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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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Electrode Surface Geometry. Four electrodes, labeled above, were tested on mice to determine the effect of surface geometry of DNA expression following electroporation into skeletal muscle.
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Figure 1: Electrode Surface Geometry. Four electrodes, labeled above, were tested on mice to determine the effect of surface geometry of DNA expression following electroporation into skeletal muscle.

Mentions: After a 50 μl aliquot of saline containing 50 μg CMVLacZ plasmid was injected into the right and left gastrocnemius of the mice using a Hamilton syringe (Hamilton, Reno, NV), four different electrodes were designed with varying degrees of perforation in the plates (Figure 1). Both legs were electroporated using one of the four different electrodes (3 mice per electrode, Groups 4, 5, 6 and 7 in Table 1).


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)

Electrode Surface Geometry. Four electrodes, labeled above, were tested on mice to determine the effect of surface geometry of DNA expression following electroporation into skeletal muscle.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Electrode Surface Geometry. Four electrodes, labeled above, were tested on mice to determine the effect of surface geometry of DNA expression following electroporation into skeletal muscle.
Mentions: After a 50 μl aliquot of saline containing 50 μg CMVLacZ plasmid was injected into the right and left gastrocnemius of the mice using a Hamilton syringe (Hamilton, Reno, NV), four different electrodes were designed with varying degrees of perforation in the plates (Figure 1). Both legs were electroporated using one of the four different electrodes (3 mice per electrode, Groups 4, 5, 6 and 7 in Table 1).

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