<td>30145369</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Ambion</td>
			<td>254707</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>GIBCO</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>BP1421</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25%</td>
			<td>Gibco-Life Technology Corporation</td>
			<td>2085459</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (DEPC treated and nuclease free)</td>
			<td>Fisher Bioreagents</td>
			<td>186163</td>
			<td></td>
		</tr>
		<tr>
			<td>Western blotting apparatus</td>
			<td>Biorad</td>
			<td>Mini Protean Tetra Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher Bioreagents</td>
			<td>BP1422</td>
			<td></td>
		</tr>
	</tbody>
</table>

stable knockdown of genes encoding extracellular matrix proteins in the c2c12 myoblast cell line using small-hairpin (sh)rna

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.

Selection of puromycin-resistant C2C12 can be achieved in 10&#8722;14 days after transfection due to efficient elimination of non-resistant, i.e., untransfected cells (Figure 1B). Typically, more than 80% of the cells detach from the cell culture dish and these cells are removed during routine cell maintenance. Puromycin-resistant C2C12 cells expressing the control (scrambled) shRNA retain the spindle-shape, elongated cell morphology at low cell density and the capability to...

Watch this Scientific Journal Video about Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA at JoVE.com

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin shRNA

We provide a protocol to stably knock down genes encoding extracellular matrix (ECM) proteins in C2C12 myoblasts using small-hairpin (sh) RNA. Targeting ADAMTSL2 as an example, we describe the methods for the validation of the knockdown efficiency on the mRNA, protein, and cellular level during C2C12 myoblast to myotube differentiation.

Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA

Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in ...

