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Mar 2021
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Electroporation of Small Interfering RNAs into Tibialis Anterior Muscles of Mice
将小干扰 RNA 电穿孔到小鼠胫骨前肌中   

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Abstract

Aging and wasting of skeletal muscle reduce organismal fitness. Regrettably, only limited interventions are currently available to address this unmet medical need. Many methods have been developed to study this condition, including the intramuscular electroporation of DNA plasmids. However, this technique requires surgery and high electrical fields, which cause tissue damage. Here, we report an optimized protocol for the electroporation of small interfering RNAs (siRNAs) into the tibialis anterior muscle of mice. This protocol does not require surgery and, because of the small siRNA size, mild electroporation conditions are utilized. By inducing target mRNA knockdown, this method can be used to interrogate gene function in muscles of mice from different strains, genotypes, and ages. Moreover, a complementary method for siRNA transfection into differentiated myotubes can be used for testing siRNA efficacy before in vivo use. Altogether, this streamlined protocol is instrumental for basic science and translational studies in muscles of mice and other animal models.

Keywords: Electroporation (电穿孔), Skeletal muscle (骨骼肌), Tibialis anterior (胫骨前肌), Small interfering RNAs (小干扰 RNA), Myofiber (肌纤维), Sarcopenia (少肌症), Aging (老化)

Background

Skeletal muscle is an important tissue with many fundamental functions (Wolfe, 2006; Nair, 2005). Consistently, diseases that affect skeletal muscle profoundly impact the organism’s fitness and survival (Demontis and Perrimon, 2009, 2010; Demontis et al., 2013a, 2014; Piccirillo et al., 2014; Rai and Demontis, 2016; Robles-Murguia et al., 2020; Rai et al., 2021a). Among the many muscle diseases, muscle wasting is a debilitating condition associated with aging and with diseases such as cancer, infections, kidney failure, sepsis, and neuromuscular disorders (Demontis et al., 2013b; Bonaldo and Sandri, 2013; Tsoli and Robertson, 2013; Piccirillo et al., 2014). Several studies have demonstrated that muscle wasting worsens disease outcome and decreases patient survival, whereas preserving skeletal muscle mass and function is protective (Zhou et al., 2010, Johnston et al., 2015). Skeletal muscles are composed of multinucleated syncytial cells known as fibers or myofibers. Myofiber types with distinct metabolic and contractile properties are differently abundant in muscles, and this varies in accordance with anatomical location and function (Schiaffino and Reggiani, 2011; Schiaffino et al., 2013). During muscle wasting, these myofiber types are differently susceptible and impacted by atrophic stimuli (Demontis et al., 2013b; Piccirillo et al., 2014; Bonaldo and Sandri, 2013; Ciciliot et al., 2013). Catabolic stimuli induce muscle wasting primarily via the induction of myofiber atrophy, whereas changes in the number of myofibers are not common. Mechanistically, a decrease in the size of myofibers occurs as a result of decreased synthesis and increased degradation of muscle protein, which is mediated by the autophagy/lysosome and ubiquitin-proteasome systems (Demontis et al., 2013b; Piccirillo et al., 2014; Bonaldo and Sandri, 2013). As for other muscle diseases, there are currently no therapies available in the clinic to prevent or cure age- and disease-associated muscle wasting. To address this unmet medical need, many experimental disease models and techniques for probing gene function in skeletal muscles have been developed over the years.


Electroporation has been used as a general method for gene delivery that is potentially applicable to all organisms and cell types (Ugen and Heller, 2003; Young and Dean, 2015). By using electrical fields, electroporation transiently destabilizes the plasma membrane and facilitates the electrophoretic movement and intracellular delivery of DNA plasmids (Ugen and Heller, 2003; Young and Dean, 2015). In skeletal muscle, this technique has been extensively used for the expression of plasmid-encoded transgenes and the subsequent assessment of gene function in skeletal muscle homeostasis (Aihara and Miyazaki, 1998; Rizzuto et al., 1999; Hoover and Magne Kalhovde, 2000; Sandri et al., 2003; Bertrand et al., 2003; Peng et al., 2005; Tevz et al., 2008). Moreover, this experimental approach has found translational application in improving the delivery of DNA plasmids, for mounting immune responses (Widera et al., 2000; Zucchelli et al., 2000; Khan et al., 2014; Vandermeulen et al., 2014; Haidari et al., 2019; Mpendo et al., 2020; Edupuganti et al., 2020), and for gene therapy in humans (Aihara and Miyazaki, 1998; Rizzuto et al., 1999; Mizui et al., 2004 ;Wong et al., 2005; Brolin et al., 2015).


Here, we report an optimized protocol for the electroporation of small interfering RNAs (siRNAs) into the tibialis anterior (TA) skeletal muscle of mice (protocol #1). We propose that this method can help to interrogate gene function in skeletal muscle, by inducing the acute knockdown of the intended target mRNAs in animal models. Moreover, we also report a protocol for siRNA-mediated knockdown in cultured myotubes (protocol #2), to complement in vivo studies with siRNA-mediated electroporation.


Knowledge gained with this siRNA electroporation technique could be similarly applied for improving the intramuscular delivery of other RNA species and RNA-based vaccines, as well as for other translational and basic science applications.


Experimental design and controls
In this protocol, the TA muscle in one hind leg is electroporated with siRNAs for targeting the intended mRNA, whereas the contralateral hind leg serves as control, and is electroporated with non-targeting (NT) siRNAs. Therefore, this experimental design provides a robust internal control unhindered by differences between individual animals. Nonetheless, to ensure that the results obtained from different animals in the same cohort can be cross-compared, it is important to utilize animals of the same age and sex, and reared in a consistent manner (siblings from the same litter should be utilized whenever possible).


A major advantage of the electroporation technique described here is that it allows probing gene function in animals of different ages. Previous studies have used 6-month-old mice as “young” controls (Sheard and Anderson, 2012) because, while postnatal muscle development mostly occurs in the first 3 months of age, growth still occurs in subsequent months (Ebert et al., 2015). However, whenever comparing electroporation experiments done at different ages, especially if these include ages at which postnatal development is not concluded, it is important to normalize the TA mass to the length of the tibia bone. This accounts for differences in whole body size when comparing muscles from different mice, because the tibia is typically static in fully-grown mice, and is not influenced by muscle atrophic and hypertrophic stimuli (Rowland, 2007; Shavlakadze et al., 2010; Puppa et al., 2014; Winbanks et al., 2016).


In addition to testing gene function in wild-type mice, siRNA electroporation can also be used to test the impact of mRNA knockdown in disease settings (e.g., models of cancer cachexia). In this scenario, the electroporation technique provides a suitable intervention for testing the requirement of a certain gene in disease progression. For example, the electroporation of siRNAs can be used to probe whether impeding the upregulation of a cancer-induced gene can preserve myofiber size and prevent cancer-induced TA mass loss. However, in this case, appropriate controls will consist of the electroporation of gene-targeting and control NT siRNAs, in contralateral legs of animals, with and without cancer (or another disease model).


Similarly, siRNA electroporation can be performed not only in wild-type animals but also concomitantly to another genetic intervention [e.g., Cre-Lox–mediated ablation of another gene in muscle (McCarthy et al., 2012)]. In this scenario, the electroporation probes the genetic interaction between the siRNA–targeted and the Cre–targeted genes. However, appropriate controls in this case will consist of the electroporation of gene–targeting and NT siRNAs in contralateral legs of animals with and without Cre–mediated ablation of the second gene tested.


Similar experimental design and controls are also used with the siRNA–mediated knockdown of target genes in cultured mouse C2C12 myotubes. Specifically, qRT-PCR and other cellular/molecular assays are used to test the outcome of siRNAs targeting the intended gene compared to control NT siRNAs. Although the percentage of mRNA knockdown needed to uncover a phenotype varies depending on the mRNA targeted, a previous estimate based on large-scale RNAi testing in Drosophila melanogaster suggests that phenotypes are typically uncovered when the reduction in mRNA levels is above 50% (Sopko et al., 2014 ; Graca et al., 2021).


We propose that testing siRNA efficacy of target gene knockdown in cultured myotubes may constitute an important step towards validation of siRNA reagents, before their use in vivo for TA muscle electroporation.


Limitations and comparison to other models
Because this protocol focuses on the electroporation of siRNAs, it is impacted by the known limitations associated with RNA interference, including the possibility that siRNAs target unintended mRNAs (RNAi off-target effects), and that the mRNA knockdown achieved via siRNAs is partial and insufficient to uncover a phenotype. These initial impediments of RNAi are largely surpassed by late-generation reagents (e.g., siRNA SMARTpools used here) (Setten et al., 2019; Neumeier and Meister, 2020).


Another limitation of siRNA delivery via electroporation is that it induces some, albeit minimal, muscle damage (McMahon and Wells, 2004; Skuk et al., 2013). In fact, the conditions reported in this protocol have been optimized to avoid extensive muscle damage, which has been possible because delivery of siRNAs requires remarkably lower electric fields and plasma membrane perturbation, when compared to the delivery of bulkier DNA plasmids (Aihara and Miyazaki, 1998; Rizzuto et al., 1999; Hoover and Magne Kalhovde, 2000; Sandri et al., 2003; Bertrand et al., 2003; Peng et al., 2005; Tevz et al., 2008), that have nonetheless lead to many landmark discoveries in muscle biology (Murgia et al., 2000; Sandri et al., 2004; Menzies et al., 2010). Specifically, the electrotransfer voltages used here have previously been demonstrated to be optimal (McMahon et al., 2001; Schertzer et al., 2006). Although the higher voltages may induce muscle damage when the skin is removed, and electrodes are placed directly on the muscle, the electroporation efficacy and damage are lower when the electrodes are placed on the skin without surgical incision, as reported here. Moreover, the method used here, by which the electrical field orientation is alternated (from lateral-medial to anterior-posterior), has been demonstrated to transduce more efficiently than the single orientation of the electrical field (Golzio et al., 2012). Therefore, in our opinion, the limitations deriving from the RNAi technology and electroporation are minimal due to the extensive optimization of siRNA reagents for this established technology, and because of the mild and non-surgical electroporation conditions that are used for siRNA delivery.


However, a major limitation of electroporation is that it allows for only a transient knockdown of gene function, which has been reported to be significant for up to 2–3 weeks (Tevz et al., 2008). Therefore, it is not possible to probe the long-term consequences of gene perturbation, as the electroporation only transiently affects mRNA levels. For the same reason, because some proteins are extremely long-lived (Savas et al., 2012; Toyama et al., 2013; Krishna et al., 2021), it may not be possible to impact their levels in the few-weeks timeframe of electroporation.


A further limitation of this protocol is that it allows for the analysis of gene function only in TA muscles. Therefore, it does not provide a means for testing gene function in muscles with different anatomical locations and physiological functions (Schiaffino and Reggiani, 2011; Schiaffino et al., 2013). Therefore, rather than surgery-mediated direct apposition to muscles, electroporation with electrodes apposed to the skin does not allow probing gene function in internal muscles, such as the soleus. Alternative methods for gene delivery to skeletal muscle are available, including adeno–associated viruses and sonodelivery (Decker et al., 2020; Manini et al., 2021), and they may overcome limitations of electroporation–mediated delivery if needed.


Advantages
Nonetheless, compared to other interventions, the siRNA electroporation protocol reported here offers several advantages. Some of the previously noted limitations (see paragraph above) can turn out to be advantageous under certain conditions. For example, the partial knockdown of the intended mRNA might be an issue and impede the uncovering of a phenotype. However, this might be advantageous when targeting a gene with fundamental functions, which would result in the death of the animal if completely ablated and/or impacted across all skeletal muscles.


It has been previously noted that, over generations, knockout animals accumulate compensatory background mutations that can reduce the phenotypic manifestation, and even lead to the complete disappearance of the phenotype (Rossi et al., 2015). In this respect, the electroporation of siRNAs provides an acute intervention for perturbing gene function, and it is therefore unencumbered by the compensatory adjustments that occur in classical genetic mutants over time. Similarly, the genetic background (i.e., the genetic makeup of the animal beyond the mutation in the intended gene) (Ungerer et al., 2003; Burnett et al., 2011; Lucanic et al., 2017; Hou et al., 2019; Koh et al., 2020), the cytoplasmic background (derived from the oocyte, with its set of mitochondria and cytoplasmic pathogens) (Toivonen et al., 2007; Joseph et al., 2013), and the microbiota (Ulgherait et al., 2016 ; Kim et al., 2017; Mamantopoulos et al., 2017; Poussin et al., 2018; Vujkovic- Cvijin et al., 2020) are examples of biological variables that are normally controlled only with a careful experimental design. For example, to ensure that all experimental animals do not differ in their cytoplasmic background, all experimental animals should be derived from the same mother, or at least from related mothers. To ensure that the animals are isogenic and do not differ in their genetic background, they should be back-crossed several times (typically 10×) against the same genetic reference strain.


The protocol reported here is based on the electroporation of siRNAs for the intended gene into the TA muscle of one hind leg, and the electroporation of control non-targeting (NT) siRNAs into the contralateral hind leg of the same animal. On this basis, any observed phenotype obtained with siRNAs for the intended gene versus NT siRNAs is not due to inter-individual differences in the genetic background, cytoplasmic background, or microbiota. Therefore, the protocol described here provides ideal settings for testing gene function in the TA muscle, without confounding biological variables.


Although skeletal muscle primarily consists of syncytial muscle cells (known as fibers or myofibers), there are many infiltrating and associated cells that are important for muscle homeostasis. These include immune cells, endothelial cells, fibroadipogenic progenitors, and satellite muscle stem cells. The electroporation likely targets all these cell types (Wong et al., 2005; Dean, 2013), and hence allows for the investigation of the function of a certain gene, not only in myofibers, but also in other muscle-associated cells.


Potential applications of this protocol and future directions
Due to its simplicity, this protocol can be applied to many different mouse strains and disease models. Moreover, it is potentially applicable to other rodent species that are generally used in research (such as rats), and other emerging rodent disease models, such as the long-lived naked-mole rat, and Octodon degus, a rodent that has been reported to develop spontaneous Alzheimer’s-like disease (Buffenstein, 2005; van Groen et al., 2011; Valenzano et al., 2017). Although these rodents radically differ from each other in regards to many features, including their life trajectories and disease predisposition, they all have TA muscles that are easily accessible for electroporation, without the need of any surgery. However, the TA muscle is bigger in rats; thus, the procedure reported here for mice would need to be scaled up. Alternatively, only a localized area of the TA could be electroporated and eventually sampled with a localized biopsy. Indeed, with appropriate adjustments, the siRNA electroporation described here could prove useful in investigating skeletal muscle biology also in larger model organisms, such as marmosets and monkeys (upon appropriate ethical approvals). In this case, the impact of siRNA electroporation into TA muscles could be tested by obtaining a biopsy of the electroporated area of the TA, without requiring sacrificing the animal (Joyce et al., 2012; Cotta et al., 2021). Beyond the TA, it may also be possible to adapt this method to test the impact of gene knockdown in other skeletal muscles. However, such additional testing would be limited to skeletal muscles located beneath the skin, and hence susceptible to electroporation without surgical incision.


In addition to RNAi–mediated knockdown, this electroporation method can also be used in conjunction with gene editing technologies such as CRISPR, to obtain target gene knockout and overexpression (Gaj et al., 2013; Doudna and Charpentier, 2014; Jiang and Doudna, 2017). For example, electroporation of short-guide RNAs (sgRNAs) into TA muscles of mouse models that express Cas9 or Cas9 fused with a transcriptional activator could lead to target gene deletion or overexpression, respectively, whereas electroporation of the contralateral leg with mock sgRNAs would provide a matched control.


