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

Year 2020, Volume: 42 Issue: 2, 140 - 147, 17.03.2020

Gamze Bora

https://doi.org/10.20515/otd.497543

Cited By: 1

Abstract

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. 

Keywords

Cell line, PC12, NSC34, RNA interference

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

Abstract

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.

Keywords

Hücre hatları, PC12, NSC34, RNA interferans

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