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>
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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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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. Dit protocol is belangrijk omdat het ons in staat stelt om de functie van eiwitten te bestuderen, zoals extracellulaire matrixeiwitten, die in latere stadia van myotubevorming of rijping zijn up-regulated. Deze methode kan worden gebruikt om elk gen dat van belang is in C2C12-cellen neer te halen met behulp van specifieke shRNAs en de respectieve analytische instrumenten voor fenotypering. Het belangrijkste voordeel van deze methode is dat we C2C12-cellen hebben gegenereerd die onze genen van...

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

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 ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Cancer Research

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, ...

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

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Biology

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 ...

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided ...

Immunology and Infection

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis.
There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation.
Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy.
We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress.
This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) ...

Neuroscience

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI). Viral particles were concentrated using ultracentrifugation, which resulted in average titers above 5 x 107. Both hESCs and NPCs could be infected at high efficiencies using these lentiviral particles, as shown by puromycin selection and stable expression in hESCs, as well as transient GFP expression in NPCs. Furthermore, western blot analysis showed a significant reduction in the expression of genes targeted by shRNAs. In addition, the cells retained their pluripotency as well as differentiation potential, as evidenced by their subsequent differentiation into different lineages of CNS. The current protocol deals with the delivery of shRNAs; however, the same approach could be used for the ectopic expression of cDNAs for overexpression studies.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine PEI

Using the low-cost cationic polymer polyethylenimine (PEI), we produced lentiviral particles for stable expression of shRNAs in H9 human embryonic stem cells (hESCs) and transiently transduced H9-derived neural progenitor cells (NPCs) at high efficiency.

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) ...

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

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