Beyond its use in experimental animal models, it has been proposed that TA electroporation in humans may constitute a means for gene therapy, by improving the intramyofiber delivery of DNA vaccines (Widera et al., 2000; Zucchelli et al., 2000; Khan et al., 2014; Vandermeulen et al., 2014; Haidari et al., 2019; Mpendo et al., 2020; Edupuganti et al., 2020), and potentially also of recently-developed RNA-based vaccines, and for the production of cytokines, growth factors, and other therapeutic factors by the skeletal muscle of patients with a number of diseases (Aihara and Miyazaki, 1998; Rizzuto et al., 1999; Mizui et al., 2004; Wong et al., 2005 ;Brolin et al., 2015).

Materials and Reagents

Biological materials (protocol #1)

  1. Laboratory animals:

    C57BL/6J mice (The Jackson Laboratory, catalog number: 000664). However, this protocol can be used with any mouse strain, and it can be similarly applied also to other experimental rodent models.

    CRITICAL: For electroporation, the animals should be 6 months of age minimum, to ensure postnatal muscle growth has been completed. However, younger animals can be used, if the intent is to probe the role of a gene in postnatal muscle growth.


Biological materials (protocol #2)

  1. C2C12 murine myoblast cells:

    The cells were obtained from the ATCC (ATCC, catalog number: CRL-1772), and were cultured in DMEM + GlutaMax with 10% (v/v) FBS media and 1% (v/v) penicillin/streptomycin at 37°C, with an atmosphere of 5% (v/v) CO2. For differentiation into myotubes, cells were grown up in DMEM + GlutaMax with 2% (v/v) HS media and 1% (v/v) penicillin/streptomycin, under the same conditions.

    CRITICAL: All cells were tested every 6 months for Mycoplasma sp. contamination. The maximum passage for optimal results is 10.


Reagents (protocol #1 and #2)

Electroporation (protocol #1)

  1. Hyaluronidase – Type IV-S from Bovine Testes (Sigma-Aldrich, catalog number: 4272)

  2. 5× siRNA Buffer (Horizon, catalog number: B-002000-UB-100)

  3. 1× PBS (Gibco, catalog number: 10010-023)

  4. 20 nM ON-TARGETplus siRNAs (delivered as individual siRNAs or as a SMARTpool of 4 siRNAs), such as the Non-targeting Control (Horizon, catalog number: D-001810-10-20) or siRNAs to target a gene of interest (Note: Other types of RNAi reagents could also be used in this protocol in place of the ON-TARGETplus siRNAs). Fluorophore-labeled siRNAs (siGLO reagents, such as the siGLO Red Transfection Indicator, D-001630-02-05) can also be used to monitor siRNA delivery when testing this protocol.

  5. Isoflurane (Piramel Critical Care, catalog number: 66794-013-25)

  6. Medical Oxygen [100% (v/v) O2]

  7. Nair Depilatory Cream (Church & Dwight, or equivalent)


Cryopreservation (protocol #1)

  1. Tragacanth Gum (Sigma-Aldrich, catalog number: G1128)

  2. 2-Methylbutane, i.e., isopentane (Sigma-Aldrich, catalog number: 277258)

  3. Ethyl Alcohol, 140 Proof (Pharmco by Greenfield Global, catalog number: 111000140)


Cryo-sectioning (protocol #1)

  1. Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., catalog number: 4583)

  2. Hematoxylin Stain (Cancer Diagnostic, catalog number: SH5777)

  3. Mount Quick Aqueous Mounting, 30 mL (Research Products International, catalog number: 195705)


Immunohistochemistry (protocol #1)

  1. Bovine Serum Albumin (BSA; GoldBio, catalog number: A-420-1)

  2. Triton X-100 (Sigma-Aldrich, catalog number: 9002-93-1)

  3. SC-71-s Primary Antibody (Developmental Studies Hybridoma Bank, catalog number: SC-71)

  4. BF-F3-s Primary Antibody (Developmental Studies Hybridoma Bank, catalog number: BF-F3)

  5. Laminin α2 Antibody (Santa Cruz Biotechnology, catalog number: SC-59854)

  6. Alexa Fluor 488 goat anti-mouse IgG1 (Thermo Fisher, Invitrogen, catalog number: A21121)

  7. Alexa Fluor 555 goat anti-mouse IgM (Thermo Fisher, Invitrogen, catalog number: A21426)

  8. Alexa Fluor 647 goat anti-rat IgG (Thermo Fisher, Invitrogen, catalog number: A21247)

  9. DAPI (Sigma-Aldrich, catalog number: 10-236-276-001)

  10. Slow Fade Gold Antifade (Thermo Fisher, Invitrogen, catalog number: S36937)

  11. Rapid Dry Topcoat Polish (Electron Microscopy Sciences, catalog number: 72180)


Cell Culture (protocol #2)

  1. DMEM, high glucose, GlutaMax Supplement (Gibco, catalog number: 10566106)

  2. Fetal Bovine Serum (FBS; Gibco, catalog number: 10438-026)

  3. 1× Penicillin/Streptomycin 10,000 U/mL (P/S; Gibco, catalog number: 15140122)

  4. Horse Serum (HS; Gibco, catalog number: 26050070)

  5. Opti-MEM Reduced Serum Media (Gibco, catalog number: 31985062)

  6. Lipofectamine 2000 (Invitrogen, catalog number: 11668019)

  7. Cytosine β-D-arabinofuranoside (Sigma-Aldrich, catalog number: C1768)

  8. 0.25% Trypsin-EDTA (w/vol), phenol red (Gibco, catalog number: 25200056)


Myotube Immunostaining (protocol #2)

  1. 16% Paraformaldehyde (PFA) Aqueous Solution, EM Grade (Fisher Scientific, catalog number: 50-980-487)

  2. Myosin 4 Monoclonal Antibody MF20 (Thermo Fisher Scientific, catalog number: 14-6503-82)

  3. Alexa Fluor 555 goat anti-mouse IgG2b (Thermo Fisher, Invitrogen, catalog number: A21147)


Reagent setup (protocol #1 and #2) (see Recipes)

  1. 10% Tragacanth

  2. Hyaluronidase stock solution

  3. Hyaluronidase working solution

  4. 50 μM siRNA stock

  5. 2% BSA blocking buffer

  6. Primary antibody staining solution (frozen slides)

  7. Secondary antibody staining solution (frozen slides)

  8. 10% FBS

  9. 2% HS

  10. 1% P/S

  11. 4% PFA

  12. Primary antibody staining solution (cell culture myotubes)

  13. Secondary antibody staining solution (cell culture myotubes)

Equipment

For protocols #1 and #2

  1. Induction chamber with nose cone (VetEquip V-10 Mobile Unit)

  2. Isoflurane vaporizer (VetEquip, catalog number: 911103)

  3. 29 ½-gauge needles (Fisher Scientific, catalog number: 14-841-32) and U-100 insulin syringes (0.5 mL 0.33 × 12.7 mm; Exel INT, catalog number: 26028)

  4. Electro Square Porator (ECM830 BTX Harvard Apparatus) and electrodes (Genetrodes, Straight, 10mm Gold Tip, catalog number: 45-0114, BTX Harvard Apparatus)

  5. Petri dish, stackable lid 100 mm × 15 mm Sterile (Fisher Scientific, catalog number: FB0875712)

  6. Exel International stainless steel disposable scalpel #11 (Fisher Scientific, catalog number: 14-840-01)

  7. Carbon fiber digital caliper (Fisher Scientific, catalog number: 15-077-957)

  8. Moloney forceps (Roboz Surgical Store, catalog number: RS-8254)

  9. Graefe forceps (Roboz Surgical Store, catalog number: RS-5139)

  10. Dissecting scissors, 4.5” Straight (Roboz Surgical Store, catalog number: RS-5912)

  11. Operating scissors, 4.5” Straight (Roboz Surgical Store, catalog number: RS-6802)

  12. Dressing forceps (World Precision Instruments, catalog number: 500365)

  13. Cork sheets (Fisher Scientific, catalog number: 07-840-10)

  14. Benchtop liquid nitrogen container, 2 L (Thermo Fisher Scientific, catalog number: 2123)

  15. Versi-Dry Dispenser Roll, 20” by 100’ (Thermo Fisher Scientific, catalog number: 62070)

  16. Insulated foam cooler

  17. Precision balance (such as Sartorius Secura Analytical Balance 0.1mg)

  18. 250 mL beaker (DWK Life Sciences, Kimble)

  19. Leica CM3050S Cryostat (Leica Biosystems, catalog number: 14903050S)

  20. Superfrost plus microscope slides (Fisher Scientific, catalog number: 22-037-246)

  21. Specimen disc, 30 mm (Leica Biosystems, catalog number: 14037008587)

  22. Leica disposable blades low-profile 819 (Leica Biosystems, catalog number: 14035838925)

  23. 3 mL Transfer pipet (Falcon, catalog number: 357524)

  24. Coverglass 22 × 30 mm, 1.5 thickness (Fisher Scientific, catalog number: NC1272771)

  25. Single edge industrial razor blade (VWR, catalog number: 55411-050)

  26. Microscope slide box 100p cork (Fisher Scientific, catalog number: 22-267294)

  27. Kimwipes (Fisher Scientific, catalog number: 06-666A)

  28. Wax pen Dako (Agilent, catalog number: S2002)

  29. Laser-scanning confocal microscope (e.g., Nikon C2)

  30. Nikon Elements software (Advanced Research version)

  31. CO2 incubator (5% (vol/vol) CO2, 37°C)

  32. Class II, Type A2 Biological Safety Cabinet

  33. High capacity, benchtop centrifuge (Sorvall T6000D)

  34. T150 cell culture flask (MidSci, catalog number: TP90151)

  35. 6-well Corning Costar Flat Bottom cell culture plates (Corning, catalog number: 3516)

  36. Falcon 50-mL conical tubes (Fisher Scientific, catalog number: 352070)

  37. Corning Costar 10-mL serological pipettes (Corning, catalog number: 4488)

  38. Corning Costar 25-mL serological pipettes (Corning, catalog number: 4489)

  39. Disposable 9-in Pasteur pipet (Fisher Scientific, catalog number: NC9496627)

  40. Portable Pipette Controller (Drummond Scientific Company, catalog number: 4-000-101)

  41. Hausser Scientific Hemocytometer (Fisher Scientific, catalog number: S17036)

  42. Hand Tally counter (Fisher Scientific, catalog number: 07-905-6)

  43. Inverted phase contrast microscope

  44. Biohazardous waste container

  45. Conical tube rack

  46. Fluorescence microscope (Keyence BZ-X700)


Equipment setup


Surgical room (protocol #1)

All animal experiments and dissections should be performed in a designated procedure room with access to an isoflurane anesthesia chamber and oxygen. In the procedure room, sterilize the workstation with 70% alcohol and place down a surgical absorbent pad. Dress in proper personal protective equipment, including full gown, hair cover, shoe covers, face mask, and sterile gloves.


Preparation for dissecting tissues (protocol #1)

Prior to entering the procedure room, prepare for each sample: a 1.5-mL RNAse-free microcentrifuge tube, and a 2-cm × 2-cm cork pad for muscle mounting and cryopreservation. Gather all reagents necessary, including liquid nitrogen, isopentane, and tragacanth. Prepare a plastic beaker with 100 mL of isopentane, insert it into a Styrofoam pad, and float in liquid nitrogen inside an appropriate insulated container with a tight-closing lid. Allow isopentane to reach optimal temperature (-160°C) before freezing any tissue. Sterilize all surgical tools with 70% alcohol.


Cell culture preparation (protocol #2)

All cell culture experiments should be performed in a sterile Class II Type A2 Biological Safety cabinet, in a designated procedure room. All personnel should be trained according to BSL-2 protocols. Experiments should be performed using aseptic technique, and 70% ethanol used to sterilize the cell culture hood, all equipment, and reagents. Biohazard waste should be discarded in the appropriate manner according to institutional and state laws. The appropriate PPE, including gloves and a clean laboratory coat, must be worn. Prior to all experiments, the necessary reagents should be warmed in a 37°C water bath for a minimum of 30 min, unless otherwise stated in the protocol.

Software

  1. GraphPad Prism (version 7 or higher)

  2. ImageJ

Procedure

  1. Electroporation of muscle • Timing ~2.5 h per mouse

    1. Place the mouse in the induction chamber and deliver 3.0% (v/v) isoflurane at a 2.5-L/min flow rate, until the animal is fully anesthetized. Monitor the mouse’s breathing rate and test the efficiency of the anesthesia via the toe-pinch reflex.

      CAUTION: Isoflurane is hazardous if vapor is inhaled. Ensure proper ventilation.

    2. Remove the mouse from the induction chamber and transfer it to a surgical pad. Place the animal in the nose cone apparatus and maintain isoflurane levels.

    3. Add a dime-sized amount of depilatory lotion to the leg for 10 s and wipe it with 70% ethanol.

    4. Inject 30 μL of hyaluronidase working solution directly into the TA muscle. To do so, position the needle with a ~10° tilt onto the desired hind leg, oriented so that you can see the opening of the needle. Insert the needle starting from the lower attachment of the TA until reaching the upper part of the muscle. Slowly release all the solution while pulling the needle out from the muscle (Figure 1a–b). Ensure that all content is delivered. As a result, the muscle will inflate, but you should not see any subcutaneous liquid.

      CRITICAL: Hyaluronidase degrades hyaluronic acid, a component of the extracellular matrix, and is crucial for helping the siRNAs spread through the TA.

    5. Once injected, return the mouse to its cage, and allow it to recover for 2 h before anesthetizing it again.

      CRITICAL: Ensure the mouse has access to food and water during recovery.

    6. While the mouse is recovering, set up the electroporation machine and adjust the settings to read 20 ms, 1 Hz, and 80V. Test the electrodes by pulsing the tips into water. The pulse will produce bubbles if working properly.

      CAUTION: The electrodes can cause damage to the skin if touched directly.

    7. After 2 h from the hyaluronidase injection, anesthetize the mouse again and place it back in the nose cone, maintaining the same levels of isoflurane as before. Inject 30 μL of 50 μM siRNA directly into the TA muscle, in a similar manner as done for the hyaluronidase solution (step 4; Figure 1a–b).

    8. Electroporate the muscle injected with siRNA by placing the Genetrodes electrodes on the previously-shaved skin parallel to the TA muscle, and pulsing four times (each pulse consisting of 1 Hz, 80 V, for 20 ms) with 1 s interval in-between the pulses (Figure 1c). Repeat this process with the electrodes placed perpendicular to the TA muscle (Figure 1d–e). Positioning the electrodes onto any area of the shaved skin on top of the TA will work, as long as firmly pressed on it.

      CAUTION: Make sure the electrodes are not touching each other or your hands. Keep the electrodes 0.2 cm apart from each other.

    9. Return the mouse to the cage for recovery. Plan to dissect the muscle for subsequent analyses 7 days post electroporation.



    Figure 1. Electroporation of siRNAs into the myofibers of tibialis anterior muscles.

    A. Location of the tibialis anterior (TA) muscle and associated tibia bone in the hind leg. b. Injection of hyaluronidase and siRNAs into the TA: these solutions are slowly released while pulling out the needle. c, Set up of the electroporator apparatus. d–e. Parallel (d) and perpendicular (e) placement of electrodes on the skin on top of the TA for the electroporation of siRNAs.


