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.

<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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrillin+protein+pleiotropy:+Acromelic+dysplasias.">Fibrillin protein pleiotropy: Acromelic dysplasias.</a> Matrix Biology. 80, 6-13 (2019).</li><li>Iozzo, R. V., Gubbiotti, M. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+ADAMTS+proteases+and+ADAMTS-like+proteins+from+mammalian+genetics.">Insights on ADAMTS proteases and ADAMTS-like proteins from mammalian genetics.</a> Matrix Biology. 44-46, 24-37 (2015).</li><li>Koo, B. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Muscle+Stem+Cell+Niche+in+Health+and+Disease.">The Muscle Stem Cell Niche in Health and Disease.</a> Current Topics in Developmental Biology. 126, 23-65 (2018).</li><li>Hindi, L., McMillan, J. D., Afroze, D., Hindi, S. M., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culturing,+and+Differentiation+of+Primary+Myoblasts+from+Skeletal+Muscle+of+Adult+Mice.">Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice.</a> Bio-protocol. 7 (9), 2248 (2017).</li><li>Krauss, R. S., Joseph, G. A., Goel, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keep+Your+Friends+Close:+Cell-Cell+Contact+and+Skeletal+Myogenesis.">Keep Your Friends Close: Cell-Cell Contact and Skeletal Myogenesis.</a> Cold Spring Harbor Perspectives in Biology. 9 (2), 029298 (2017).</li><li>Lawson, M. A., Purslow, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+myoblasts+in+serum-free+media:+effects+of+modified+media+are+cell+line-specific.">Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific.</a> Cells Tissues Organs. 167 (2-3), 130-137 (2000).</li><li>Fujita, H., Endo, A., Shimizu, K., Nagamori, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+serum-free+differentiation+conditions+for+C2C12+myoblast+cells+assessed+as+to+active+tension+generation+capability.">Evaluation of serum-free differentiation conditions for C2C12 myoblast cells assessed as to active tension generation capability.</a> Biotechnology and Bioengineering. 107 (5), 894-901 (2010).</li><li>Cheng, C. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+efficiency+of+nonviral+gene+transfer+reagents.">Comparison of transfection efficiency of nonviral gene transfer reagents.</a> Molecular Biotechnology. 46 (3), 287-300 (2010).</li><li>Luo, J., 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=An+efficient+method+for+in+vitro+gene+delivery+via+regulation+of+cellular+endocytosis+pathway.">An efficient method for in vitro gene delivery via regulation of cellular endocytosis pathway.</a> International Journal of Nanomedicine. 10, 1667-1678 (2015).</li><li>Sandri, M., Bortoloso, E., Nori, A., Volpe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotransfer+in+differentiated+myotubes:+a+novel,+efficient+procedure+for+functional+gene+transfer.">Electrotransfer in differentiated myotubes: a novel, efficient procedure for functional gene transfer.</a> Experimental Cell Research. 286 (1), 87-95 (2003).</li><li>Yi, C. E., Bekker, J. M., Miller, G., Hill, K. L., Crosbie, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+potent+RNA+interference+in+terminally+differentiated+myotubes.">Specific and potent RNA interference in terminally differentiated myotubes.</a> Journal of Biological Chemistry. 278 (2), 934-939 (2003).</li><li>Antolik, C., De Deyne, P. G., Bloch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transfection+of+cultured+myotubes.">Biolistic transfection of cultured myotubes.</a> Science's STKE. 2003 (192), 11 (2003).</li><li>Shintaku, J., 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=MyoD+Regulates+Skeletal+Muscle+Oxidative+Metabolism+Cooperatively+with+Alternative+NF-kappaB.">MyoD Regulates Skeletal Muscle Oxidative Metabolism Cooperatively with Alternative NF-kappaB.</a> Cell Reports. 17 (2), 514-526 (2016).</li></ol>

The authors have nothing to disclose.

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...

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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 ...

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Research

JoVE Journal

Genetics

7.7K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

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 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 ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...