Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation

Jennifer L. Kirby, Ling Yang, Jacquelyn C. Labus, R. John Lye, Nelson Hsia, Richard Day, Gail A. Cornwall, Barry T. Hinton

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 γ-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.

Original languageEnglish (US)
Pages (from-to)613-619
Number of pages7
JournalBiology of Reproduction
Volume71
Issue number2
DOIs
StatePublished - Aug 2004
Externally publishedYes

Fingerprint

Electroporation
Base Pairing
Epithelial Cells
Epididymis
Gene Expression
gamma-Glutamyltransferase
Genes
Reporter Genes
Ligation
Cystatins
Sperm Maturation
Androgens
In Vitro Techniques
Messenger RNA

Keywords

  • Epididymis
  • Gene regulation
  • Male reproductive tract

ASJC Scopus subject areas

  • Cell Biology
  • Developmental Biology
  • Embryology

Cite this

Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation. / Kirby, Jennifer L.; Yang, Ling; Labus, Jacquelyn C.; Lye, R. John; Hsia, Nelson; Day, Richard; Cornwall, Gail A.; Hinton, Barry T.

In: Biology of Reproduction, Vol. 71, No. 2, 08.2004, p. 613-619.

Research output: Contribution to journalArticle

Kirby, JL, Yang, L, Labus, JC, Lye, RJ, Hsia, N, Day, R, Cornwall, GA & Hinton, BT 2004, 'Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation', Biology of Reproduction, vol. 71, no. 2, pp. 613-619. https://doi.org/10.1095/biolreprod.103.025635
Kirby, Jennifer L. ; Yang, Ling ; Labus, Jacquelyn C. ; Lye, R. John ; Hsia, Nelson ; Day, Richard ; Cornwall, Gail A. ; Hinton, Barry T. / Characterization of epididymal epithelial cell-specific gene promoters by in vivo electroporation. In: Biology of Reproduction. 2004 ; Vol. 71, No. 2. pp. 613-619.
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