  2. Muscle dissection and preservation • Timing ~7 min per mouse (protocol #1)

    1. Place the mouse into the euthanasia chamber, and deliver 100% (v/v) CO2 at a 3-L/min flow rate for 2 min. Observe the animal for lack of breathing, and maintain the CO2 level for an additional minute. Remove the animal from the chamber, and perform cervical dislocation to confirm the animal’s death. Record its weight (relevant if comparing results obtained from animals of different ages, as explained in the background section), and immediately transfer to the surgical absorbent pad.

    2. Position the animal on its back and spray the hind leg with 70% ethanol. Remove the skin surrounding the leg by making a small cut above the ankle with the operating scissors. Pull the skin back towards the thigh with the Moloney forceps to expose the TA.

      CRITICAL: Carefully remove the fascia (i.e., the connective sheet that covers the muscle) by using the tips of the Moloney forceps.

      CAUTION: All surgical tools are extremely sharp and could cause injury if not used properly.

    3. Sever the distal tendon with a scalpel to release the muscle from the ankle. Using the Moloney forceps, pull the muscle toward the thigh, and hold the tissue in place. Detach the muscle from the proximal attachment and associated bone (i.e., the tibia) using the micro-dissecting scissors. Weigh the muscle and record the measurement.

    4. To preserve the tissue for analysis, cut the TA in half at the mid-belly (the widest section of the muscle) with a scalpel. Prepare the sample for histology by spreading tragacanth on a prepared cork square. Using the Graefe forceps, drag the distal half of the TA muscle, tendon side down, into the tragacanth. Place the cork into the isopentane suspended in liquid nitrogen for a minimum of 30–45 s, to cryopreserve the tissue.

      CAUTION: Isopentane is hazardous if inhaled. Avoid skin contact and wear protective equipment when handling.

    5. Cut the remaining piece of the TA muscle in half again. Collect all the tissue in the prepared 1.5-mL microcentrifuge tube, and place in liquid nitrogen to snap-freeze for further analyses (such as western blot, RNA-seq, proteomics, etc.), following similar procedures as previously described (Meng et al., 2014).

      CRITICAL: Perform an RNA extraction using one of the tissue pieces, to test the knockdown efficiency of the siRNA via qRT-PCR.

    6. Using the operating scissors, cut at the middle of the knee to detach the leg from the animal. Once detached, locate the junction of the knee and tibia bone, and cut again. To expose the bone, press the foot of the animal towards the surgical pad, and pull the skin down. Measure the length of the tibia (in mm) using the digital caliper, and record. The measurement of the tibia length is used to normalize the weight of the TA muscle, in order to account for variability in body and muscle size among experimental animals.

    7. Repeat steps 11–15 on the opposite leg.

      Note: In order to provide an app0ropriate robust control, one TA is electroporated with siRNAs targeting a gene of interest, whereas the other TA in the contralateral leg is electroporated with control non-targeting siRNAs (Figure 2).

    8. Temporarily store the cork and tubes on dry ice during dissections. Store all samples at -80°C for future analysis.



    Figure 2. Dissection of the tibialis anterior muscle and tibia.

    a–i. Step-by-step visual representation of the dissection of the tibialis anterior (TA) muscle from the front of the hind leg. a. A small incision is made at the ankle using operating scissors. b. The skin is removed to expose the underlying TA muscle. c. The fascia is removed. d. The proximal tendon is severed, releasing the TA muscle. e. The TA muscle is pulled towards the thigh. f. The muscle is detached from the tibia bone. g. A cut is made at the level of the knee to detach the lower part of the hind leg. h. The junction between the knee and tibia bone is cut. i. The tibia is placed on the surgical pad, and the foot of the animal is pulled down to expose the bone, so that the full length of the tibia bone can be correctly measured.


  3. Cryo-sectioning of tissue • Timing ~15 min per sample (protocol #1)

    1. Remove the tissue from the -80°C freezer, and place it in the cryostat chamber 30 min prior to sectioning. Adjust the specimen and chamber temperatures to -20°C.

      CRITICAL: Specimen temperature varies depending on specimen type. For skeletal muscle, it is recommended a temperature range between -25°C and -15°C.

    2. Place the specimen disc and brushes into the chamber, and align the disposable blade into the holder.

      CAUTION: The disposable blade is sharp; use caution when handling to avoid injury. The cryostat handwheel should always be in the locked position when not in use.

    3. Apply a sufficient amount of O.C.T. onto the 30-mm specimen disc, and mount the sample. Prepare four microscope slides per sample while the tissue freezes.

    4. Once the tissue is frozen, insert the disc into the specimen head. Using the attached levers, orient the tissue into the desired position, and clamp down.

    5. Adjust the trimming thickness to 10 µm, and bring the specimen towards the disposable blade, by unlocking the handwheel and using the motorized coarse feed. Begin trimming the muscle manually by rotating the handwheel clockwise until the tissue is smooth.

    6. Trim the tissue again with the anti-roll plate down. Using a brush, smooth out the tissue and collect the sample onto a microscope slide.

      TROUBLESHOOTING: If the sections curl or shred, adjust the knob attached to the anti-roll plate, to change its distance from the blade.

    7. To test if the section is trimmed correctly, apply hematoxylin stain to the slide with a dropper, and wash with water after 10 s. Mount the slide and observe under a microscope. If correct, trim and collect the tissue until each additional microscope slide has up to three sections of tissue for subsequent immunostaining.

    8. When finished, lock the handwheel and remove the specimen from the specimen head. Insert a razor blade between the disc and the tissue cork, to detach the sample from the freezing medium. Temporarily store the tissue on dry ice.

      CAUTION: The razor blade is sharp, be careful when using to avoid injury.

    9. Return the specimen head to the home position, and remove the disposable blade. Sweep all sectioning waste into the waste tray, and discard it in the biohazard waste.

      CRITICAL: Do not leave the specimen in the cryostat chamber, as the chamber defrosts every 24 h.

    10. Disinfect the chamber using 70% ethanol, close the chamber sliding window, and turn off the illumination.

    11. Preserve the microscope slides in a slide-box and keep them at -20°C until needed. The slides can be stored at -20°C indefinitely.


  4. Immunostaining slides • Timing ~1.5 h for day one and ~3 h for day two (protocol #1)

    1. Remove the slides from -20°C storage and thaw them in a dry slide box for 10 min at room temperature.

    2. Once thawed, outline a square around the desired tissue on the slide using a hydrophobic wax marker. Hydrate the slides with 1× PBS for 10 min, being careful not to touch the tissue.

    3. Aspirate the PBS from the slides and add 2% BSA-0.1% Triton X-100 blocking solution for 1 h at room temperature.

    4. Aspirate the blocking solution and add the primary antibody staining solution to the slides. Incubate overnight at 4°C. To identify TA myofiber types, the primary antibody solution should include anti-laminin α2, to detect myofiber boundaries, SC-71 antibodies, to immunostain for type IIA myofibers, and BF-F3 antibodies, to immunostain for type IIB myofibers. To minimize evaporation of the antibody solution, add water or soaked paper to the box containing the slides.

    5. The next day, remove the slides from 4°C and aspirate the antibody solution. Wash the slides three times, using 1× PBS at room temperature. Incubate the slides with secondary antibodies at room temperature for 2 h. For the immunostaining of TA myofiber types, the secondary antibody solution should contain appropriate Alexa Fluor-conjugated antibodies, including AF647 goat anti-rat IgG to detect laminin, AF488 goat anti-mouse IgG1 to detect type IIA fibers, and AF555 goat anti-rabbit IgM to detect type IIB fibers. DAPI can also be included to stain the nuclei of myofibers.

    6. Wash the slides twice with 1× PBS. After the final wash, prepare each slide individually by aspirating the 1× PBS from the slides, and removing the wax coating with a kimwipe.

    7. To preserve for imaging, add two drops of antifade mounting medium to the slide, and place a cover glass over the tissue. Allow the slides to air dry for 24 h at room temperature.

      CRITICAL: Fluorescent dyes are light-sensitive, store the slides in a dark place.

    8. After 24 h, seal the slides by coating the edges with clear nail polish, and store at 4°C.

      PAUSE POINT: Samples can be stored at 4°C for weeks or even months, but the signal intensity will decline over time.

    9. Analyze the samples using a laser-scanning confocal microscope (Figure 3).



    Figure 3. Outcome of siRNA electroporation into tibialis anterior muscles.

    a. Representative images of tibialis anterior (TA) skeletal muscles following electroporation of siGLO red and non-fluorescent control non-targeting (NT) siRNAs. Red fluorescence is detected in TA muscles electroporated with siGLO red, whereas no fluorescence is detected with NT siRNA electroporation. b. Red fluorescence is also detected in the myofibers of the TA muscle electroporated with siGLO red. c. Cross-sections of TA muscles 7 days after electroporation with control NT siRNAs and with siRNAs targeting the myokine Cyr61. Immunostaining for different myosin heavy chain isoforms identifies distinct myofiber types: type IIA (green), presumed IIX (black, i.e., lack of staining), and IIB (red) myofibers. Note the lack of damage/regeneration in the muscle electroporated with Cyr61 siRNAs, whereas only minor inflammatory infiltration and small regenerated fibers are marginally seen on the periphery to the right side (yellow asterisk) in the TA muscle electroporated with NT siRNAs. Cyr61 siRNAs induce a shift from type IIX to type IIB myofibers. Data in a–b is reproduced from Hunt LC et al. Cell Reports (2019), and data in c is from Hunt LC et al. Cell Reports (2021a).


  5. Confocal Imaging • Timing ~7 min per slide (protocol #1)

    1. Turn on the Nikon C2 microscope. Open the imaging software program “NIS-Elements-Confocal”. For each laser channel, open the pinhole, and change the pixel size to 1024 × 1024 pixels.

      NOTE: Equivalent laser scanning confocal microscopes and associated image acquisition software can be used for these analyses.

    2. Mount the sample slide on the stage, with the coverslip down. Turn on the “Eye Port”, and use the 4× objective to observe the slide through the eyepiece. Adjust the stage until the lens is centered on the muscle section.

    3. Turn off the eye port, and switch to the 10× objective. Select the Cy5 channel (405 nm), and scan the image live. Adjust the focus until the laminin-stained cell boundaries are clear. For each individual laser channel, optimize the signal amplification by adjusting the gain.

      CRITICAL: Set the gain of the Cy5 channel using a control sample, and keep consistent for all samples.

    4. Under the ND Acquisition tab, select lambda to individually check the confocal single channels: Cy5, FITC, TRITC, and DAPI. Then, select Larger Image, and scan the area in 4 × 4 fields, with an optimal path overlap of 10% stitching.

    5. Select “start run” and save the image as both a ND2 and JPEG file for image analysis.


  6. Image Analysis – Nikon Elements • Timing ~10 min per image (protocol #1)

    1. Open the Nikon Elements – Advanced Research software. Select the analysis controls from the View pulldown menu, and dock the binary layers, thresholding, and object count functions.

    2. Open the image as an ND2 file, and use the polygonal region of interest (ROI) option to draw an outline around the muscle section, to carefully establish a boundary for analysis. Under the result table, record the total area of the muscle, which will be later used to determine the total cross-sectional area (CSA).

      CRITICAL: Outline as close to the border of the muscle as possible, to avoid detection of background by the analysis software.

    3. Under the object count control, observe the restrictions box. Select both Area and MinFeret, and set the desired restrictions. For skeletal muscle, the restriction range is 100–10,000 μm2 and 10–150 μm, for area and Feret’s minimal diameter, respectively. Once set, select keep updating count.

      CRITICAL: Restrictions and ranges can be adjusted according to the experimental model. Parameters can also be added under the results table.

    4. Observe three working layers on the binary layers control: Cy5, FITC, and TRITC, representing laminin, type IIA fibers, and type IIB fibers, respectively. Set the threshold by selecting each layer individually, and adjusting the low/high option. Specifically, the software will display the detected fibers in a different color from the rest of the muscle based on the threshold limit. Using the reference layers, adjust the threshold to accurately represent the fibers by ensuring the threshold is identifying individual myofibers, based on the corresponding immunostaining (i.e., anti-IIA immunostaining only identifies IIA fibers). Once set, keep the thresholds the same for all images.

      CRITICAL: For skeletal muscle, the low threshold for the Cy5 (Alexa 635) channel is always 0, and the high threshold for the FITC/TRITC (Alexa 488/555) channels is always 4095.

    5. To determine type IIA myofibers, open the binary operations dialog box and create an intersection (AND) between the Cy5 and FITC layers. The software will recognize the fibers using the inverse threshold of laminin α2, to determine myofiber boundaries.

    6. After the intersection is created, review the area of each fiber under the results table on the object count control. Use the reference layer for FITC as a guide, and delete any intersection that does not represent a fiber. Export the data into Excel, and label the sheet as the sample name and fiber type.

    7. To determine type IIB myofibers, repeat steps 47–48 for the TRITC channel.

    8. To find non-stained, presumed type IIX fibers, open the binary operations dialog box and create a subtraction (second layer from first layer) between the Cy5 and FITC layers. Repeat this step by using the newly created subtraction layer, and the TRITC layer. Review the area as before, deleting any non-fiber, and export the data to Excel.

    9. De-select keep updating count, and close the image. Do not save changes to the image.

    10. Repeat steps 47–51 for each sample (Figure 4).




    Figure 4. Analysis of TA myofiber size with the Nikon Elements software.

    a. The Nikon Elements software is opened, and the thresholding, binary layers, and object count functions are docked. b. The image is opened, and a polygonal region of interest (ROI) is drawn around the muscle border. c. On the object count function, the Area and MinFeret options are selected, and the restrictions are set to the desired range. The “keep updating count” function is selected. d. Three working layers (Cy5, FITC, and TRITC) are observed, and the thresholds are adjusted to accurately represent the fiber types (IIA, IIB, and IIX), and the myofiber boundary (indicated by anti-Laminin immunostaining). e. An intersection is created between the Cy5 and FITC channels, by using the binary operations dialog box to identify IIA fibers. f. An intersection is created between the Cy5 and TRITC channels, using the binary operations dialog box to identify IIB fibers. g. A subtraction is created between the Cy5 and FITC channels, using the binary operations dialog box. The step is repeated, using the newly created subtraction and the TRITC channel. Non-stained, presumed IIX fibers are detected. After steps e–g, the resulting data is exported to Excel for analysis.


  7. Average Feret’s Minimal Diameter and Percentages of Fibers Analysis using Excel and GraphPad Prism software • Timing ~15 min per sample group (protocol #1)

    1. Add a new sheet to the Excel file with the Nikon Elements analysis data. Create two tables, one for the average Feret’s minimal diameter (FMD), and one for the percentage of fibers. Label the columns with the fiber types (IIA, IIB, and IIX), and the rows with the sample name.

    2. To determine the FMD for each fiber type per sample, take the average of the Feret’s minimal diameter from the exported data. This analysis can also be performed for the cross-sectional area (CSA) of each fiber, by averaging the total area number recorded previously.

    3. To obtain the percentage of fibers, count the total number of fibers for each fiber type per sample from the exported data. Sum the fiber counts (IIA, IIB, and IIX) together for the sample, and divide the individual fiber type count by the sum. Multiply that number by 100 to display the data in percentage.

    4. Graph and analyze the results using GraphPad Prism, or an equivalent graphing software, by creating a new Grouped table, and selecting to “enter number of replicates in side-by-side sub columns”. Title each column as the name of the sample group, and label each row with the fiber type. The number of replicates corresponds to the number of samples per experiment group.

