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) the density of the cultured cells during routine maintenance, and iii) nutrient availability, requiring frequent and regular replenishment of the cell culture medium11,12,13. Due to some unknown mechanisms, higher passage number C2C12 cells also lose the potential for further myoblast fusion11. The instructions from the provider of the C2C12 cells suggest maintaining these cells up to passage number 15. Thereafter, the differentiation potential may be reduced and experiments with such cells may result in less consistent myotube formation. On the other hand, the cell density during maintenance can result in similar effects9. Reaching confluent C2C12 cell densities during routine cell culture promote initiation of C2C12 myoblast differentiation and thus may negatively influence the differentiation potential of the cell population. Therefore, it is of critical importance to prevent C2C12 cells from reaching high cell densities during routine C2C12 cell maintenance. This can be achieved by already sub-culturing C2C12 cells at low cell densities (&#60;50&#8722;60% confluence). Serum starvation is used to induce C2C12 cell differentiation into myotubes. Therefore, maintaining cells for longer times without replenishing medium severely exhaust the nutrient and serum levels. Replenishing with fresh serum containing medium at least every two days can prevent the onset of unwanted premature differentiation due to nutrient and serum deprivation.
C2C12 differentiation is typically induced by serum starvation. The percentage of serum used to induce C2C12 differentiation can greatly influence the results, specifically the time it takes to form MyHC positive myotubes14,15. Several protocols show successful induction of differentiation under various serum concentrations. Use of 2&#8722;10% FBS or horse serum and complete serum deprivation has been reported and all conditions result in C2C12 myotube formation. The serum percentage or change in the serum lot can significantly alter markers for differentiation. In addition, the source of the serum may affect the experimental outcome, because the country of origin may or may not allow certain additives during bovine serum production8. The serum level could be adjusted to achieve the differentiation rate according to specific experimental requirements. Insulin can be added to the culture medium of C2C12 cells to accelerate differentiation and myotube formation16.
The ability to deliver plasmids encoding shRNA or recombinant proteins into C2C12 cells via transfection is an attractive feature of C2C12 cells. Several commercial liposome-based transfection reagents have been used previously to deliver plasmid DNA into C2C12 cells with variable reported transfection efficiencies17,18,19,20,21. PEI also shows reasonable transfection efficiency and is a cost-effective alternative to liposome-based transfection reagents. A comparison between transfection with a commercial liposome-based transfection reagent and PEI showed no considerable change in the transfection efficiency and slightly higher efficiency was found with PEI22. In our hands, transfection efficiency with PEI was sufficient to generate stable puromycin-resistant C2C12 cells. However, a critical step in this protocol is to keep the incubation time of the transfection reagent short. As mentioned above, C2C12 cells are sensitive to serum withdrawal and prolonged exposure to low serum conditions during transfection may favor C2C12 cell differentiation. Since transfection is performed in the absence of serum, the incubation time after transfection was kept short to resupply quickly serum-containing medium to prevent premature differentiation. Puromycin was added to the culture medium 24 h after transfection to initiate the selection of puromycin-resistant C2C12 cells. Methods for introducing plasmid DNA into differentiated myotubes include transfection (up to 85% efficiency reported), electroporation, or a biolistic approach23,24,25. Alternatively, stable C2C12 cells harboring an inducible shRNA plasmid could be generated to knock down genes at later stages of myotube differentiation or during myotube maturation26.
The method described here resulted in stable knockdown of ADAMTSL2 in C2C12 cells where its function during differentiation into myotubes can now be determined. The generation of stable knockdown cells may be especially important to study the function of proteins that are induced during myotube formation or maturation. Transient transfection with siRNA for example would not be sufficient to efficiently knockdown these genes, since the transient knockdown effect of siRNA may wean off after 5&#8722;7 days. Alternatively, the knockout of an ECM protein using CRISPR/Cas9 is technically more challenging and individual clones need to be selected and sequenced to ensure knockout of the desired gene. However, the evolving line of reagents may render CRISPR/Cas9 the method of choice for loss-of-function experiments in the future.

