Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation.
The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.
Kirby
Jennifer L
JL
Department of Cell Biology, University of Virginia Health System, Charlottesville, VA 22908, USA.
Yang
Ling
L
Labus
Jacquelyn C
JC
Lye
R John
RJ
Hsia
Nelson
N
Day
Richard
R
Cornwall
Gail A
GA
Hinton
Barry T
BT
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D018274:Electroporation / Q000379:methods*; D004822:Epididymis / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D008297:Male; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012670:Seminiferous Epithelium / Q000166:cytology / Q000502:physiology*
D049452:Green Fluorescent Proteins