This protocol is significant because it allows us to study the function of proteins, such as extracellular matrix proteins, that are up-regulated in later stages of myotube formation or maturation. This method can be used to knock down any gene of interest in C2C12 cells by using specific shRNAs and the respective analytical tools for phenotyping. The main advantage of this method is that we have generated C2C12 cells that can continuously suppress our genes of interest, even in mature myotubes.The most important aspect of this procedure is to maintain C2C12 cells in the undifferentiated state, which means at a low cell density during routine cell culture. The day before transfection, seed five times 10 to the fourth C2C12 cells per milliliter in two milliliters of complete DMEM per well in a six-well plate to achieve a 40 to 50%confluence after overnight incubation at 37 degrees Celsius and 5%CO2. The next morning, combine 100 microliters of 25-millimolar sodium chloride in one 1.5-milliliter reaction tube per culture well with 25.5 microliters PEI stock solution of an undifferentiated C2C12 cell culture for a five-minute incubation at 37 degrees Celsius.Next, combine three micrograms of plasmid DNA with 100 microliters of 25-millimolar sodium chloride per culture well for a five-minute incubation at 37 degrees Celsius. At the end of the incubations, combine the entire volume of each diluted PEI reagent with each diluted plasmid solution with gentle pipetting for a 25-minute incubation at 37 degrees Celsius. During the incubation, replace the supernatants of the C2C12 cell cultures with DMEM without antibiotics or serum.At the end of the incubation, add the entire volume of each transfection mix to each well in droplets, while continuously moving the cell culture dish. After six hours in the cell culture incubator, replace the medium in each well with complete DMEM. 24 hours after the transfection, replace the complete DMEM in each well with selection medium supplemented with five micrograms per milliliter of puromycin, and return the cells to the cell culture incubator for 10 to 14 days or until stable C2C12 cells are observed.Then expand the puromycin-resistant C2C12 cells in low cell density cultures in the presence of five micrograms per milliliter of puromycin, and cryopreserve six to 10 vials of cells for future use. To prepare the C2C12 cells for differentiation, seed 1.5 times 10 to the fifth stable puromycin-resistant C2C12 cells in one milliliter of selection medium per well in a 12-well plate, and incubate at 37 degrees Celsius and 5%carbon dioxide until the cultures are 95%confluent. To induce differentiation, replace the medium in each well with serum-free DMEM every two days following the myotube formation on an inverted bright-field microscope equipped with a camera.For myosin heavy chain immunostaining of the differentiated cells, seed five times 10 to the fourth puromycin-resistant C2C12 cells in 500 microliters of complete DMEM per chamber onto an eight-well chamber slide, and return the cells to the cell culture incubator. When the cells reach 95%confluency, replace the selection medium in each well with 500 microliters of serum-free DMEM. At the appropriate experimental time points, rinse the differentiating cells in each well three times with 5 milliliters of PBS per wash before fixing the cells with 200 microliters of 4%paraformaldehyde in PBS per well.After 15 minutes, wash the cells three times with fresh PBS per wash as just demonstrated followed by incubation with 200 microliters of 5-molar glycine in PBS per well for five minutes. At the end of the incubation, wash the cells three times before permeabilizing the cells with 200 microliters of 1%non-ionic surfactant in PBS for 10 minutes. Next, block the nonspecific antibody binding sites with 200 microliters of 5%bovine serum albumin in PBS per well for one hour followed by three washes in PBS.After the last wash, label the cells with 200 microliters of myosin heavy chain antibody for two hours at room temperature. After three PBS washes, incubate the cells with the appropriate secondary antibody for two hours at room temperature, protected from light. After three final washes, aspirate the PBS, and mount the cells with one drop of mounting medium supplemented with DAPI and a coverslip.Then observe each chamber on a fluorescence microscope using the appropriate filter set. To assess the knockdown efficiency in the cell cultures by western blotting, first seed three times 10 to the fifth puromycin-resistant C2C12 cells in two milliliters of complete DMEM per well in a six-well plate. After 24 hours, rinse the cultures with PBS, and initiate differentiation of the cells with two milliliters of serum-free DMEM as demonstrated.For analysis of the secreted proteins at the appropriate experimental time points, collect one milliliter of serum-free conditioned medium from each well into individual 1.5-milliliter reaction tubes for centrifugation. After centrifugation, transfer one milliliter of the conditioned medium supernatants into new 1.5-milliliter reaction tubes, and precipitate the proteins with 391 microliters of trichloroacetic acid in non-ionic surfactant per tube. After 30 seconds of vortexing, incubate the mixtures on ice for 10 minutes.At the end of the incubation, pellet the precipitated proteins by centrifugation, and wash the protein pellets three times with fresh ice-cold acetone and one centrifugation per wash. After the last wash, air-dry the pellets for three to four minutes at room temperature before resuspension in 50 microliters of SDS-PAGE sample buffer per tube. Then boil the samples for five minutes at 95 degrees Celsius before western blot analysis according to standard protocols.For intracellular and membrane-bound protein analysis, rinse the cell layer in each well with two milliliters of PBS per well, and use a cell scraper to carefully dislodge the cells in one-milliliter volumes of PBS. Transfer the cells into individual 1.5-milliliter tubes for centrifugation followed by three washes in one milliliter of PBS per tube per wash via centrifugation. After the last wash, resuspend the cell pellets in 200 microliters of lysis buffer supplemented with EDTA-free protease inhibitor cocktail reagent for a 30-minute incubation on ice.At the end of the incubation, ultrasonicate the samples for 15 seconds on ice with a power output setting of 10 at an operating frequency of 23 kilohertz, and remove the cell debris by centrifugation. Transfer the supernatants into new 1.5-milliliter reaction tubes, and determine the protein concentrations according to standard protocols. Combine 100 micrograms of protein with 5X SDS-PAGE sample buffer in a total volume of 60 microliters per sample, and boil the samples for five minutes at 95 degrees Celsius.Then analyze the samples by western blotting for the presence of the desired target protein. The selection of puromycin-resistant C2C12 cells can be achieved within 10 to 14 days of transfection due to an efficient elimination of the non-resistant cells. Typically, more than 80%of the C2C12 cells detach from the cell culture dish during this time period and are removed during routine cell maintenance.Puromycin-resistant C2C12 cells expressing the control or the ADAMTSL2 shRNA retain their spindle-shaped, elongated cell morphology at a low cell density, as well as their ability to differentiate into myotubes. C2C12 differentiation upon serum withdrawal can be monitored by bright-field microscopy and immunostaining for the myotube marker myosin heavy chain. Myosin heavy chain-positive myotubes are observed between three to five days after differentiation initiation and are multinucleated, as observed by the presence of more than one DAPI-positive nucleus within the cell boundaries.As the gene expression data in proliferation conditions demonstrate, the knockdown efficiency of the transfection is 40 to 60%Western blot analysis can be performed to confirm the success of the knockdown in cell lysates obtained, for example, from C2C12 cells stably expressing shRNA that targets ADAMTSL2 compared to control shRNA expressing cells. Be sure to maintain the puromycin concentration during the selection process high enough to eliminate all of the non-transfected C2C12 cells, or the non-transfected cells may outgrow the transfected cells. This assay allows us to determine the function of extracellular matrix proteins that are expressed later in muscle development, even if they are induced five or 10 days after C2C12 differentiation.Remember that the RNA extraction reagent and beta-mercaptoethanol should be handled in a fume hood and to handle the trichloroacetic acid according to the appropriate safety protocols. Thanks for watching, and good luck with your experiment.

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Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

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 ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...