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BMC Biotechnology

, 14:71

Genetic engineering in animals and animal cell systems


BackgroundMonkey 20α-hydroxysteroid dehydrogenase 20α-HSD is a catabolic enzyme responsible for converting progesterone into biologically inactive 20α-hydroxyprogesterone, thereby playing a key role in the estrous cycle or pregnancy and allowing ovulation and parturition to occur in most mammalian animals. Monkey 20α-HSD was highly abundant in ovarian and placental tissues during the pre-ovulation and pre-parturition phase and was primarily localized in the syncytiotrophoblast of the placenta. In this study, we focused on the molecular characterization of the monkey 20α-HSD promoter region by conducting reporter assays in Chinese hamster ovary CHO K1 cells.

ResultsA reporter assay using constructs of various lengths of the 5′-flanking region -890-Luc -513-Luc -306-Luc -273-Luc, and -70-Luc revealed that a region corresponding to the activator protein 1 Ap-1 located between -281 and -274 bp was essential for the transcriptional activity. Absence of the Ap-1 site in -273-Luc dramatically decreased the transcription levels to the control levels. When the reporter constructs were co-transfected with Ap-1 Jun and specificity protein Sp-1 genes, the transcription activities of the constructs increased with the exception of -273 and -70, while that of the double construct was reduced compared to that of Ap-1 alone. Furthermore, mutational analysis demonstrated that a putative Ap-1 site played an important role in the expression of the reporter gene. These findings were confirmed by EMSA examining the interactions of the protein Ap-1 in a nuclear extract from CHO-K1 cells and the expression levels of the Ap-1 transcription factor in pre-parturition placenta and CHO-K1 cells. Although mut-1 and mut-2 of Ap-1 bound with nuclear extracts from CHO-K1 cells, the transcriptional activity of mut-3 was almost completely suppressed.

ConclusionsOur results indicate that the Ap-1 site -281 → -274 5′-TGTCTCAT-3′ plays a crucial role in the activation of the monkey 20α-HSD gene. Thus, we demonstrated that monkey 20α-HSD promoter activity is regulated by the transcription factor Ap-1 in CHO-K1 cells.

KeywordsMacaque monkey 20α-HSD Transcription factor Ap-1 Sp-1 CHO-K1 cells Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-14-71 contains supplementary material, which is available to authorized users.

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Autor: Tsevelmaa Nanjidsuren - Kwan-Sik Min


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