Hiroki Kubota1, Yutaro Hayashi, Yasue Kubota, Kevin Coward, John Parrington. 1. Department of Surgical Medicine, Nephrourology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. kubochin@med.nagoya-cu.ac.jp
Abstract
OBJECTIVE: To evaluate two contrasting methods of in vivo gene transfer into testicular cells using electroporation, with regard to efficiency of transfer and damage to the testes. DESIGN: Controlled animal study. SETTING: Research laboratory at a university medical school. ANIMAL(S): 8-10-week-old male mice. INTERVENTION(S): The reporter construct pCAGGS-LacZ consisting of a cytomegalovirus enhancer/chicken beta-actin promoter attached to the LacZ gene was introduced into the testes in vivo using electroporation. For eight weeks, the efficiency and extent of LacZ gene expression, and the extent to which the testis was damaged by the technique, were investigated. MAIN OUTCOME MEASURE(S): Beta-galactosidase activity resulting from expression of the LacZ transgene was verified by X-gal staining, and LacZ mRNA expression was determined by RT-PCR analysis. Potential disorders associated with seminiferous tubular sperm formation were evaluated using the Johnsen score. RESULT(S): Long-lasting beta-galactosidase activity was detected in spermatogenic cells up to eight weeks postelectroporation. Apparent damage to spermatogenesis was evident but was transient in nature and recovered with time; this plasticity was particularly evident following rete testes injection. CONCLUSION(S): Injection into the rete testis appears to be more suitable for in vivo gene transfer by electroporation than direct intratesticular injection.
OBJECTIVE: To evaluate two contrasting methods of in vivo gene transfer into testicular cells using electroporation, with regard to efficiency of transfer and damage to the testes. DESIGN: Controlled animal study. SETTING: Research laboratory at a university medical school. ANIMAL(S): 8-10-week-old male mice. INTERVENTION(S): The reporter construct pCAGGS-LacZ consisting of a cytomegalovirus enhancer/chickenbeta-actin promoter attached to the LacZ gene was introduced into the testes in vivo using electroporation. For eight weeks, the efficiency and extent of LacZ gene expression, and the extent to which the testis was damaged by the technique, were investigated. MAIN OUTCOME MEASURE(S): Beta-galactosidase activity resulting from expression of the LacZ transgene was verified by X-gal staining, and LacZ mRNA expression was determined by RT-PCR analysis. Potential disorders associated with seminiferous tubular sperm formation were evaluated using the Johnsen score. RESULT(S): Long-lasting beta-galactosidase activity was detected in spermatogenic cells up to eight weeks postelectroporation. Apparent damage to spermatogenesis was evident but was transient in nature and recovered with time; this plasticity was particularly evident following rete testes injection. CONCLUSION(S): Injection into the rete testis appears to be more suitable for in vivo gene transfer by electroporation than direct intratesticular injection.