    5. Copy the average FMD or percentage of fibers data calculated in the Excel file, and paste into the corresponding data table.

    6. Graph the data as an individual interleaved scatter plot, and measure the variability by using the mean with standard deviation (SD).

    7. Analyze the data using the 2-way ANOVA test under grouped analyses, to observe significant changes in fiber size or percentage of fibers. Change the parameters by selecting “within each row, compare columns” under the multiple comparisons tab.


  8. Histogram Analysis using GraphPad Prism of the Feret’s Minimal Diameter data • Timing ~20 min (protocol #1)

    1. Open the GraphPad Prism software and create a new Column table, choosing to “enter replicate values, stacked into columns”. Title each column as the name of the sample group, and duplicate the table for each additional fiber type.

    2. For each fiber and sample, copy all the FMD data from the Nikon Elements Excel file and paste into the corresponding data tables.

    3. Using the analyze function, select the frequency distribution option under the Column analyses. Change the parameters to “relative frequency (percentages)” and adjust the bin width and range accordingly. Graph the results as an interleaved bar graph.

    4. Analyze the frequency distribution results, and select non-linear regression (curve fit) under the XY analyses. Under the parameters, use the Gaussian equation, keeping the default settings. This analysis can also be performed for the CSA of the fibers, using the “Area” data from the Nikon Elements Excel file.


  9. C2C12 Cell Culture • Timing ~several days for growth and ~1 h for cell splitting (protocol #2)

    1. Grow the cells in 10% (v/v) FBS DMEM + GlutaMax media at 37°C with 5% (v/v) CO2. When the cells reach 80% confluence, split them, and prepare plates for transfection.

    2. To split the cells, remove the media from the flask, and wash twice with 1× PBS.

      CRITICAL: Avoid adding PBS directly to the cells, as they could detach and be lost.

    3. Detach the cells using 0.25% Trypsin-0.53mM EDTA (w/v) for 3 min at 37°C. To ensure detachment, tap the sides of the flask, and visualize under the microscope.

    4. Neutralize the trypsinization by adding 10 mL of DMEM+10% (v/v) FBS back to the flask. Collect the cells in a 50-mL conical tube and centrifuge the cells at 2,000 × g for 10 min. To keep the cells growing, add 20 mL of 10% FBS media back to the flask, and place in the incubator.

    5. Observe the pellet after centrifugation and aspirate the remaining media. Resuspend the pellet with 3 mL of growth media, adding 1 mL at a time, to ensure proper resuspension.

    6. Count the cells by adding 10 µL of resuspended cells to both chambers of a hemocytometer. Visualize the central area of the chamber (1 mm2) under the microscope, and count the cells with a cell counter. Repeat the step for both chambers of the hemocytometer and average the two counts.

      CRITICAL: Multiply the average count by the conversion factor 10,000 or 104 cells.

    7. Prepare the appropriate amount of 6-well cell culture plates, depending on the number of siRNAs to be tested. Using the total cell number, calculate the volume needed from the suspension to have a final concentration of 20,000 cells/mL per well, with a total well volume of 2 mL.

      CRITICAL: A minimum of 3 wells per siRNA are needed. Also include a siRNA-treated plate (one siRNA per well) for staining, imaging, and analyzing myotube diameter size.

    8. Prepare a mixture of DMEM growth media and resuspended cells, and add the appropriate volume to the cell culture plates. Incubate the plates in 5% (v/v) CO2 at 37°C. Replace the growth media every other day, being careful not to detach the cells.

    9. When the myoblasts reach 80–100% confluence, differentiate the cells to myotubes, by replacing the 10% (v/v) FBS growth media with 2% (v/v) HS differentiation media.

    10. After four days of differentiation, treat the cells with Ara-c for two days in 10% FBS growth media, to kill any remaining myoblasts. The concentration of Ara-c on the first day of treatment should be 4.0 µg/mL, and 0.4 µg/mL on the second day.


  10. Myotube siRNA Transfection • Timing ~45 min (protocol #2)

    1. Prior to transfecting the cells, pre-warm Opti-MEM to 22°C and thaw the appropriate siRNAs on ice. Calculate the amount of reagent needed to have a total volume of 200 µL Opti-MEM, 8 µL of Lipofectamine 2000, and 8 µL of 50 µM siRNA per well.

      CRITICAL: Include a non-targeting (NT) siRNA to use as control. The mixture volumes for Opti-MEM, Lipofectamine 2000, and siRNA can be adjusted based on plate size.

    2. Dilute 8 µL of 50 µM siRNAs into 100 µL of Opti-MEM, and mix gently by pipetting. Repeat this step by adding 8 µL of Lipofectamine 2000 into 100 µL of Opti-MEM. Combine the two solutions, pipetting to mix, and incubate for 10 min.

    3. During the incubation period, remove the media on the prepared 6-well plates, and add 1.8 mL of fresh 10% (v/v) FBS growth media.

    4. After 10 min, add 200 µL of the transfection mixture to each well, and mix gently by rocking the plate back and forth. Incubate the plates with 5% (v/v) CO2 at 37°C for 48 h.

    5. After 24 h, change the media to no serum media (containing 1% P/S).

    6. To check the knockdown induced by the siRNAs, perform an RNA extraction 48 h post transfection, by using the Invitrogen TRIzol reagent and accompanying protocol. Run a qRT-PCR with mRNA-specific oligonucleotides, to quantify the knockdown of the siRNA-targeted mRNA, using the NT siRNA-treated samples as control.


  11. Myotube Immunostaining and Imaging • Timing ~4 h for staining and ~1 h for imaging (protocol #2)

    1. Add 1 mL of 4% PFA to each mL of culture media, and incubate for 10 min at room temperature (the final PFA concentration is 2%).

      CRITICAL: Add the solution to the side of the well to avoid lifting the cells. For the same reason, the fixing solution is added directly to the cell culture medium.

    2. Remove the PFA solution and wash the wells three times with 1× PBS. Aspirate the remaining 1× PBS, and block the wells by adding 1 mL of 2% BSA blocking buffer (2% BSA Triton X-100). Leave the plate gently shaking for 1 h at room temperature.

    3. After blocking the wells, add 300 µL of the primary antibody solution, and gently shake the plate overnight at 4°C.

    4. The following day, remove the antibody, and wash the wells three times with 1× PBS. Add 300 µL of the secondary antibody solution for 2 h at room temperature, gently shaking.

    5. Wash the wells once with 1× PBS, then replace with 1 mL of fresh 1× PBS and store at 4°C, protecting the plate from light, until ready for imaging.

      PAUSE POINT: The cells can remain in PBS for ~1 month, but the signal intensity will decline.

    6. Image the plate using a fluorescent microscope with access to a 532-nm laser channel (TRITC). Using the 10× objective, image each well individually for further analysis.

      CRITICAL: Image a hemocytometer with the brightfield setting, using the same objective as a scale (Figure 5).



    Figure 5. Outcome of siRNA electroporation into C2C12 myoblasts and myotubes.

    a. Representative images of C2C12 myoblasts transfected with siGLO red demonstrate cellular uptake of the fluorescent siRNAs, whereas no fluorescence is seen in myoblasts transfected with non-fluorescent NT siRNAs. b. Similar results are found with transfection of siGLO red into C2C12 myotubes. Data in a–b is reproduced from Hunt LC et al. Cell Reports (2019).


  12. Myotube Diameter Measurement •Timing ~30 min per image (protocol #2)

    1. Open ImageJ. Prior to measuring the myotubes, open the hemocytometer image. Using the straight-line tool, draw a line inside one of the 25 smaller squares (0.25 mm) located in the four corners of the hemocytometer.

    2. Select the set scale option from the Analyze pulldown menu, and enter the size of the square into the “known distance” box as “250” and the unit of length as “µm”. Check the “global” option, and press OK. The scale should be 0.648pixels/µm.

      CRITICAL: All images need to be set to the same scale for accurate analysis.

    3. Open the stitched myotube image and zoom in on individual myotubes. Using the straight-line tool, draw three lines across the diameter, to indicate the beginning, middle, and end of each myotube. After each line, select the measurement option from the Analyze pulldown menu.

    4. Repeat this until a minimum of 50 myotubes per group have been measured. Export the measurement data to Excel.

    5. To determine the myotube diameter, average the three measurements per myotube. Graph the average of each myotube, compared to control, in GraphPad Prism or an equivalent analysis software. Perform appropriate statistical tests to determine whether a significant effect in myotube diameter size is induced by siRNAs (Figure 6).



    Figure 6. Determination of C2C12 myotube diameter with the ImageJ analysis software.

    a. The hemocytometer image is opened in ImageJ to establish a scale. b. By using the straight line tool, a line is drawn inside one of the 25 small squares located in one of the four corners of the hemocytometer. c. The set scale option is selected from the Analyze pulldown menu, and 250 μm is entered as the known pixel distance and unit, respectively. The global option is selected to set the scale as 0.648 pixels/μm. d. The stitched myotube image is opened. e. Individual myotubes are zoomed in. By using the straight line tool, three lines are drawn horizontally across the width of the myotube at the two ends, and in the middle of the myotube. f. After each straight line is drawn, the measurement option is selected from the Analyze pulldown menu. g. A minimum of 50 myotubes are measured, and the data is exported to Excel. The three measurements per myotube are averaged to determine the mean diameter size.


    Anticipated results. With the analyses reported above, TA muscles with a target gene knockdown can be analyzed to determine the resulting impact on myofiber size and myofiber type composition, but also for determining mRNA and protein changes, via follow-up applications such as RNA sequencing and western blot analysis of detergent-soluble and insoluble fractions (Rai et al., 2021b; Hunt et al., 2021a). To analyze myofiber size, muscle cryosections are stained with an anti-laminin antibody, to delineate the boundaries of all myofibers, and antibodies specific for myosin heavy chain isoforms are used to identify distinct myofiber types present in the TA muscle, i.e., fast glycolytic type 2B or IIB myofibers (MHC- 2B- positive) and smaller, more oxidative, type 2A or IIA myofibers (MHC-2A-positive), and 2X or IIX myofibers (MHC-2A-negative and 2B-negative), as previously done (Hunt et al., 2015 , 2021a, 2021b). With these analyses, it is possible to determine whether siRNA-mediated knockdown of a target mRNA in TA muscles leads to changes in myofiber size and in the proportion of distinct myofiber types (Hunt et al., 2015, 2021a, 2021b). When measuring myofiber size in electroporated TA muscles, the Feret’s minimal diameter can be used, as this geometrical parameter enables reliable measurements of myofiber cross-sectional areas, unhindered by cellular distortion introduced by cryosectioning (Bloemberg and Quadrilatero, 2012). Similarly, estimation of the size of cultured C2C12 myotubes transfected with siRNAs provides insight into whether the siRNA-targeted mRNA has a general role in myotube size determination, compared to control NT siRNA transfection [however, myofiber type analysis is routinely not done in cell culture (Huang et al., 2021)]. Hunt et al. (2019) provides an example of similar results obtained with knockdown of the same protein in cultured C2C12 myotubes, and in mouse TA skeletal muscles in vivo: in both cases, knockdown of the E3 ubiquitin ligase UBR4 induces myofiber hypertrophy (Hunt et al., 2019).

      Beyond these anticipated results, the protocol reported above can be coupled with the immunostaining of informative antigens and additional biochemical/molecular assays, to determine whether the acute knockdown of a target mRNA impacts any aspects of muscle homeostasis. This can consist in the estimation of myofiber size, when studying disease-associated muscle wasting, and in the determination of the abundance of organelles or markers associated with optimal muscle function or with muscle disease. In addition to determining cellular and molecular features, TA muscles that have been electroporated can also be analyzed functionally, to determine whether siRNAs targeting certain mRNAs lead to corresponding changes in the isometric and tetanic force (Hunt et al., 2019, 2021b).

      Lastly, this method can overcome logistic limitations of other technical approaches. For example, the study of sarcopenia (i.e., the age-associated decline in skeletal muscle mass and strength) has been hindered by the relatively long time necessary to obtain old mice of a desired genotype. The protocol and optimized conditions reported here for siRNA electroporation into TA muscles may help overcome this limitation. Specifically, we propose that this approach may provide a rapid and robust method for achieving target gene knockdown in the TA muscles of old wild-type mice, and hence for rapidly assessing gene function in the context of sarcopenia.

Recipes

Reagent setup (protocol #1 and #2)

  1. 10% Tragacanth

    Mix 10 mg of tragacanth gum in 100 mL of distilled water. Store at 4°C for two weeks.

  2. Hyaluronidase stock solution

    Mix 10 mg of hyaluronidase powder in 1 mL of 1× PBS. Store at -20°C for up to 1 year.

  3. Hyaluronidase working solution

    Dilute stock solution with 1× PBS to a final concentration of ~0.4 units/mL. Store at -20°C for up to 6 months.

  4. 50 μM siRNA stock

    Resuspend 20 nmol siRNAs in 1× siRNA buffer for a final concentration of 50 μM. Store at -20°C for up to 6 months.

  5. 2% BSA blocking buffer

    Mix 0.4 g of BSA powder (2% (wt/vol) final concentration) and 20 μL of Triton X-100 (0.1% (vol/vol) final concentration) in 20 mL of 1× PBS. Store at 4°C for up to 1 month.

  6. Primary antibody staining solution (frozen slides)

    Dilute primary antibody to a final concentration of 1:150 in 1× PBST (for 1.5 mL: 1.5 mL PBS, 1.5 μL Tween-20, 10 μL of primary antibody)

  7. Secondary antibody staining solution (frozen slides)

    Dilute secondary antibody to a final concentration of 1:200 in 1× PBST (for 2 mL: 2 mL PBS, 2 μL Tween-20, 10 μL of secondary antibody)

  8. 10% Fetal Bovine Serum (FBS)

    Mix 50 mL of FBS with 500 mL DMEM + GlutaMax media.

  9. 2% Horse Serum (HS)

    Mix 10 mL of HS with 500 mL DMEM + GlutaMax media.

  10. 1% Penicillin/Streptomycin

    Add 5.55 mL of P/S to 550 mL DMEM + GlutaMax serum media.

  11. 4% PFA

    Dilute 16% PFA with 1x PBS for a final concentration of 4% (4 mL of 16% PAF and 12 mL of PBS).

  12. Primary antibody staining solution (cell culture myotubes)

    Dilute primary antibody to a final concentration of 1:200 in 1× PBST (for 2 mL: 2 mL PBS, 2 μL Tween-20, 10 μL of primary antibody).

  13. Secondary antibody staining solution (cell culture myotubes)

    Dilute secondary antibody to a final concentration of 1:400 in 1× PBST (for 2 mL: 2 mL PBS, 2 μL Tween-20, 5 μL of secondary antibody).

Acknowledgments

Schemes were drawn with BioRender. The anti-MHC antibodies used to identify myofiber types were obtained from the Developmental Studies Hybridoma Bank. This work was supported by research grants to F.D. from the National Institute on Aging (R01AG055532 and R56AG63806). Research at St. Jude Children’s Research Hospital is supported by the American Lebanese Syrian Associated Charities (ALSAC). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author contributions: A.S. and F.D. wrote the manuscript. F.A.G. and L.C.H. provided feedback. All authors read and edited the manuscript.

Competing interests

The authors declare no competing interests.