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><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 differentiate into myotubes. C2C12 differentiation upon serum withdrawal can be monitored by bright field microscopy and by immunostaining for the myotube marker myosin heavy chain (MyHC) (Figure 2). MyHC-positive myotubes are observed between 3&#8722;5 days after differentiation initiation. Myotubes are multinucleated as shown by the presence of more than one DAPI-positive nucleus within the MyHC-positive cell boundaries. Figure 3A shows bright field images of stable C2C12 cells cultured in complete DMEM. The knockdown efficiency presented here ranges from 40&#8722;60% (Figure 3B). Since the mRNA was harvested in the proliferative state where little Adamtsl2 is expressed, the knockdown efficiency appears low, but the knockdown efficiency is expected to be larger at later time points during C2C12 differentiation, where endogenous Adamtsl2 is induced and thus expressed at much higher levels. Western blot analysis confirmed the successful knockdown of ADAMTSL2 in the cell lysate obtained from C2C12 cells stably expressing shRNA 3086 compared to control shRNA (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60824/60824fig1.jpg" /> 
Figure 1: Selection of stable C2C12 cells after transfection with shRNA-encoding plasmid DNA. (A) Table showing the target region (CDS, coding sequence; 3&#39;-UTR, 3&#39;-untranslated region), clone ID (hereafter referred to as 1977, 3086, and 972), and sequence of the shRNAs used to target Adamtsl2. (B) The panels show the selection of C2C12 cells transfected with the control shRNA and the three Adamtsl2-targeting shRNAs. C2C12 cells were transfected with the shRNA plasmids and puromycin was added to the medium after 24 h. Puromycin-sensitive cells appear round and eventually detach during routine cell culture maintenance (red arrows). In contrast, puromycin-resistant cells harboring the integrated shRNA plasmids appear spindle-shaped, slightly elongated, attached, and viable (blue arrows). Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60824/60824fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60824/60824fig2.jpg" /> 
Figure 2: C2C12 myoblast to myotube differentiation. Bright field images of differentiating C2C12 cells show dense cobblestone appearance of the cells at the beginning of the differentiation (day 0&#8722;1) and multinucleated myotubes were observed after day 5 (upper row). Immunostaining of differentiating C2C12 cells with myosin heavy chain (MyHC, red), which is a marker for myotubes, is induced at day 3 of differentiation (middle panel). Nuclei were stained with DAPI and the merged image is shown in the lower panels. Scale bars = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60824/60824fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60824/60824fig3.jpg" /> 
Figure 3: Validation of stable knockdown in proliferating C2C12 cells. (A) Bright field images of stable C2C12 cells cultured in complete DMEM. Scale bars = 300 &#181;m. (B) qRT-PCR analysis of Adamtsl2 mRNA expression in stable C2C12 cells. Ct values were normalized to the housekeeping gene Hprt1. mRNA was harvested before the onset of differentiation. Error bars represent standard deviation. (C) Western blot analysis showing reduced ADAMTSL2 protein in the cell lysate/ECM fraction from C2C12 cells stably expressing shRNA 3086. Endogenous ADAMTSL2 was detected using a custom-made polyclonal peptide antibody (available upon request). <a href="https://www.jove.com/files/ftp_upload/60824/60824fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

stable-knockdown-genes-encoding-extracellular-matrix-proteins-c2c12

Nanyang Technological University

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

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Developmental Biology

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biology

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Bioengineering

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Medicine

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression

Source: Hsiao, Y. L., et al. <a href="https://www.jove.com/v/62730">Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro.</a> J. Vis. Exp. (2022).
In this video, we demonstrate a method to perform transduction of shRNA lentiviral vectors to obtain stable knockdown cell lines in immortalized hematopoietic stem and progenitor cells (iHSPCs).

Short Hairpin RNA Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based ShRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression and Generation of Stable Knockdo

Source: Hsiao, Y. L., et al.  Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell ...

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

Summary

Explore More Videos

Chapters in this video

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

TGF-β-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression