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Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation.

Taylor J, Babbs CF, Alzghoul MB, Olsen A, Latour M, Pond AL, Hannon K - BMC Biotechnol. (2004)

Bottom Line: We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers.Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo.When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Medical Sciences, Purdue University, W Lafayette, IN 47907, USA. jat4@cec.wustl.edu

ABSTRACT

Background: Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums.

Results: We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo.

Conclusions: When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

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Related in: MedlinePlus

Ectopic DNA expression decreases with increasing tissue damage following electroporation. LacZ expressing cDNA was electroporated into the gastrocnemius/soleus skeletal muscle. DNA expression was quantitated by measuring the amount of b-gal staining per area total muscle, and is reported as a percent of the gastrocnemius/soleus muscles. Tissue damage was measured and is reported as a percent of damage per total gastrocnemius/soleus muscle area. A second order polynomial was fitted to the data and it was found that there was a correlation coefficient of 0.826, which corresponds to a statistical significance of p < 0.01. This polynomial demonstrates that with increasing skeletal muscle damage, the amount of ectopic DNA expression decreases.
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Figure 2: Ectopic DNA expression decreases with increasing tissue damage following electroporation. LacZ expressing cDNA was electroporated into the gastrocnemius/soleus skeletal muscle. DNA expression was quantitated by measuring the amount of b-gal staining per area total muscle, and is reported as a percent of the gastrocnemius/soleus muscles. Tissue damage was measured and is reported as a percent of damage per total gastrocnemius/soleus muscle area. A second order polynomial was fitted to the data and it was found that there was a correlation coefficient of 0.826, which corresponds to a statistical significance of p < 0.01. This polynomial demonstrates that with increasing skeletal muscle damage, the amount of ectopic DNA expression decreases.

Mentions: We found that as the amount of muscles damage (reported as percent necrosis) increased, the level of β-Gal expression significantly decreased (Figure 2). A second order polynomial was fit to the data with a correlation coefficient of 0.826 (p < .01). Furthermore, there was no evidence of β-Gal expression in damaged fibers (Figure 3).


Optimization of ectopic gene expression in skeletal muscle through DNA transfer by electroporation.

Taylor J, Babbs CF, Alzghoul MB, Olsen A, Latour M, Pond AL, Hannon K - BMC Biotechnol. (2004)

Ectopic DNA expression decreases with increasing tissue damage following electroporation. LacZ expressing cDNA was electroporated into the gastrocnemius/soleus skeletal muscle. DNA expression was quantitated by measuring the amount of b-gal staining per area total muscle, and is reported as a percent of the gastrocnemius/soleus muscles. Tissue damage was measured and is reported as a percent of damage per total gastrocnemius/soleus muscle area. A second order polynomial was fitted to the data and it was found that there was a correlation coefficient of 0.826, which corresponds to a statistical significance of p < 0.01. This polynomial demonstrates that with increasing skeletal muscle damage, the amount of ectopic DNA expression decreases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC425588&req=5

Figure 2: Ectopic DNA expression decreases with increasing tissue damage following electroporation. LacZ expressing cDNA was electroporated into the gastrocnemius/soleus skeletal muscle. DNA expression was quantitated by measuring the amount of b-gal staining per area total muscle, and is reported as a percent of the gastrocnemius/soleus muscles. Tissue damage was measured and is reported as a percent of damage per total gastrocnemius/soleus muscle area. A second order polynomial was fitted to the data and it was found that there was a correlation coefficient of 0.826, which corresponds to a statistical significance of p < 0.01. This polynomial demonstrates that with increasing skeletal muscle damage, the amount of ectopic DNA expression decreases.
Mentions: We found that as the amount of muscles damage (reported as percent necrosis) increased, the level of β-Gal expression significantly decreased (Figure 2). A second order polynomial was fit to the data with a correlation coefficient of 0.826 (p < .01). Furthermore, there was no evidence of β-Gal expression in damaged fibers (Figure 3).

Bottom Line: We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers.Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo.When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Medical Sciences, Purdue University, W Lafayette, IN 47907, USA. jat4@cec.wustl.edu

ABSTRACT

Background: Electroporation (EP) is a widely used non-viral gene transfer method. We have attempted to develop an exact protocol to maximize DNA expression while minimizing tissue damage following EP of skeletal muscle in vivo. Specifically, we investigated the effects of varying injection techniques, electrode surface geometry, and plasmid mediums.

Results: We found that as the amount of damage increased in skeletal muscle in response to EP, the level of beta-galactosidase (beta-gal) expression drastically decreased and that there was no evidence of beta-gal expression in damaged fibers. Two specific types of electrodes yielded the greatest amount of expression. We also discovered that DNA uptake in skeletal muscle following intra-arterial injection of DNA was significantly enhanced by EP. Finally, we found that DMSO and LipoFECTAMINE, common enhancers of DNA electroporation in vitro, had no positive effect on DNA electroporation in vivo.

Conclusions: When injecting DNA intramuscularly, a flat plate electrode without any plasmid enhancers is the best method to achieve high levels of gene expression.

Show MeSH
Related in: MedlinePlus