Ethics

All experiments were performed in accordance with federal and local regulations. The St. Jude Children’s Research Hospital (SJCRH) animal care and use committee (IACUC) approved all protocols performed. Animals were housed in a ventilated, temperature-controlled facility in the Animal Resource Center at SJCRH in Memphis, Tennessee, USA.

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简介

[摘要] 骨骼肌的老化和消瘦会降低机体的适应性。遗憾的是,目前只有有限的干预措施可用于解决这一未满足的医疗需求。已经开发了许多方法来研究这种情况,包括 DNA 质粒的肌内电穿孔。然而,这种技术需要手术和高电场,这会导致组织损伤。在这里,我们报告了将小干扰 RNA (siRNA) 电穿孔到小鼠胫骨前肌中的优化方案。该协议不需要手术,并且由于 siRNA 尺寸小,使用温和的电穿孔条件。通过诱导目标 mRNA 敲低,该方法可用于询问来自不同品系、基因型和年龄的小鼠肌肉中的基因功能。此外,一种将 siRNA 转染到分化肌管中的补充方法可用于在体内使用之前测试 siRNA 功效。总而言之,这种简化的协议有助于小鼠和其他动物模型肌肉的基础科学和转化研究。

[背景]骨骼肌是具有许多基本功能的重要组织(Wolfe,2006;Nair,2005) 。始终如一地,影响骨骼肌的疾病深刻影响生物体的健康和生存(Demontis and Perrimon, 2009, 2010; Demontis et al. , 2013a, 2014; Piccirillo et al. , 2014; Rai and Demontis, 2016; Robles-Murguia et al . . , 2020; Rai等人, 2021a) .在众多肌肉疾病中,肌肉萎缩是一种与衰老以及癌症、感染、肾衰竭、败血症和神经肌肉疾病等疾病相关的衰弱状况(Demontis等人,2013b;Bonaldo 和 Sandri,2013;Tsoli 和 Robertson, 2013 年;Piccirillo等人,2014 年) 。几项研究表明,肌肉萎缩会恶化疾病结果并降低患者存活率,而保留骨骼肌质量和功能具有保护作用(Zhou et al. , 2010, Johnston et al. , 2015) 。骨骼肌由称为纤维或肌纤维的多核合胞细胞组成。具有不同代谢和收缩特性的肌纤维类型在肌肉中含量不同,这根据解剖位置和功能而有所不同(Schiaffino 和 Reggiani,2011;Schiaffino等人,2013) 。在肌肉萎缩期间,这些肌纤维类型对萎缩性刺激具有不同的敏感性和影响(Demontis等人,2013b;Piccirillo等人,2014;Bonaldo 和 Sandri,2013;Ciciliot等人,2013) 。分解代谢刺激主要通过诱导肌纤维萎缩来诱导肌肉萎缩,而肌纤维数量的变化并不常见。从机制上讲,肌纤维尺寸减小是由于肌肉蛋白合成减少和降解增加,这是由自噬/溶酶体和泛素-蛋白酶体系统介导的(Demontis et al. , 2013b; Piccirillo et al. , 2014) ;博纳尔多和桑德里,2013) 。至于其他肌肉疾病,目前临床上没有可用于预防或治愈与年龄和疾病相关的肌肉萎缩的疗法。为了解决这种未满足的医疗需求,多年来已经开发了许多用于探测骨骼肌基因功能的实验性疾病模型和技术。
电穿孔已被用作基因递送的通用方法,该方法可能适用于所有生物体和细胞类型(Ugen 和 Heller,2003;Young 和 Dean,2015) 。通过使用电场,电穿孔会瞬时破坏质膜并促进DNA 质粒的电泳运动和细胞内递送(Ugen 和 Heller,2003;Young 和 Dean,2015) 。在骨骼肌中,该技术已广泛用于表达质粒编码的转基因和随后评估骨骼肌稳态中的基因功能(Aihara 和 Miyazaki,1998;Rizzuto等,1999;Hoover 和 Magne Kalhovde,2000; Sandri等人,2003;Bertrand等人,2003;Peng等人,2005;Tevz等人,2008) 。此外,该实验方法已在改善 DNA 质粒的递送方面发现了转化应用,以增强免疫反应(Widera等人,2000;Zucchelli等人,2000;Khan等人,2014;Vandermeulen等人,2014; Haidari等人,2019;Mpendo等人,2020;Edupuganti等人,2020),以及用于人类基因治疗(Aihara 和 Miyazaki,1998;Rizzuto等人,1999;Mizui等人,2004;Wong等人,2005;布罗林等人,2015) 。
在这里,我们报告了将小干扰 RNA (siRNA) 电穿孔到小鼠胫骨前 (TA) 骨骼肌中的优化方案(方案 #1)。我们建议这种方法可以通过在动物模型中诱导预期目标 mRNA 的急性敲低来帮助询问骨骼肌中的基因功能。此外,我们还报告了 siRNA 介导的培养肌管敲低的协议(协议 #2),以补充siRNA 介导的电穿孔的体内研究。
通过这种 siRNA 电穿孔技术获得的知识可以类似地应用于改善其他 RNA 种类和基于 RNA 的疫苗的肌肉内递送,以及其他转化和基础科学应用。

实验设计和控制
在该协议中,一条后腿的 TA 肌肉用 siRNA 电穿孔,用于靶向预期的 mRNA,而对侧后腿作为对照,并用非靶向 (NT) siRNA 电穿孔。因此,该实验设计提供了不受个体动物之间差异阻碍的强大内部控制。尽管如此,为了确保从同一队列中的不同动物获得的结果可以进行交叉比较,重要的是使用相同年龄和性别的动物,并以一致的方式饲养(无论何时都应使用来自同一窝的兄弟姐妹)可能的)。
这里描述的电穿孔技术的一个主要优点是它可以探测不同年龄动物的基因功能。以前的研究使用 6 个月大的小鼠作为“年轻”对照(Sheard 和 Anderson,2012) ,因为虽然出生后肌肉发育主要发生在前 3 个月大,但生长仍然发生在随后的几个月(Ebert等人, 2015) 。然而,每当比较在不同年龄进行的电穿孔实验时,特别是如果这些实验包括出生后发育尚未结束的年龄,将 TA 质量标准化为胫骨长度是很重要的。这解释了在比较不同小鼠的肌肉时全身大小的差异,因为成年小鼠的胫骨通常是静止的,并且不受肌肉萎缩和肥大刺激的影响(Rowland,2007;Shavlakadze等,2010;Puppa等人,2014 年;Winbanks等人,2016 年) 。
除了测试野生型小鼠的基因功能外,siRNA 电穿孔还可用于测试 mRNA 敲低在疾病环境中的影响(例如,癌症恶病质模型)。在这种情况下,电穿孔技术提供了一种合适的干预措施,用于测试某种基因在疾病进展中的需求。例如,siRNA 的电穿孔可用于探测阻碍癌症诱导基因的上调是否可以保持肌纤维大小并防止癌症诱导的 TA 质量损失。然而,在这种情况下,适当的对照将包括基因靶向和对照 NT siRNA 的电穿孔,在动物的对侧腿中,有和没有癌症(或其他疾病模型)。
类似地,siRNA 电穿孔不仅可以在野生型动物中进行,还可以与另一种基因干预同时进行[例如,Cre-Lox 介导的肌肉中另一个基因的消融(McCarthy等人,2012 年)]。在这种情况下,电穿孔探测靶向 siRNA 的基因和靶向 Cre 的基因之间的遗传相互作用。然而,在这种情况下,适当的控制将包括基因靶向和 NT siRNA 在动物对侧腿中的电穿孔,有和没有 Cre 介导的第二个测试基因的消融。
类似的实验设计和对照也用于培养的小鼠 C2C12 肌管中 siRNA 介导的靶基因敲低。具体而言, qRT -PCR 和其他细胞/分子测定用于测试靶向预期基因的 siRNA 与对照 NT siRNA 相比的结果。尽管发现表型所需的 mRNA 敲低百分比因靶向 mRNA 而异,但先前基于黑腹果蝇大规模 RNAi 测试的估计表明,当 mRNA 水平降低超过 50% 时,通常会发现表型(Sopko等等人,2014 年;格拉卡等人,2021 年) 。
在体内用于 TA 肌肉电穿孔之前,在培养的肌管中测试靶基因敲低的 siRNA 功效可能构成验证 siRNA 试剂的重要一步。

限制和与其他模型的比较
由于该协议侧重于 siRNA 的电穿孔,因此它受到与 RNA 干扰相关的已知限制的影响,包括 siRNA 靶向非预期 mRNA(RNAi 脱靶效应)的可能性,以及通过 siRNA 实现的 mRNA 敲低是部分且不足的发现表型。 RNAi 的这些初始障碍在很大程度上被晚期试剂(例如,此处使用的 siRNA SMARTpools )所超越(Setten等人,2019;Neumeier 和 Meister,2020) 。
通过电穿孔传递 siRNA 的另一个限制是它会诱导一些肌肉损伤,尽管是最小的损伤(McMahon 和 Wells,2004;Skuk等人,2013) 。事实上,该协议中报告的条件已经过优化,以避免广泛的肌肉损伤,这是可能的,因为与交付更大的 DNA 质粒(Aihara 和宫崎骏, 1998;Rizzuto等人,1999;Hoover 和 Magne Kalhovde,2000;Sandri等人,2003;Bertrand等人,2003;Peng等人,2005;Tevz等人, 2008)肌肉生物学领域的许多里程碑式发现(Murgia等人,2000;Sandri等人,2004;Menzies等人,2010) 。具体而言,此处使用的电转移电压先前已被证明是最佳的(McMahon等人,2001;Schertzer等人,2006) 。尽管在去除皮肤时较高的电压可能会导致肌肉损伤,并且电极直接放置在肌肉上,但当电极放置在皮肤上而不进行手术切口时,电穿孔效率和损伤较低,如本文所述。此外,这里使用的方法是交替电场方向(从外侧-内侧到前-后),已被证明比单一方向的电场更有效地转换(Golzio等人,2012) 。因此,在我们看来,由于 siRNA 试剂针对这项已建立的技术进行了广泛优化,并且由于用于 siRNA 递送的温和和非手术电穿孔条件,RNAi 技术和电穿孔产生的限制是最小的。
然而,电穿孔的一个主要限制是它只允许基因功能的短暂敲除,据报道,这在长达 2-3 周内是显着的(Tevz et al. , 2008) 。因此,不可能探测基因扰动的长期后果,因为电穿孔只会暂时影响 mRNA 水平。出于同样的原因,由于某些蛋白质的寿命极长(Savas等人,2012;Toyama等人,2013;Krishna等人,2021) ,因此可能无法在几周内影响它们的水平电穿孔的时间范围。
该协议的另一个限制是它只允许分析 TA 肌肉中的基因功能。因此,它没有提供一种方法来测试具有不同解剖位置和生理功能的肌肉中的基因功能(Schiaffino 和 Reggiani,2011;Schiaffino等人,2013) 。因此,不是手术介导的直接与肌肉接触,而是将电极放置在皮肤上的电穿孔不允许探测内部肌肉(例如比目鱼肌)中的基因功能。可以使用其他方法将基因递送至骨骼肌,包括腺相关病毒和超声递送 (Decker等人,2020 年;Manini等人,2021 年) ,如果需要,它们可以克服电穿孔介导递送的限制。

优点
尽管如此,与其他干预措施相比,这里报道的 siRNA 电穿孔协议提供了几个优点。前面提到的一些限制(参见上文段落)在某些条件下可能是有利的。例如,预期 mRNA 的部分敲低可能是一个问题,并阻碍了表型的揭示。然而,当靶向具有基本功能的基因时,这可能是有利的,如果完全消融和/或影响所有骨骼肌,这将导致动物死亡。
之前已经注意到,经过几代人的淘汰,基因敲除动物积累了可以减少表型表现的补偿性背景突变,甚至导致表型完全消失(Rossi等人,2015) 。在这方面,siRNA 的电穿孔为扰乱基因功能提供了一种急性干预,因此不受经典遗传突变体随时间推移发生的补偿性调整的影响。同样,遗传背景(即超出预期基因突变的动物的遗传构成) (Ungerer et al. , 2003; Burnett et al. , 2011; Lucanic et al. , 2017; Hou et al. , 2019 ; Koh等人,2020 年) 、细胞质背景(源自卵母细胞,及其线粒体和细胞质病原体) (Toivonen等人,2007 年;Joseph等人,2013 年)和微生物群(Ulgherait等人) . , 2016; Kim et al. , 2017; Mamantopoulos et al. , 2017; Poussin et al. , 2018; Vujkovic-Cvijin et al. , 2020)是通常仅通过仔细的实验设计才能控制的生物变量的示例。例如,为确保所有实验动物的细胞质背景没有差异,所有实验动物都应来自同一母体,或至少来自相关母体。为确保这些动物是同基因的并且它们的遗传背景没有差异,它们应该与相同的遗传参考菌株回交数次(通常为 10倍)。
此处报告的协议是基于将预期基因的 siRNA 电穿孔到一条后腿的 TA 肌肉中,以及将对照非靶向 (NT) siRNA 电穿孔到同一动物的对侧后腿中。在此基础上,针对预期基因的 siRNA 与 NT siRNA 相比,任何观察到的表型都不是由于遗传背景、细胞质背景或微生物群的个体间差异。因此,此处描述的协议为测试 TA 肌肉中的基因功能提供了理想的设置,而不会混淆生物变量。
尽管骨骼肌主要由合胞肌细胞(称为纤维或肌纤维)组成,但仍有许多对肌肉稳态很重要的浸润细胞和相关细胞。这些包括免疫细胞、内皮细胞、纤维脂肪生成祖细胞和卫星肌肉干细胞。电穿孔可能针对所有这些细胞类型(Wong等人,2005;Dean,2013),因此可以研究特定基因的功能,不仅在肌纤维中,而且在其他肌肉相关细胞中。

该协议的潜在应用和未来方向
由于其简单性,该协议可应用于许多不同的小鼠品系和疾病模型。此外,它可能适用于研究中常用的其他啮齿动物物种(如大鼠),以及其他新兴的啮齿动物疾病模型,如长寿裸鼹鼠和已报道的啮齿动物Octodon degus发展自发性阿尔茨海默病样疾病(Buffenstein,2005;van Groen等人,2011;Valenzano等人,2017) 。尽管这些啮齿动物在许多特征(包括它们的生活轨迹和疾病易感性)方面彼此完全不同,但它们都具有易于电穿孔的 TA 肌肉,无需任何手术。然而,大鼠的 TA 肌肉更大;因此,这里报告的小鼠程序需要扩大。或者,可以仅对 TA 的局部区域进行电穿孔,并最终通过局部活检进行取样。事实上,通过适当的调整,这里描述的 siRNA 电穿孔可以证明在研究更大的模型生物中的骨骼肌生物学方面也很有用,例如狨猴和猴子(在适当的伦理批准下)。在这种情况下,可以通过对 TA 的电穿孔区域进行活检来测试 siRNA电穿孔对 TA 肌肉的影响,而无需牺牲动物(Joyce等人,2012;Cotta等人,2021) 。除了 TA,也有可能采用这种方法来测试基因敲除对其他骨骼肌的影响。然而,这种额外的测试将仅限于位于皮肤下方的骨骼肌,因此容易在没有手术切口的情况下进行电穿孔。
除了 RNAi 介导的敲除之外,这种电穿孔方法还可以与 CRISPR 等基因编辑技术结合使用,以获得目标基因敲除和过表达(Gaj等,2013;Doudna 和 Charpentier,2014;Jiang 和 Doudna, 2017) 。例如,将短导 RNA (sgRNA) 电穿孔到表达 Cas9 或与转录激活因子融合的 Cas9 的小鼠模型的 TA 肌肉中可能分别导致靶基因缺失或过表达,而用模拟 sgRNA 电穿孔对侧腿将提供一个匹配的控件。
除了在实验动物模型中的应用之外,已经提出通过改善DNA 疫苗的肌纤维内递送,人体中的 TA 电穿孔可以构成基因治疗的一种手段(Widera等人,2000;Zucchelli等人,2000;Khan等人)。等人,2014 年;Vandermeulen等人,2014 年;Haidari等人,2019 年;Mpendo等人,2020 年;Edupuganti等人,2020),以及最近开发的基于 RNA 的疫苗,并可能用于生产多种疾病患者骨骼肌对细胞因子、生长因子和其他治疗因子的影响(Aihara 和 Miyazaki,1998;Rizzuto等,1999;Mizui等,2004;Wong等,2005;Brolin等人,2015) 。

