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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

Quantification of miR-206 reporter activity in mdx4cv mice. (A) miR-206 expression measured in the gastrocnemius muscles of 3 month-old mdx4cv animals in comparison to C57Bl/6. miR-206 expression is normalized to sno202 ( n = 4 animals/group), mean values are displayed; error bars = standard error of the mean (SEM), *P ≤0.05. (B) Bioluminescent imaging of mdx4cv animals showing CBG99 luciferase and mKATE expression. Left hindlimbs were co-injected with pCBG99-2x-miR-206 and pcDNA-mKATE, and right hindlimbs were co-injected with pCBG99-Control and pcDNA-mKATE. (C) Region of interest (ROI) analysis of (B). The same region size was used for all animals (n= 4 animals/group; mean values are displayed; error bars = SEM, *P≤0.05.
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Figure 4: Quantification of miR-206 reporter activity in mdx4cv mice. (A) miR-206 expression measured in the gastrocnemius muscles of 3 month-old mdx4cv animals in comparison to C57Bl/6. miR-206 expression is normalized to sno202 ( n = 4 animals/group), mean values are displayed; error bars = standard error of the mean (SEM), *P ≤0.05. (B) Bioluminescent imaging of mdx4cv animals showing CBG99 luciferase and mKATE expression. Left hindlimbs were co-injected with pCBG99-2x-miR-206 and pcDNA-mKATE, and right hindlimbs were co-injected with pCBG99-Control and pcDNA-mKATE. (C) Region of interest (ROI) analysis of (B). The same region size was used for all animals (n= 4 animals/group; mean values are displayed; error bars = SEM, *P≤0.05.

Mentions: In order to measure miRNA activity in vivo, we inserted two perfectly complementary miRNA binding sites downstream of the CBG99 luciferase coding sequence, creating pCBG99-2x-miR-206. We next co-injected 100 μg of pcDNA-mKATE along with either pCBG99-2x-miR-206 (left hindlimbs) or pCBG99-Luc-Control (right hindlimbs) into C57Bl/6 or dystrophic mdx4cv mice. Seven days later, we measured the signal using in vivo BLI followed by ROI analysis (Figure 4B). To ensure that we minimized the effect of differential plasmid distribution and injection efficiency, the same region size was used for quantification in all animals, and the luciferase signal was normalized to mKATE. After normalizing, the CBG99:mKATE ratio from left hindlimbs was compared to control right hindlimbs. In agreement with a 5-fold increase in miR-206 expression in mdx4cv animals by qRT-PCR (P = 0.0001) (Figure 4A), the normalized bioluminescent signal in the left hindlimbs of mdx4cv mice was reduced 3.3-fold (P = 0.02) (Figure 2C). While the decreased average signal in C57Bl/6 did not reach significance (P = 0.15), the downward trend is likely due to the high abundance of miR-206 in skeletal muscle, and the decreased miR-206 reporter signal in mdx4cv animals is due to increased miR-206 expression. Given that miR-206 is highly expressed in the regenerating fibers of dystrophic mice [13], this result suggests that pDNA injected using HLV is also expressed in these fibers. To test this, we injected mdx4cv animals with 100 μg of pCMV-eGFP and collected tissues for immunofluorescence 7 days later. As expected, we found GFP expression in small, desmin-positive, regenerating myofibers with centrally located nuclei (Figure 5, arrowheads), indicating that the decrease in luciferase signal measured using BLI likely corresponds to a loss of luciferase activity in these fibers.


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)

Quantification of miR-206 reporter activity in mdx4cv mice. (A) miR-206 expression measured in the gastrocnemius muscles of 3 month-old mdx4cv animals in comparison to C57Bl/6. miR-206 expression is normalized to sno202 ( n = 4 animals/group), mean values are displayed; error bars = standard error of the mean (SEM), *P ≤0.05. (B) Bioluminescent imaging of mdx4cv animals showing CBG99 luciferase and mKATE expression. Left hindlimbs were co-injected with pCBG99-2x-miR-206 and pcDNA-mKATE, and right hindlimbs were co-injected with pCBG99-Control and pcDNA-mKATE. (C) Region of interest (ROI) analysis of (B). The same region size was used for all animals (n= 4 animals/group; mean values are displayed; error bars = SEM, *P≤0.05.
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Figure 4: Quantification of miR-206 reporter activity in mdx4cv mice. (A) miR-206 expression measured in the gastrocnemius muscles of 3 month-old mdx4cv animals in comparison to C57Bl/6. miR-206 expression is normalized to sno202 ( n = 4 animals/group), mean values are displayed; error bars = standard error of the mean (SEM), *P ≤0.05. (B) Bioluminescent imaging of mdx4cv animals showing CBG99 luciferase and mKATE expression. Left hindlimbs were co-injected with pCBG99-2x-miR-206 and pcDNA-mKATE, and right hindlimbs were co-injected with pCBG99-Control and pcDNA-mKATE. (C) Region of interest (ROI) analysis of (B). The same region size was used for all animals (n= 4 animals/group; mean values are displayed; error bars = SEM, *P≤0.05.
Mentions: In order to measure miRNA activity in vivo, we inserted two perfectly complementary miRNA binding sites downstream of the CBG99 luciferase coding sequence, creating pCBG99-2x-miR-206. We next co-injected 100 μg of pcDNA-mKATE along with either pCBG99-2x-miR-206 (left hindlimbs) or pCBG99-Luc-Control (right hindlimbs) into C57Bl/6 or dystrophic mdx4cv mice. Seven days later, we measured the signal using in vivo BLI followed by ROI analysis (Figure 4B). To ensure that we minimized the effect of differential plasmid distribution and injection efficiency, the same region size was used for quantification in all animals, and the luciferase signal was normalized to mKATE. After normalizing, the CBG99:mKATE ratio from left hindlimbs was compared to control right hindlimbs. In agreement with a 5-fold increase in miR-206 expression in mdx4cv animals by qRT-PCR (P = 0.0001) (Figure 4A), the normalized bioluminescent signal in the left hindlimbs of mdx4cv mice was reduced 3.3-fold (P = 0.02) (Figure 2C). While the decreased average signal in C57Bl/6 did not reach significance (P = 0.15), the downward trend is likely due to the high abundance of miR-206 in skeletal muscle, and the decreased miR-206 reporter signal in mdx4cv animals is due to increased miR-206 expression. Given that miR-206 is highly expressed in the regenerating fibers of dystrophic mice [13], this result suggests that pDNA injected using HLV is also expressed in these fibers. To test this, we injected mdx4cv animals with 100 μg of pCMV-eGFP and collected tissues for immunofluorescence 7 days later. As expected, we found GFP expression in small, desmin-positive, regenerating myofibers with centrally located nuclei (Figure 5, arrowheads), indicating that the decrease in luciferase signal measured using BLI likely corresponds to a loss of luciferase activity in these fibers.

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