Bilge OZSAİT SELCUK, Evrim KOMURCU BAYRAK, Muhammed Abdulvahid KALKAN, Gizem ÇELEBİ, Nihan ERGİNEL ÜNALTUNA

KALBE ÖZGÜ “SUBTRACTIVE” HIBRIDIZASYON CDNA KÜTÜPHANESINDEN ELDE EDILEN MFN2, MT-ATP6, MIDN VE KPNB1 GENLERININ YETIŞKIN, NEONATAL VE EMBRIYONIK DOKULARDAKI EKSPRESYON ANALIZI

Amaç: Eş zamanlı eksprese olan genlerin belirlenmesinde ve farklılaşmış gen ekspresyonunun tanımlanmasında dokuya özgü moleküler yolakların belirlenmesi önem taşımaktadır. Daha önceki çalışmalarımızda, “subtractive” hibridizasyon cDNA kütüphanesi (SHL) oluşturulmuş ve ardından bu transkriptlerin kalp dokusundaki rollerinin tanımlanabilmesi amacı ile çok sayıda fonksiyonel çalışma yürütülmüştür. Bu çalışmadaki amacımız, SHL’den seçilen dört transkriptin çoklu doku ve farklı evrelerdeki total embriyo dokularındaki ekspresyon özelliklerinin araştırılmasıdır. Gereç ve Yöntem: Toplam 16 farklı yetişkin BALB/c fare dokusu, neonatal kalp ve iskelet kası dokusu ve 5 farklı embriyonik döneme ait dokularda total RNA izolasyonu ve cDNA sentezi gerçekleştirildi. SHL’den elde edilen dört genin, mitofusin-2 (Mfn2), mitokondriyal ATP sentaz 6 (mt-Atp6), midnolin (Midn) ve karyoferin (importin) beta 1 (Kpnb1) genlerinin qRT-PCR yöntemi ile gen ekspresyon analizi yapıldı. Ek olarak, mt-Atp6 geninin ekspresyonu çoklu doku Northern blot analizi ile araştırıldı. Bulgular: Araştırılan dört transkriptin neonatal dönemde iskelet kasına oranla kalp dokusundaki ekspresyon seviyelerinin daha fazla olduğu tespit edildi. Yetişkin kalp ve iskelet dokularında da benzer yapı gözlendi. Diğer yandan, mt-Atp6 ve Mfn2 genlerinin diğer tüm dokular ile karşılaştırıldığında kalp dokusunda daha fazla ekspresyonunun olduğu belirlendi. Sonuç: Farklı dokular ve embriyonik dönemler arasında farklılaşmış gen ekspresyonlarının olması, bu dört transkriptin hücre fizyolojisinde önemli bir role sahip olduğunu düşündürmektedir. SHL’den izole edilen bu transkriptlerin seviyelerinin sağlıklı dokularda ve embriyonik gelişim dönemlerinde gösterilmesi, kalp fizyolojisi ile ilişkili metabolik yolaklardaki işlevlerinin tanımlanması için önem taşımaktadır.

Anahtar Kelimeler:

“subtractive” hibridizasyon, qRT-PCR, Mfn2, mt-Atp6, Midn, Kpn1b

EXPRESSION ANALYSIS OF MFN2, MT-ATP6, MIDN AND KPNB1 GENES ISOLATED FROM HEART-SPECIFIC SUBTRACTIVE HYBRIDIZATION CDNA LIBRARY IN ADULT, NEONATAL AND EMBRYONIC TISSUES

Objective: Identification of differential gene expression and determination of co-expressed genes is important in the characterization of tissue specific molecular pathways. In our previous studies, we constructed a heart-specific subtractive hybridization cDNA library (SHL) and conducted several functional studies in order to analyze the role of the isolated transcripts in heart tissue. In this study, our aim was to investigate the expression patterns of four selected transcripts from SHL in multiple tissues and, total embryos tissues of different stages.Materials and Methods: Total RNA isolation and cDNA synthesis were performed from 16 different tissues, 5 different stages of total embryos and neonatal heart and skeletal muscle of BALB/c mice. The expression profiles of SHL isolated four genes, mitofusin-2 (Mfn2), mitochondrial ATP synthase 6 (mt-Atp6), midnolin (Midn) and karyopherin (importin) beta 1 (Kpnb1) were analyzed by quantitative real time PCR. Additionally, Northern blot analysis of mt-Atp6 in multiple tissues was performed.Results: Gene expression levels of the four transcripts were higher in neonatal heart than in neonatal skeletal muscle. Similar pattern was observed in adult heart and skeletal muscle. However, mt-Atp6 and Mfn2 genes were over-expressed in heart tissue compared to all other tissues and embryonic stages. Conclusion: Differential expression of the transcripts among different tissues as well as in different embryonic stages suggests that these four genes have crucial roles in cellular physiology. Demonstration of the gene expression levels of the transcripts in the SHL in healthy tissues and embryonic development will contribute to the identification of the function of the genes in related metabolic pathways.

Keywords:

subtractive hybridization, qRT-PCR, Mfn2, mt-Atp6, Midn, Kpnb1,

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