关键字:电穿孔, 骨骼肌, 胫骨前肌, 小干扰 RNA, 肌纤维, 少肌症, 老化

材料和试剂
生物材料(协议#1)
1.实验动物:
C57BL/6J 小鼠(杰克逊实验室,目录号:000664)。然而,该协议可用于任何小鼠品系,它也可以类似地应用于其他实验啮齿动物模型。
关键:对于电穿孔,动物应至少 6 个月大,以确保完成产后肌肉生长。然而,如果目的是探索基因在出生后肌肉生长中的作用,可以使用更年轻的动物。


生物材料(协议#2)
1.C2C12 鼠成肌细胞:
在 37°C 下在含有 10% (v/v) FBS 培养基和 1% (v/v) 青霉素/链霉素的 DMEM + GlutaMax中培养,在 5% (v/v) CO 2的气氛中。为了分化成肌管,细胞在含有 2% (v/v) HS 培养基和 1% (v/v) 青霉素/链霉素的 DMEM + GlutaMax中培养,在相同的条件下。
关键:每 6 个月对所有细胞进行一次支原体检测。污染。最佳结果的最大通道数为 10。


试剂(方案 #1 和 #2)
电穿孔(协议#1)
1.透明质酸酶 - 来自牛睾丸的 IV-S 型(Sigma-Aldrich,目录号:4272)
2.5 × siRNA缓冲液(Horizon,目录号:B-002000-UB-100)
3.1 × PBS(Gibco,目录号:10010-023)
4.20 nM ON- TARGETplus siRNA(作为单个 siRNA 或作为 4 个 siRNA 的SMARTpool提供),例如非靶向对照(Horizon,目录号:D-001810-10-20)或靶向感兴趣基因的 siRNA(注意:本协议中也可以使用其他类型的 RNAi 试剂代替 ON- TARGETplus siRNA)。荧光团标记的siRNA( siGLO试剂,例如siGLO Red Transfection Indicator,D-001630-02-05)也可用于在测试此方案时监测 siRNA 的传递。
5.异氟醚( Piramel Critical Care,目录号:66794-013-25)
6.医用氧气 [100% (v/v) O 2 ]
7.Nair 脱毛膏(Church & Dwight 或同等产品)


冷冻保存(协议#1)
1.黄蓍胶(Sigma-Aldrich,目录号:G1128)
2.2-甲基丁烷,即异戊烷(Sigma-Aldrich,目录号:277258)
3.乙醇,140 Proof( Greenfield Global的Pharmco ,目录号:111000140)


冷冻切片(协议 #1)
1.Tissue-Tek OCT 化合物(Sakura Finetek USA,Inc.,目录号:4583)
2.苏木精染色(Cancer Diagnostic,目录号:SH5777)
3.Mount Quick Aqueous Mounting,30 mL(Research Products International,目录号:195705)


免疫组织化学(方案 #1)
1.牛血清白蛋白(BSA; GoldBio ,目录号:A-420-1)
2.Triton X-100(Sigma-Aldrich,目录号:9002-93-1)
3.SC-71-s 一抗(Developmental Studies Hybridoma Bank,目录号:SC-71)
4.BF-F3-s 一抗(Developmental Studies Hybridoma Bank,目录号:BF-F3)
5.层粘连蛋白α2抗体(Santa Cruz Biotechnology,目录号:SC-59854)
6.Alexa Fluor 488山羊抗小鼠IgG1( Thermo Fisher,Invitrogen,目录号:A21121)
7.Alexa Fluor 555山羊抗小鼠IgM( Thermo Fisher,Invitrogen,目录号:A21426)
8.Alexa Fluor 647山羊抗大鼠IgG( Thermo Fisher,Invitrogen,目录号:A21247)
9.DAPI(Sigma-Aldrich,目录号:10-236-276-001)
10.Slow Fade Gold Antifade( Thermo Fisher,Invitrogen,目录号:S36937)
11.快速干燥面漆抛光剂(电子显微镜科学,目录号:72180)


细胞培养(方案#2)
1.DMEM,高葡萄糖, GlutaMax补充剂(Gibco,目录号:10566106)
2.胎牛血清(FBS;Gibco,目录号:10438-026)
3.1 ×青霉素/链霉素10,000 U/mL(P/S;Gibco,目录号:15140122)
4.马血清(HS;Gibco,目录号:26050070)
5.Opti-MEM 减少血清培养基(Gibco,目录号:31985062)
6.Lipofectamine 2000(Invitrogen,目录号:11668019)
7.胞嘧啶β-D-阿拉伯呋喃糖苷(Sigma-Aldrich,目录号:C1768)
8.0.25% 胰蛋白酶-EDTA(w/vol),酚红(Gibco,目录号:25200056)


肌管免疫染色(方案 #2)
1.16%多聚甲醛(PFA)水溶液,EM级(Fisher Scientific,目录号:50-980-487)
2.肌球蛋白 4 单克隆抗体 MF20( Thermo Fisher Scientific,目录号:14-6503-82)
3.Alexa Fluor 555山羊抗小鼠IgG2b( Thermo Fisher,Invitrogen,目录号:A21147)


试剂设置(方案 #1 和 #2)(参见配方)
1.10% 黄蓍胶
2.透明质酸酶原液
3.透明质酸酶工作液
4.50 μM siRNA 原液
5.2% BSA 封闭缓冲液
6.一抗染色液(冷冻载玻片)
7.二抗染色液(冷冻载玻片)
8.10% 胎牛血清
9.2% 恒生
10.1% P/S
11.4% PFA
12.一抗染色液(细胞培养肌管)
13.二抗染色液(细胞培养肌管)


设备 


对于协议 #1 和 #2
1.带鼻锥的感应室( VetEquip V-10 移动装置)
2.异氟醚蒸发器( VetEquip ,目录号:911103)
3.29 ½ 号针头(Fisher Scientific,目录号:1 4-841-32)和U-100 胰岛素注射器(0.5 mL 0.33 × 12.7 mm; Exel INT,目录号:26028)
4.Electro Square Porator (ECM830 BTX Harvard Apparatus)和电极( Genetrodes ,直,10mm Gold Tip,目录号:45-0114,BTX Harvard Apparatus)
5.培养皿,可堆叠盖 100 mm × 15 mm 无菌(Fisher Scientific,目录号:FB0875712)
6.Exel International不锈钢一次性手术刀#11(Fisher Scientific,目录号:14-840-01)
7.碳纤维数显卡尺(Fisher Scientific,目录号:15-077-957)
8.莫洛尼镊子( Roboz Surgical Store,目录号:RS-8254)
9.Graefe镊子( Roboz Surgical Store,目录号:RS-5139)
10.解剖剪刀,4.5 英寸直( Roboz Surgical Store,目录号:RS-5912)
11.手术剪刀,4.5 英寸直( Roboz Surgical Store,目录号:RS-6802)
12.敷料钳(World Precision Instruments,目录号:500365)
13.软木板(Fisher Scientific,目录号:07-840-10)
14.台式液氮容器,2 L( Thermo Fisher Scientific,目录号:2123)
15.Versi -Dry Dispenser Roll,20 英寸 x 100 英寸( Thermo Fisher Scientific,目录号:62070)
16.绝缘泡沫冷却器
17.精密天平(如 Sartorius Secura Analytical Balance 0.1mg)
18.250 mL 烧杯(DWK Life Sciences,Kimble)
19.Leica CM3050S 低温恒温器(Leica Biosystems,目录号:14903050S)
20.Superfrost plus 显微镜载玻片(Fisher Scientific,目录号:22-037-246)
21.样品盘,30 mm(Leica Biosystems,目录号:14037008587)
22.Leica 一次性刀片低剖面 819(Leica Biosystems,目录号:14035838925)
23.3 mL 移液管(Falcon,目录号:357524)
24.盖玻片22 × 30 mm,1.5厚度(Fisher Scientific,目录号:NC1272771)
25.单刃工业剃须刀片(VWR,目录号:55411-050)
26.显微镜载玻片盒100p软木塞(Fisher Scientific,目录号:22-267294)
27.Kimwipes(Fisher Scientific,目录号:06-666A)
28.蜡笔Dako (Agilent,目录号:S2002)
29.激光扫描共聚焦显微镜(例如,尼康 C2)
30.Nikon Elements 软件(高级研究版)
31.CO 2培养箱 (5% (vol/vol) CO 2 , 37°C)
32.II级,A2型生物安全柜
33.大容量台式离心机( Sorvall T6000D)
34.T150细胞培养瓶( MidSci ,目录号:TP90151)
35.6孔康宁Costar平底细胞培养板(康宁,目录号:3516)
36.Falcon 50-mL 锥形管(Fisher Scientific,目录号:352070)
37.Corning Costar 10-mL血清移液管(Corning,目录号:4488)
38.Corning Costar 25-mL血清移液管(Corning,目录号:4489)
39.一次性 9 英寸巴斯德吸管(Fisher Scientific,目录号:NC9496627)
40.便携式移液器控制器(Drummond Scientific Company,目录号:4-000-101)
41.Hausser Scientific血细胞计数器(Fisher Scientific,目录号:S17036 )
42.手动计数计数器(Fisher Scientific,目录号:07-905-6)
43.倒置相差显微镜
44.生物危险废物容器
45.锥形管架
46.荧光显微镜(Keyence BZ-X700)


设备设置


手术室(方案#1)
所有动物实验和解剖都应在指定的手术室进行,该手术室可以使用异氟醚麻醉室和氧气。在手术室中,用 70% 的酒精对工作站进行消毒,并放置一个手术吸水垫。穿着适当的个人防护装备,包括长袍、发套、鞋套、面罩和无菌手套。


准备解剖组织(协议 #1)
在进入手术室之前,准备好每个样品:一个 1.5-mL不含 RNAse的微量离心管,和一个 2-cm × 2-cm 用于肌肉安装和冷冻保存的软木垫。收集所有必要的试剂,包括液氮、异戊烷和黄蓍胶。准备一个装有 100 mL 异戊烷的塑料烧杯,将其插入聚苯乙烯泡沫塑料垫中,然后在液氮中漂浮在带有密封盖的适当绝缘容器内。在冷冻任何组织之前,让异戊烷达到最佳温度 (-160°C)。用 70% 的酒精对所有手术工具进行消毒。


细胞培养制备(协议 #2)
所有细胞培养实验都应在无菌 II 类 A2 型生物安全柜中的指定程序室中进行。所有人员都应根据 BSL-2 协议进行培训。应使用无菌技术进行实验,并使用 70% 乙醇对细胞培养罩、所有设备和试剂进行消毒。应根据机构和州法律以适当的方式丢弃生物危害废物。必须穿戴适当的 PPE,包括手套和干净的实验室外套。在所有实验之前,必要的试剂应在 37°C 水浴中加热至少 30 分钟,除非方案中另有说明。


软件


1.GraphPad Prism(版本 7 或更高版本)
2.图像J


程序_


A.肌肉电穿孔 • 时间每只小鼠约 2.5 小时
1.将鼠标放在感应室中,以 2.5 升/分钟的流速输送 3.0% (v/v) 异氟醚,直到动物完全麻醉。监测鼠标的呼吸频率,并通过脚趾捏反射测试麻醉效率。
注意:如果吸入蒸气,异氟醚是危险的。确保适当的通风。
2.从感应室中取出鼠标并将其转移到手术垫上。将动物放在鼻锥装置中并保持异氟醚水平。
3.在腿上加入一角硬币大小的脱毛乳液 10 秒,然后用 70% 乙醇擦拭。
4.30 μL的透明质酸酶工作溶液直接注入 TA 肌肉。为此,将针头倾斜约 10 °放置在所需的后腿上,定向以便您可以看到针头的开口。从 TA 的下部附件开始插入针头,直到到达肌肉的上部。将针从肌肉中拉出时缓慢释放所有溶液(图 1a - b)。确保交付所有内容。结果,肌肉会膨胀,但你不应该看到任何皮下液体。
关键:透明质酸酶降解透明质酸,透明质酸是细胞外基质的一种成分,对于帮助 siRNA 通过 TA 扩散至关重要。
5.注射后,将鼠标放回笼子,让它恢复 2 小时,然后再次麻醉。
关键:确保鼠标在恢复过程中能够获得食物和水。
6.在鼠标恢复时,设置电穿孔机并调整设置以读取 20 ms 、1 Hz 和 80V。通过将尖端脉冲到水中来测试电极。如果工作正常,脉冲会产生气泡。
注意:如果直接接触电极,可能会对皮肤造成伤害。
7.注射透明质酸酶 2 小时后,再次麻醉小鼠并将其放回鼻锥中,保持与以前相同水平的异氟醚。以与透明质酸酶溶液类似的方式将30 μL的 50 μM siRNA 直接注入 TA 肌肉(步骤 4;图 1a-b)。
8.Genetrodes电极放置在与 TA 肌肉平行的先前剃过的皮肤上,对注射了 siRNA 的肌肉进行电穿孔,脉冲四次(每个脉冲由 1 Hz、80 V 组成,持续 20 ms ),中间间隔 1 s脉冲(图 1c)。使用垂直于 TA 肌肉放置的电极重复此过程(图 1d-e)。将电极放置在 TA 顶部剃光皮肤的任何区域都可以,只要用力按压即可。
注意:确保电极没有相互接触或您的手。使电极彼此相距 0.2 厘米。
9.将鼠标返回笼子进行恢复。计划在电穿孔后 7 天解剖肌肉以进行后续分析。


图 1. 将 siRNA 电穿孔到胫骨前肌的肌纤维中。
一个。胫骨前肌 ( TA) 肌肉和相关胫骨在后腿中的位置。湾。将透明质酸酶和 siRNA 注入 TA:这些溶液在拔出针头的同时缓慢释放。 c,电穿孔仪的设置。 d - e。将电极平行(d)和垂直(e)放置在 TA 顶部的皮肤上,用于电穿孔 siRNA。


