Korean J Urol.  2015 Mar;56(3):197-204. 10.4111/kju.2015.56.3.197.

Optimizing in vivo gene transfer into mouse corpus cavernosum by use of surface electroporation

Affiliations
  • 1National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon, Korea. jksuh@inha.ac.kr
  • 2Department of Urology, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon, Korea.

Abstract

PURPOSE
Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum.
MATERIALS AND METHODS
Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue.
RESULTS
Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis.
CONCLUSIONS
We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

Keyword

Electroporation; Erectile dysfunction; Gene therapy; Penis

MeSH Terms

Animals
Diabetes Mellitus, Experimental/complications
Electroporation/*methods
Erectile Dysfunction/*therapy
Gene Expression
Gene Transfer Techniques
Genes, Reporter
Genetic Therapy/*methods
Luciferases/metabolism
Male
Mice
Mice, Inbred C57BL
Penile Erection/physiology
Penis/*physiopathology
Transfection
Luciferases

Figure

  • Fig. 1 Luciferase expression driven by pCMV-Luc with electroporation (EP) in the corpus cavernosum of normal mice. pCMV-Luc (100 µg/40 µL) was injected into the midportion of the corpus cavernosum and then each animal received EP at the injection sites with eight 40-ms pulses at voltages of 5, 10, 30, or 50 V. The penis was harvested and gene expression was measured by luciferase assay 3 days after injection. The luciferase activity of the no EP group was arbitrarily set equivalent to 1. The data are expressed as mean values (±standard deviation) for 5 animals per group. **p<0.01 vs. no EP group. RLU, relative light unit.

  • Fig. 2 Luciferase expression driven by pCMV-Luc with electroporation (EP) in the corpus cavernosum of normal mice. pCMV-Luc (100 µg/40 µL) was injected into the midportion of the corpus cavernosum and then each animal received EP at the injection sites with the following stimulation settings: eight 40-ms pulses, sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. The penis was harvested and gene expression was measured by luciferase assay 3 days after injection. The luciferase activity of the eight 40-ms pulses group was arbitrarily set equivalent to 1. The data are expressed as mean values (±standard deviation) for 5 animals per group. **p<0.01 vs. eight 40-ms pulses group; *p<0.05 vs. eight 40-ms pulses group. RLU, relative light unit.

  • Fig. 3 The gross appearance of penis of normal mice after electroporation (EP). pCMV-Luc (100 µg/40 µL) was injected into the midportion of the corpus cavernosum and then each animal received EP into injection sites with following stimulation settings: no EP (A); eight 40-ms pulses at a voltage of 20 (B); eight 40-ms pulses (D), sixteen 40-ms pulses (E), eight 100-ms pulses (F), and sixteen 100-ms pulses at a voltage of 30 (G); and eight 40-ms pulses at a voltage of 50 (C). Note area of electrical burn scar in the penis (arrows).

  • Fig. 4 Hematoxylin-eosin (H&E) staining of penis of normal mice after electroporation (EP). pCMV-Luc (100 µg/40 µL) was injected into the midportion of the corpus cavernosum and then each animal received EP into injection sites with following stimulation settings: no EP (A); eight 40-ms pulses at voltages of 30 (B); and eight 40-ms pulses at voltages of 50 (C). Note area of extensive necrosis in the corpus cavernosum of mice that received EP at 50 V (arrow).

  • Fig. 5 Luciferase expression driven by pCMV-Luc with electroporation (EP) in the corpus cavernosum of streptozotocin-induced diabetic mice. At 8 weeks after the induction of diabetes, pCMV-Luc (100 µg/40 µL) was injected into the midportion of the corpus cavernosum of diabetic mice or their age-matched controls. The animals then each received EP at the injection sites with eight 40-ms pulses at a voltage of 30 V. (A) The penis was harvested and gene expression was measured by luciferase assay 3 days after injection. The luciferase activity of the normal mice without EP was arbitrary set equivalent to 1. The data are expressed as mean values (±standard deviation) for 5 animals per group. **p<0.01 vs. no EP group. (B) The gross appearance of the penis of diabetic mice after EP. RLU, relative light unit; DM, diabetes mellitus.


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