A Key Enzyme in Placental Steroidogenesis: Detection of 3β-Hydroxysteroid Dehydrogenase/∆5-4 İsomerase (3β-HSD)’ın With Cloned Probes on Eukaryotic Vector

Year 2010, Volume: 29 Issue: 1, 7 - 10, 01.06.2010

Gözde R.özalp Gerhard Schuler

Abstract

3β-hydroxysteroid dehydrogenase/∆5-4 isomerase (3β-HSD) is a key enzyme in placental steroidogenesis. To form steroid hormones, pregnenolone requires enzymes 3βhydroxysteroiddehydrogenase/∆5-4 isomerase (3β-HSD), which belongs to the ∆5 and ∆4 steroidogenic pathways. The created probes will help to identify the molecular basis of enzymes which play a role in steroidogenesis. Therefore, the objective of this study was to develop probes with cloning the target gene on a eukaryotic vector. Total RNA was isolated and RT-PCR was performed. DNA bands were extracted from agarose gel and purified. 3βHSD-DNAs were transformed on E.coli strain. Vector containing 3βHSD insert were digested with the restriction enzymes NcoI and NotI. In vitro transcription of the DIG-labelled cRNA was carried out by using DIG-RNA labelling mix. In situ hybridization was carried out with the probes. Specific staining was observed in uninucleated trophoblast cells where as negative control was negative. In conclusion, specific probes were generated with cloning the target gene on a eukaryote vector, whic are sitable for detection of steroidogenic enzymes with in situ hybridization.

Keywords

3β-HSD, placenta, in situ hybridization

Plasental Steroidogeneziste Kilit Enzim: 3β-Hidroksisteroid Dehidrogenaz/∆5-4 İzomeraz (3β-HSD)’ın Ökaryon Vektörde Klonlanmış Prob’larla Belirlenmesi

Abstract

3β-hidroksisteroid dehidrogenaz/∆5-4 izomeraz (3β-HSD) plasental steroidogenesiz’de kilit enzim olarak
kabul edilir. Steroid hormonların oluşumu için, pregnenolon ∆5 ve ∆4 sentez yollarında kullanılan 3β-
hidroksisteroid dehidrogenaz/∆5-4 izomeraz (3β-HSD)’a ihtiyaç duyar. Çalışmanın amacı, hedef genin, bir
ökaryon vektor üzerinde klonlanmasıyla prob elde edilmesi ve in situ hibridizasyonda kullanılabilirliğinin gösterilmesidir.
Bu amaçla total RNA izolasyonundan sonra RT-PCR yapıldı. DNA bantları agaroz jelden ayrılarak
saflaştırıldı. 3βHSD-DNA’ların E.coli suşuna transformasyonu tamamlandıktan sonra 3βHSD insert içeren vektörden
NcoI ve NotI restriksiyon enzimleriyle kesildi. DIG-işaretli cRNA’ların transkripsiyonu sonrasında in situ
hibridizasyon yapıldı. Spesifik boyanmalar tek nukleuslu trofoblast hücrelerinde gözlenirken negatif kontrol
grubunda boyanma gözlenmedi. Sonuç olarak bir ökaryon vector üzerinde, hedef gen klonlanarak steroidogenik
enzimlerin belirlenmesinde kullanılmak üzere, in situ hibridizasyon tekniği için oldukça spesifik prob’lar elde
edilmiştir.

Keywords

3β-HSD, plasenta, in situ hibridizasyon

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