B.肌肉解剖和保存• 时间为每只小鼠约 7 分钟(协议 #1)
10.将鼠标放入安乐死室,以 3-L/min 的流速提供 100% (v/v) CO 2 2 分钟。观察动物是否有呼吸困难,并将 CO 2水平再维持一分钟。将动物从房间中取出,并进行颈椎脱位以确认动物的死亡。记录其重量(如果比较从不同年龄的动物获得的结果相关,如背景部分所述),并立即转移到手术吸水垫。
11.将动物放在其背上,用 70% 乙醇喷洒后腿。用手术剪刀在脚踝上方做一个小切口,去除腿部周围的皮肤。用莫洛尼镊子将皮肤拉回大腿以暴露 TA。
关键:使用 Moloney 镊子的尖端小心地去除筋膜(即覆盖肌肉的结缔层)。
注意:所有手术工具都非常锋利,如果使用不当可能会造成伤害。
12.用手术刀切断远端肌腱,从脚踝释放肌肉。使用莫洛尼钳,将肌肉拉向大腿,并将组织固定到位。使用微解剖剪刀将肌肉与近端附件和相关骨骼(即胫骨)分离。称量肌肉并记录测量值。
13.为了保存组织进行分析,用手术刀在中腹部(肌肉最宽的部分)将 TA 切成两半。通过在准备好的软木广场上传播黄蓍胶来准备组织学样品。使用Graefe钳,将 TA 肌肉的远端半部(肌腱侧向下)拖入黄蓍。将软木塞放入悬浮在液氮中的异戊烷中至少 30-45 秒,以冷冻保存组织。
注意:吸入异戊烷是危险的。处理时避免皮肤接触并穿戴防护设备。
14.再次将剩余的 TA 肌肉切成两半。收集准备好的 1.5-mL 微量离心管中的所有组织,并按照前面描述的类似程序( Meng et等人,2014 年) 。
关键:使用其中一个组织块进行 RNA 提取,以通过qRT - PCR 测试 siRNA 的击倒效率。
15.使用手术剪刀,在膝盖中间剪开,将腿与动物分离。一旦分离,找到膝盖和胫骨的交界处,然后再次切割。为了暴露骨头,将动物的脚压向手术垫,然后将皮肤向下拉。使用数字卡尺测量胫骨的长度(以毫米为单位) ,并记录。胫骨长度的测量用于标准化 TA 肌肉的重量,以解释实验动物的身体和肌肉大小的变异性。
16.在另一条腿上重复步骤 11 - 15。
注意:为了提供适当的稳健控制,一个 TA 用靶向感兴趣基因的 siRNA 进行电穿孔,而对侧腿中的另一个 TA 用对照非靶向 siRNA 进行电穿孔(图 2)。
17.在解剖过程中将软木塞和管子暂时存放在干冰上。将所有样品储存在 -80 °C以供将来分析。




图 2. 胫骨前肌和胫骨的解剖。
一-我。从后腿前部解剖胫骨前 (TA) 肌肉的分步视觉表示。一个。使用手术剪刀在脚踝处做一个小切口。湾。去除皮肤以暴露下面的 TA 肌肉。 C。筋膜被移除。 d。切断近端肌腱,释放 TA 肌。 e. TA 肌肉被拉向大腿。 F。肌肉与胫骨分离。克。在膝盖水平进行切割以分离后腿的下部。 H。膝盖和胫骨之间的连接被切断。我。将胫骨放在手术垫上,将动物的脚向下拉露出骨头,这样就可以正确测量胫骨的全长。


C.组织的冷冻切片 • 每个样本的时间约为 15 分钟(协议 #1)
18.从 -80°C 冰箱中取出组织,切片前 30 分钟将其放入低温恒温器室中。将样品和腔室温度调整至 -20°C。
批判的: 试样温度因试样类型而异。对于骨骼肌,建议温度范围在 -25 °C和 -15 °C之间。
19.将样品盘和刷子放入腔室,并将一次性刀片对准支架。
注意:一次性刀片很锋利;搬运时要小心,以免受伤。不使用时,低温恒温器手轮应始终处于锁定位置。
20.在 30 毫米样品盘上涂抹足量的 OCT,然后安装样品。在组织冻结时为每个样品准备四张显微镜载玻片。
21.组织冷冻后,将圆盘插入样品头。使用附加的杠杆,将组织定位到所需位置,然后向下夹。
22.将修整厚度调整为 10 µm,通过解锁手轮并使用电动粗进刀将样品带到一次性刀片。 通过顺时针旋转手轮开始手动修剪肌肉,直到组织光滑。
23.用防卷板向下再次修剪纸巾。使用刷子将组织弄平并将样品收集到显微镜载玻片上。
故障排除:如果切片卷曲或切碎,请调整连接到防倾板的旋钮,以改变其与刀片的距离。
24.要测试切片是否正确修剪,请用滴管将苏木精染色剂涂抹在载玻片上,10 秒后用水清洗。安装载玻片并在显微镜下观察。如果正确,则修剪并收集组织,直到每个额外的显微镜载玻片具有多达三个组织部分,用于随后的免疫染色。 
25.完成后,锁定手轮并从样品头中取出样品。在圆盘和软木塞之间插入刀片,将样品从冷冻介质中分离出来。暂时将组织存放在干冰上。
注意:刀片很锋利,使用时要小心,以免受伤。
26.将样品头放回原位,取下一次性刀片。将所有切片废物清扫到废物托盘中,并将其丢弃在生物危害废物中。
关键:不要将样品留在低温恒温箱中,因为冷冻箱每 24 小时会解冻一次。
27.使用 70% 乙醇对腔室进行消毒,关闭腔室滑动窗口,然后关闭照明。 
28.将显微镜载玻片保存在幻灯片盒中,并在需要时将其保持在 -20°C。载玻片可在 -20°C 下无限期保存。


D.免疫染色载玻片 • 第一天的时间约为 1.5 小时,第二天的时间约为 3 小时(方案 #1)
29.从 -20°C 存储中取出载玻片,并在干燥的载玻片盒中在室温下解冻 10 分钟。
30.解冻后,使用疏水蜡标记在幻灯片上所需组织周围勾勒出一个正方形。 用 1 × PBS水合载玻片10 分钟,注意不要接触组织。
31.从幻灯片中吸出 PBS,并在室温下加入 2% BSA-0.1% Triton X-100 阻断溶液 1 小时。
32.吸出阻塞溶液并将一抗染色溶液添加到载玻片中。在 4 °C 下孵育过夜。为了识别 TA 肌纤维类型,一抗溶液应包括抗层粘连蛋白 α2,用于检测肌纤维边界、SC-71 抗体,用于免疫染色IIA 型肌纤维,以及 BF-F3 抗体,用于免疫染色IIB 型肌纤维。为了尽量减少抗体溶液的蒸发,请在装有载玻片的盒子中加入水或浸泡过的纸。
33.第二天,从 4°C 中取出载玻片并吸出抗体溶液。在室温下使用 1 × PBS清洗载玻片 3 次。在室温下用二级抗体孵育载玻片 2 小时。对于 TA 肌纤维类型的免疫染色,二抗溶液应包含适当的 Alexa Fluor 偶联抗体,包括用于检测层粘连蛋白的 AF647 山羊抗大鼠 IgG、用于检测 IIA 型纤维的 AF488 山羊抗小鼠 IgG1 和 AF555 山羊抗兔IgM 检测 IIB 型纤维。 DAPI 也可以用于染色肌纤维的细胞核。
34.× PBS清洗幻灯片两次。最后一次清洗后,通过从载玻片上吸出 1 × PBS 并用kimwipe去除蜡涂层来单独准备每张载玻片。
35.为了保留成像,在幻灯片上添加两滴抗褪色安装介质,并在组织上放置一个盖玻片。让幻灯片在室温下风干 24 小时。
关键:荧光染料对光敏感,请将载玻片存放在黑暗的地方。
36.24 小时后,用透明指甲油涂抹边缘以密封载玻片,并在 4°C 下储存。
暂停点:样品可以在 4 °C下保存数周甚至数月,但信号强度会随着时间的推移而下降。
37.使用激光扫描共聚焦显微镜分析样品(图 3)。




图 3. siRNA 电穿孔进入胫骨前肌的结果。
一个。 siGLO红色和非荧光对照非靶向 (NT) siRNA电穿孔后胫骨前部 (TA) 骨骼肌的代表性图像。在用siGLO red电穿孔的 TA 肌肉中检测到红色荧光,而用 NT siRNA 电穿孔没有检测到荧光。湾。 在用siGLO red电穿孔的 TA 肌肉的肌纤维中也检测到红色荧光。 C。用对照 NT siRNA 和靶向肌因子 Cyr61 的 siRNA 电穿孔后 7 天的 TA 肌肉横截面。不同肌球蛋白重链亚型的免疫染色可识别不同的肌纤维类型:IIA 型(绿色)、推测的 IIX(黑色,即缺乏染色)和 IIB(红色)肌纤维。注意在用 Cyr61 siRNA 电穿孔的肌肉中缺乏损伤/再生,而在用 NT siRNA 电穿孔的 TA 肌肉的右侧(黄色星号)外周上只能看到轻微的炎症浸润和小的再生纤维。 Cyr61 siRNA 诱导从 IIX 型到 IIB 型肌纤维的转变。 a-b 中的数据来自Hunt LC等人。 Cell Reports (2019), c 中的数据来自Hunt LC等人。细胞报告(2021a)。


E.共焦成像• 每张幻灯片的时间约为 7 分钟(协议 #1)
38.打开尼康 C2 显微镜。打开成像软件程序“NIS-Elements-Confocal”。对于每个激光通道,打开针孔,并将像素大小更改为 1024 × 1024 像素。
注:等效的激光扫描共聚焦显微镜和相关的图像采集软件可用于这些分析。
39.将样品幻灯片安装在舞台上,盖玻片向下。打开“Eye Port”,并使用 4 ×物镜通过目镜观察幻灯片。调整舞台,直到镜头位于肌肉部分的中心。
40.关闭眼端口,并切换到 10 ×目标。选择 Cy5 通道 (405 nm),并实时扫描图像。调整焦点,直到层粘连蛋白染色的细胞边界清晰。对于每个单独的激光通道,通过调整增益来优化信号放大。
CRITICAL:使用对照样本设置 Cy5 通道的增益,并为所有样本保持一致。
41.在ND 采集选项卡下,选择 lambda 以单独检查共焦单通道:Cy5、FITC、TRITC 和 DAPI。然后,选择 Larger Image,并在 4 × 4 区域中扫描该区域,最佳路径重叠为 10% 拼接。
42.选择“开始运行”并将图像另存为 ND2 和 JPEG 文件以进行图像分析。


F.图像分析 – Nikon Elements •每张图像约 10 分钟的时间(协议 #1)
43.打开 Nikon Elements – Advanced Research 软件。从视图下拉菜单中选择分析控件,并停靠二进制层、阈值和对象计数功能。
44.将图像作为 ND2 文件打开,并使用多边形感兴趣区域 (ROI) 选项在肌肉部分周围绘制轮廓,以仔细建立分析边界。在结果表下,记录肌肉的总面积,稍后将用于确定总横截面积 (CSA)。
CRITICAL:轮廓尽可能靠近肌肉边界,以避免分析软件检测到背景。
45.在对象计数控制下,观察限制框。选择 Area 和MinFeret ,并设置所需的限制。对于骨骼肌,面积和Feret最小直径的限制范围分别为 100 – 10,000 μm 2和 10 – 150 μm 。设置后,选择保持更新计数。 
CRITICAL:限制和范围可以根据实验模型进行调整。也可以在结果表下添加参数。
46.观察二进制层控件上的三个工作层:Cy5、FITC 和 TRITC,分别代表层粘连蛋白、IIA 型纤维和 IIB 型纤维。通过单独选择每一层并调整低/高选项来设置阈值。具体来说,该软件将根据阈值限制以与肌肉其余部分不同的颜色显示检测到的纤维。使用参考层,通过确保阈值根据相应的免疫染色(即抗 IIA 免疫染色仅识别 IIA纤维)来识别单个肌纤维,调整阈值以准确表示纤维。设置后,保持所有图像的阈值相同。
CRITICAL:对于骨骼肌,Cy5 (Alexa 635) 通道的低阈值始终为 0,而 FITC/TRITC (Alexa 488/555) 通道的高阈值始终为 4095。
47.要确定 IIA 型肌纤维,请打开二元运算对话框并在 Cy5 和 FITC 层之间创建交集 (AND)。该软件将使用层粘连蛋白 α2 的逆阈值识别纤维,以确定肌纤维边界。
48.在对象计数控件的结果表下查看每根纤维的面积。使用 FITC 的参考层作为指导,并删除任何不代表光纤的交点。将数据导出到 Excel 中,并将工作表标记为样品名称和纤维类型。
49.要确定 IIB 型肌纤维,请对 TRITC 通道重复步骤47-48 。
50.要查找未染色的假定 IIX 型纤维,请打开二元操作对话框并在 Cy5 和 FITC 层之间创建一个减法(第一层的第二层)。使用新创建的减法层和 TRITC 层重复此步骤。像以前一样检查该区域,删除任何非光纤,然后将数据导出到 Excel。
51.取消选择保持更新计数,并关闭图像。不要保存对图像的更改。
52.对每个样本重复步骤 47-51 (图 4)。




图 4. 使用 Nikon Elements 软件分析 TA 肌纤维尺寸。
一个。 Nikon Elements软件打开,对接阈值、二值层、物体计数功能。湾。打开图像,并在肌肉边界周围绘制一个多边形感兴趣区域 (ROI)。 C。在对象计数功能上,选择了 Area 和MinFeret选项,并将限制设置为所需的范围。选择“不断更新计数”功能。 d。观察到三个工作层(Cy5、FITC 和 TRITC),并调整阈值以准确表示纤维类型(IIA、IIB 和 IIX)和肌纤维边界(由抗层粘连蛋白免疫染色指示)。 e.通过使用二元运算对话框来识别 IIA光纤,在 Cy5 和 FITC 通道之间创建了一个交集。 F。在 Cy5 和 TRITC 通道之间创建一个交叉点,使用二进制操作对话框来识别 IIB光纤。 G。使用二进制运算对话框在 Cy5 和 FITC 通道之间创建一个减法。使用新创建的减法和 TRITC 通道重复该步骤。检测到未染色的假定 IIX纤维。在步骤 e-g 之后,将生成的数据导出到 Excel 进行分析。


G.使用 Excel 和 GraphPad Prism 软件分析Feret 的平均最小直径和纤维百分比• 每个样品组的时间约为 15 分钟(协议 #1)
53.将包含 Nikon Elements 分析数据的新工作表添加到 Excel 文件。创建两个表,一个用于平均Feret最小直径 (FMD),另一个用于纤维百分比。用纤维类型(IIA、IIB 和 IIX)标记列,用样品名称标记行。
54.要确定每个样品的每种纤维类型的 FMD,请从导出的数据中取Feret最小直径的平均值。通过平均先前记录的总面积数,还可以对每根光纤的横截面积 (CSA) 执行此分析。
55.要获得纤维的百分比,请从导出的数据中计算每个样本的每种纤维类型的纤维总数。将样本的纤维计数(IIA、IIB 和 IIX)相加,然后将单个纤维类型计数除以总和。将该数字乘以 100 以百分比形式显示数据。
56.和分析结果,方法是创建一个新的分组表,并选择“在并排子列中输入重复次数”。将每一列命名为样品组的名称,并用纤维类型标记每一行。重复次数对应于每个实验组的样本数。
57.复制Excel 文件中计算的平均 FMD 或纤维百分比数据,并粘贴到相应的数据表中。
58.将数据绘制为单个交错散点图,并使用具有标准偏差 (SD) 的平均值来测量变异性。
59.分析数据,以观察纤维尺寸或纤维百分比的显着变化。通过在多重比较选项卡下选择“在每一行内,比较列”来更改参数。


