Gene Silencing by RNA Interference in Neuron-like Cell Lines: in vitro SMA models.

Öz

In order to explain the biological mechanisms in the eukaryotic cells, cell lines are valuable tools in cases where access to primary cells is limited. Although they do not reflect primary cells exactly, cell lines are often needed and used to model diseases in vitro. In this study, two neuron-like cell lines were used for gene silencing by RNA interference to both evaluate advantages/disadvantages of cells and their potential to be used as an in vitro models for neurodegenerative diseaeses through spinal muscular atrophy. As in vitro SMA models, SMN gene was knocked-down in both PC12 and NSC34 cell lines and gene silencing was confirmed by Western blot and immunofluorescence stainings. We found that SMN knock-down efficiencies were more than 90% in both PC12 and NSC34 cell lines. We compared culture, differentiation and transfection conditions of cell lines during silencing studies. Our findings will be helpful for further studies using neuron-like PC12 and NSC34 cell lines. 

Anahtar Kelimeler

• Cell line,PC12,NSC34,RNA interference

Nöron Benzeri Hücre Hatlarında RNA Interferans Aracılı Gen İfadesi Baskılama Çalışmaları: in vitro SMA modelleri

Öz

Ökaryotik hücredeki biyolojik olayların açıklanabilmesi amacıyla primer hücrelere ulaşımın kısıtlı olduğu durumlarda hücre hatları kullanılmaktadır. Hücre hatları, primer hücreleri birebir yansıtmamalarına karşın hastalık modeli oluşturmak için sıklıkla ihtiyaç duyulan ve kullanılan hücrelerdir. Bu çalışmada genlerin fonksiyonunun baskılanması prensibine dayanan RNA interferans yöntemi iki farklı nöron benzeri hücre hatında uygulanmış, hücrelerin birbirlerine göre avantajları/dezavantajları, nörodejeneratif bir hastalık olan Spinal müsküler atrofi üzerinden karşılaştırılmıştır. In vitro SMA modeli oluşturmak üzere, nöron benzeri hücrelere farklılaştırılan PC12 ve NSC34 hücre hatlarında SMN gen ifadesi siRNA yöntemi ile baskılanmiş, baskılanma Western blot ve immünfloresan boyama yöntemleri ile gösterilmiştir.PC12 ve NSC34 hücre hatlarında SMN gen ifadesi %90’ın üzerinde baskılanabilmiş ve çalışmalar sırasında hücre hatlarının kültür, farklılaşma, transfeksiyon koşulları arasındaki farklar karşılaştırmalı olarak incelenmiştir. Elde ettiğimiz bulgular, ileride nöron benzeri PC12 ve NSC34 hücre hatları kullanılarak gerçekleştirilecek çalışmalar açısından yol gösterici olacaktır.

Anahtar Kelimeler

• Hücre hatları,PC12,NSC34,RNA interferans

Kaynakça

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