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Measuring microRNA reporter activity in skeletal muscle using hydrodynamic limb vein injection of plasmid DNA combined with in vivo imaging.

Guess MG, Barthel KK, Pugach EK, Leinwand LA - Skelet Muscle (2013)

Bottom Line: Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups.Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and BioFrontiers Institute, University of Colorado, Boulder, CO, USA. leslie.leinwand@colorado.edu.

ABSTRACT

Background: microRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread "fine-tuning" of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.

Methods: To improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.

Results: We found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.

Conclusion: Hydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

No MeSH data available.


Related in: MedlinePlus

Reporter distribution. Reporter expression after hydrodynamic limb vein injection. Left column: green fluorescent protein (GFP) epifluorescent signal displayed as inverted grayscale image, percentages are an average of GFP-positive fibers for indicated muscles. Right column: immunostaining for laminin (red) and epifluorescence for GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to show distribution of GFP-positive myofibers in indicated muscles; n=1 animal. Scale bars = 100 μm. TA, tibialis anterior.
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Figure 2: Reporter distribution. Reporter expression after hydrodynamic limb vein injection. Left column: green fluorescent protein (GFP) epifluorescent signal displayed as inverted grayscale image, percentages are an average of GFP-positive fibers for indicated muscles. Right column: immunostaining for laminin (red) and epifluorescence for GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to show distribution of GFP-positive myofibers in indicated muscles; n=1 animal. Scale bars = 100 μm. TA, tibialis anterior.

Mentions: To test the muscle distribution of reporter pDNA, we next injected C57Bl/6 mice with 100 μg/animal of pCMV-eGFP. After 7 days, we performed immunofluorescence on fixed cryosections and found that GFP expression was visible in the sarcoplasm of myofibers in the gastrocnemius, soleus, and TA muscles (Figure 2). To determine injection efficiency, we quantified GFP-positive myofibers and found that 7.7%, 15.9% and 6.1% of fibers in the soleus, gastrocnemius, and TA, respectively, expressed the reporter. We also observed an absence of centrally located nuclei in GFP-positive myofibers, further supporting the finding that HLV injection itself causes minimal muscle injury. Next, we tested whether reporter gene expression can be quantified from both bioluminescent and fluorescent reporters using in vivo imaging. To this end, we injected either 100 μg each of pCBG99-Luc-Control and pcDNA-mKATE, or pcDNA-mKATE alone into the right hindlimbs of C57Bl/6 mice and collected images 7 days later using BLI. The signal was easily visible and localized to the hindlimb skeletal muscle for both reporters, although mKATE background signal was frequently observed from the ventilation nosepiece and/or the distal parts of the hindlimb (Figure 3). We also found that mKATE fluorescence did not bleed into the luciferase channel, making it ideal for in vivo use in combination with CBG99-luciferase.


Measuring microRNA reporter activity in skeletal muscle using hydrodynamic limb vein injection of plasmid DNA combined with in vivo imaging.

Guess MG, Barthel KK, Pugach EK, Leinwand LA - Skelet Muscle (2013)

Reporter distribution. Reporter expression after hydrodynamic limb vein injection. Left column: green fluorescent protein (GFP) epifluorescent signal displayed as inverted grayscale image, percentages are an average of GFP-positive fibers for indicated muscles. Right column: immunostaining for laminin (red) and epifluorescence for GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to show distribution of GFP-positive myofibers in indicated muscles; n=1 animal. Scale bars = 100 μm. TA, tibialis anterior.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750807&req=5

Figure 2: Reporter distribution. Reporter expression after hydrodynamic limb vein injection. Left column: green fluorescent protein (GFP) epifluorescent signal displayed as inverted grayscale image, percentages are an average of GFP-positive fibers for indicated muscles. Right column: immunostaining for laminin (red) and epifluorescence for GFP (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to show distribution of GFP-positive myofibers in indicated muscles; n=1 animal. Scale bars = 100 μm. TA, tibialis anterior.
Mentions: To test the muscle distribution of reporter pDNA, we next injected C57Bl/6 mice with 100 μg/animal of pCMV-eGFP. After 7 days, we performed immunofluorescence on fixed cryosections and found that GFP expression was visible in the sarcoplasm of myofibers in the gastrocnemius, soleus, and TA muscles (Figure 2). To determine injection efficiency, we quantified GFP-positive myofibers and found that 7.7%, 15.9% and 6.1% of fibers in the soleus, gastrocnemius, and TA, respectively, expressed the reporter. We also observed an absence of centrally located nuclei in GFP-positive myofibers, further supporting the finding that HLV injection itself causes minimal muscle injury. Next, we tested whether reporter gene expression can be quantified from both bioluminescent and fluorescent reporters using in vivo imaging. To this end, we injected either 100 μg each of pCBG99-Luc-Control and pcDNA-mKATE, or pcDNA-mKATE alone into the right hindlimbs of C57Bl/6 mice and collected images 7 days later using BLI. The signal was easily visible and localized to the hindlimb skeletal muscle for both reporters, although mKATE background signal was frequently observed from the ventilation nosepiece and/or the distal parts of the hindlimb (Figure 3). We also found that mKATE fluorescence did not bleed into the luciferase channel, making it ideal for in vivo use in combination with CBG99-luciferase.

Bottom Line: Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups.Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and BioFrontiers Institute, University of Colorado, Boulder, CO, USA. leslie.leinwand@colorado.edu.

ABSTRACT

Background: microRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread "fine-tuning" of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.

Methods: To improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.

Results: We found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.

Conclusion: Hydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

No MeSH data available.


Related in: MedlinePlus