H.Feret最小直径数据的GraphPad Prism 进行直方图分析• 时间~20 分钟 (协议#1)
60.打开 GraphPad Prism 软件并创建一个新的 Column 表,选择“输入重复值,堆叠成列”。将每一列命名为样品组的名称,并为每种其他纤维类型复制该表。
61.对于每根光纤和样品,从 Nikon Elements Excel 文件中复制所有 FMD 数据并粘贴到相应的数据表中。
62.使用分析功能,选择列分析下的频率分布选项。将参数更改为“相对频率(百分比)”并相应地调整 bin 宽度和范围。将结果绘制为交错条形图。
63.分析频率分布结果,在XY分析下选择非线性回归(曲线拟合)。在参数下,使用高斯方程,保持默认设置。也可以使用 Nikon Elements Excel 文件中的“面积”数据对光纤的 CSA 执行此分析。


I.C2C12 细胞培养• 大约数天的生长时间和大约 1 小时的细胞分裂时间(协议 #2)
64.在 37°C 和 5% (v/v) CO 2的 10% (v/v) FBS DMEM + GlutaMax培养基中培养细胞。当细胞达到 80% 汇合时,将它们分开,并准备转染板。
65.要分裂细胞,请从烧瓶中取出培养基,并用 1 × PBS 洗涤两次。
关键:避免将 PBS 直接添加到细胞中,因为它们可能会脱落并丢失。
66.使用 0.25% Trypsin-0.53mM EDTA (w/v) 在 37°C 下分离细胞 3 分钟。为确保分离,轻敲烧瓶的侧面,并在显微镜下观察。
67.通过将 10 mL 的 DMEM+10% (v/v) FBS 添加回烧瓶中来中和胰蛋白酶化。将细胞收集在 50 mL 锥形管中,以 2,000 × g离心细胞10 分钟。为了保持细胞生长,将 20 mL 的 10% FBS 培养基添加回烧瓶中,然后放入培养箱中。
68.离心后观察沉淀并吸出剩余的培养基。用 3 mL 的生长培养基重新悬浮颗粒,一次添加 1 mL,以确保适当的重新悬浮。
69.血细胞计数器的两个腔室中添加 10 μL 的重新悬浮细胞来计数细胞。在显微镜下可视化腔室的中心区域(1 mm 2 ),并用细胞计数器计数细胞。对血细胞计数器的两个腔室重复该步骤并平均两个计数。
CRITICAL:将平均计数乘以转换因子 10,000 或 10 4 个细胞。
70.根据要测试的 siRNA 的数量,准备适量的 6 孔细胞培养板。使用总细胞数, 计算悬浮液所需的体积, 以使每口井的最终浓度为 20,000 个细胞/mL, 总井体积为 2 mL。 
关键:每个 siRNA 至少需要 3 个孔。还包括一个经过 siRNA 处理的板(每孔一个 siRNA),用于染色、成像和分析肌管直径大小。
71.准备 DMEM 生长培养基和重悬细胞的混合物,并在细胞培养板中加入适当的体积。在 37°C 下将板在 5% (v/v) CO 2中孵育。每隔一天更换一次生长培养基,注意不要分离细胞。
72.当成肌细胞达到 80 – 100% 汇合时,将 10% (v/v) FBS 生长培养基替换为 2% (v/v) HS 分化培养基,将细胞分化为肌管。
73.分化四天后,在 10% FBS 生长培养基中用 Ara-c 处理细胞两天,以杀死任何剩余的成肌细胞。治疗第一天 Ara-c 浓度应为 4.0 µg/mL,第二天为 0.4 µg/mL。


J.Myotube siRNA 转染• 时间约为 45 分钟(协议 #2)
74.在转染细胞之前,将 Opti-MEM 预热至 22°C,并在冰上解冻适当的 siRNA。计算总体积为 200 μL Opti-MEM、8 μL 的 Lipofectamine 2000 和 8 μL 的 50 μM siRNA 所需的试剂量。
关键:包括一个非靶向 (NT) siRNA 用作对照。 Opti-MEM、Lipofectamine 2000 和 siRNA 的混合体积可以根据板的大小进行调整。
75.将 8 μL 的 50 μM siRNA 稀释到 100 μL 的 Opti-MEM 中,并通过移液轻轻混合。重复此步骤,将 8 μL 的 Lipofectamine 2000 添加到 100 μL 的 Opti-MEM 中。将两种溶液混合,移液混合,孵育 10 分钟。
76.在潜伏期,取出准备好的 6 孔板上的介质,并添加 1.8 mL 的新鲜 10% (v/v) FBS 生长介质。
77.10 分钟后,将 200 μL 的转染混合物添加到每个孔中,并通过来回摇动板轻轻混合。将板与 5% (v/v) CO 2在 37°C 下孵育 48 小时。
78.24 小时后,将介质更改为无血清介质(含 1% P/S)。
79.要检查 siRNA 引起的击倒,请使用 Invitrogen TRIzol试剂和随附的协议在转染后 48 小时进行 RNA 提取。使用 mRNA 特异性寡核苷酸运行qRT -PCR,以量化 siRNA 靶向 mRNA 的击倒,使用 NT siRNA 处理的样品作为对照。


K.Myotub e 免疫染色和成像• 染色时间约为 4 小时,成像时间约为 1 小时(方案 #2)
80.在每毫升培养基中加入 1 mL 的 4% PFA,并在室温下孵育 10 分钟(最终 PFA 浓度为 2%)。
关键:将溶液添加到孔的一侧以避免提起细胞。出于同样的原因,固定溶液直接添加到细胞培养基中。
81.取出 PFA 溶液并用 1 × PBS 洗井 3 次。吸出剩余的 1 × PBS,并通过添加 1 mL 的 2% BSA 阻塞缓冲液(2% BSA Triton X-100)来阻塞井。让盘子在室温下轻轻晃动 1 小时。
82.封闭孔后,加入 300 µL 一抗溶液,在 4°C 下轻轻摇动板过夜。
83.第二天,去除抗体,用 1 × PBS 洗孔 3 次。在室温下加入 300 μL 的二级抗体溶液 2 小时,轻轻摇动。
84.× PBS清洗孔一次,然后用 1 mL 新鲜的 1 × PBS 替换并储存在 4°C,保护板免受光照,直到准备好成像。
暂停点:细胞可以在 PBS 中保留约 1 个月,但信号强度会下降。
使用可访问 532 nm 激光通道 (TRITC) 的荧光显微镜对板进行成像。使用 10 ×物镜,分别对每个孔进行成像以进行进一步分析。
关键:使用与刻度相同的物镜对血细胞计数器进行明场设置成像(图 5)。


图 5. siRNA 电穿孔进入 C2C12 成肌细胞和肌管的结果。
一个。用siGLO red转染的 C2C12 成肌细胞的代表性图像显示了荧光 siRNA 的细胞摄取,而在用非荧光 NT siRNA 转染的成肌细胞中没有观察到荧光。湾。将siGLO red 转染到 C2C12 肌管中也发现了类似的结果。 a-b 中的数据来自Hunt LC等人。细胞报告(2019 年) 。


L.肌管直径测量•每张图像的时间约为 30 分钟(协议 #2)
86.打开 ImageJ。在测量肌管之前,打开血细胞计数器图像。使用直线工具,在血细胞计数器四个角的 25 个小方块(0.25 毫米)之一内画一条线。
87.分析下拉菜单中选择设置比例选项,并在“已知距离”框中输入正方形的大小为“250”,长度单位为“µm”。检查“全局”选项,然后按 OK。比例应为 0.648pixels/µm。
CRITICAL:所有图像都需要设置为相同的比例才能进行准确分析。
88.打开缝合的肌管图像并放大单个肌管。使用直线工具,在直径上绘制三条线,以指示每个肌管的开始、中间和结束。在每一行之后,从分析下拉菜单中选择测量选项。
89.重复此操作,直到测量到每组至少 50 个肌管。将测量数据导出到 Excel。
90.要确定肌管直径,请平均每个肌管的三个测量值。在 GraphPad Prism 或等效分析软件中绘制每个肌管的平均值,与对照相比。执行适当的统计测试以确定 siRNA 是否会引起肌管直径大小的显着影响(图 6)。




图 6. 使用 ImageJ 分析软件测定 C2C12 肌管直径。
一个。在ImageJ 中打开血细胞计数器图像以建立比例。湾。通过使用直线工具,在血细胞计数器四个角之一的 25 个小方块之一内绘制一条线。 C。从分析下拉菜单中选择设置比例选项,并分别输入 250 μm作为已知像素距离和单位。选择全局选项将比例设置为 0.648 像素/ μm 。 d。打开缝合的肌管图像。 e.放大单个肌管。通过使用直线工具,在肌管的两端和中间的肌管宽度上水平绘制三条线。 F。绘制每条直线后,从“分析”下拉菜单中选择测量选项。 G。至少测量 50 个肌管,并将数据导出到 Excel。每个肌管的三个测量值被平均以确定平均直径大小。


预期的结果。通过上面报告的分析,可以分析具有靶基因敲除的 TA 肌肉,以确定由此产生的对肌纤维大小和肌纤维类型组成的影响,还可以通过 RNA 测序和蛋白质印迹等后续应用确定 mRNA 和蛋白质变化洗涤剂可溶性和不溶性部分的分析(Rai等人,2021b;Hunt等人,2021a) 。为了分析肌纤维大小,用抗层粘连蛋白抗体对肌肉冷冻切片进行染色,以描绘所有肌纤维的边界,并使用肌球蛋白重链异构体特异性抗体来识别 TA 肌肉中存在的不同肌纤维类型,即快速糖酵解2B 或 IIB 型肌纤维(MHC-2B-阳性)和更小、更具氧化性的 2A 型或 IIA 型肌纤维(MHC-2A 阳性)和 2X 或 IIX 型肌纤维(MHC-2A 阴性和 2B 阴性),如前所述完成(Hunt等人,2015、2021a、2021b) 。通过这些分析,可以确定 siRNA 介导的 TA 肌肉中靶 mRNA 的敲低是否会导致肌纤维大小和不同肌纤维类型比例的变化(Hunt等人,2015、2021a、2021b) 。在测量电穿孔 TA 肌肉中的肌纤维尺寸时,可以使用Feret 的最小直径,因为该几何参数能够可靠地测量肌纤维横截面积,不受冷冻切片引入的细胞变形的阻碍 (布隆伯格和 Quadrilatero,2012 年) 。类似地,与对照 NT siRNA 转染相比,对转染 siRNA 的培养的 C2C12 肌管大小的估计提供了洞察siRNA 靶向 mRNA 在肌管大小测定中是否具有一般作用[然而,肌纤维类型分析通常不在细胞中进行文化(黄等,2021 )]。亨特等人。 2019 提供了一个类似结果的例子,在培养的 C2C12 肌管中敲除相同的蛋白质,并且 在体内小鼠 TA 骨骼肌中:在这两种情况下,E3 泛素连接酶 UBR4 的敲低都会诱导肌纤维肥大(Hunt等人,2019)。
除了这些预期的结果之外,上面报告的协议可以与信息性抗原的免疫染色和额外的生化/分子测定相结合,以确定目标 mRNA 的急性敲低是否影响肌肉稳态的任何方面。这可以包括在研究与疾病相关的肌肉萎缩时估计肌纤维大小,以及确定与最佳肌肉功能或肌肉疾病相关的细胞器或标志物的丰度。除了确定细胞和分子特征外,还可以对电穿孔的 TA 肌肉进行功能分析,以确定靶向某些 mRNA 的 siRNA 是否会导致等长和强直力的相应变化(Hunt等人,2019、2021b) 。
最后,这种方法可以克服其他技术方法的后勤限制。例如,肌肉减少症(即与年龄相关的骨骼肌质量和力量下降)的研究因获得所需基因型的老年小鼠所需的相对较长时间而受到阻碍。此处报告的将 siRNA 电穿孔到 TA 肌肉中的协议和优化条件可能有助于克服这一限制。具体来说,我们建议这种方法可以提供一种快速而稳健的方法,用于在老野生型小鼠的 TA 肌肉中实现靶基因敲低,从而在肌肉减少症的背景下快速评估基因功能。


食谱


试剂设置(方案 #1 和 #2)
1.10% 黄蓍胶
在 100 mL 蒸馏水中混合 10 mg 黄蓍胶。在 4 ° C下储存两周。
2.透明质酸酶原液
在 1 mL 的 1 × PBS 中混合 10 mg 透明质酸酶粉末。在 -20 °C 下储存长达 1 年。
3.透明质酸酶工作液
× PBS稀释原液至终浓度为 ±0.4 单位/ mL。在-20 °C 下储存长达 6 个月。
4.50 μM siRNA 原液
× siRNA 缓冲液中重悬 20 nmol siRNA,最终浓度为 50 μM 。在-20 °C 下储存长达 6 个月。
5.2% BSA 封闭缓冲液
× PBS中混合 0.4 克 BSA 粉末(2%(重量/体积)最终浓度)和 20 μL的 Triton X-100(0.1%(体积/体积)最终浓度) 。在 4 °C 下储存长达 1 个月。
6.一抗染色液(冷冻载玻片)
× PBST中将一抗稀释至终浓度为 1:150 (对于 1.5 mL:1.5 mL PBS、1.5 μL Tween- 20、10 μL一抗)
7.二抗染色液(冷冻载玻片)
× PBST中稀释二抗至终浓度为 1:200 (对于 2 mL:2 mL PBS、2 μL Tween- 20、10 μL二抗)
8.10%胎牛血清(FBS)
将 50 mL 的 FBS 与 500 mL DMEM + GlutaMax介质混合。
9.2% 马血清 (HS)
将 10 mL 的 HS 与 500 mL DMEM + GlutaMax介质混合。
10.1% 青霉素/链霉素
将 5.55 mL 的 P/S 添加到 550 mL DMEM + GlutaMax血清培养基中。
11.4% PFA
用 1x PBS 稀释 16% PFA,最终浓度为 4%(4 mL 的 16% PAF 和 12 mL 的 PBS)。
12.一抗染色液(细胞培养肌管)
× PBST中将一抗稀释至 1:200 的最终浓度(对于 2 mL:2 mL PBS、2 μL Tween- 20、10 μL一抗)。
13.二抗染色液(细胞培养肌管)
× PBST中将二级抗体稀释至 1:400 的最终浓度(对于 2 mL:2 mL PBS、2 μL Tween- 20、5 μL二级抗体)。


致谢


BioRender绘制方案。 用于鉴定肌纤维类型的抗 MHC 抗体获自Developmental Studies Hybridoma Bank。这项工作得到了国家老龄化研究所 (R01AG055532 和 R56AG63806) 对 FD 的研究资助。圣裘德儿童研究医院的研究得到美国黎巴嫩叙利亚联合慈善机构 (ALSAC) 的支持。内容完全由作者负责,并不一定代表美国国立卫生研究院的官方观点。作者贡献: AS 和 FD 撰写了手稿。 FAG 和 LCH 提供了反馈。所有作者都阅读并编辑了手稿。


利益争夺


作者声明没有竞争利益。


伦理


所有实验均按照联邦和地方法规进行。圣裘德儿童研究医院 (SJCRH) 动物护理和使用委员会 (IACUC) 批准了执行的所有协议。动物被安置在美国田纳西州孟菲斯市 SJCRH 的动物资源中心的通风、温度控制设施中。


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引用:Stephan, A., Graca, F. A., Hunt, L. C. and Demontis, F. (2022). Electroporation of Small Interfering RNAs into Tibialis Anterior Muscles of Mice. Bio-protocol 12(11): e4428. DOI: 10.21769/BioProtoc.4428.
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