Download In vitro gene knock-down of Sox9 via RNA interference

In vitro gene knock-down of Sox9 via RNA interference
Sox9, a master transcription factor for chondrogenesis, is indispensable for progression of
chondrogenic differentiation and the maintenance of the differentiated state.
However there are many unknown aspects about Sox9, for example its gene regulation,
redundancies among the Sox family members and the effects of the expression level.
Due to post natal fatality in attempts for generation of Sox9 null homozygous transgenic mice, we
are developing a method for a Sox9 knockdown by RNA interference to elucidate the role of
Sox9.
RNA interference (RNAi) is a post transcriptional gene silencing technique used to knock
down the expression of specific genes via small interfering RNAs (siRNAs), small hairpin
loop RNAs (shRNAs) or micro RNAs. Briefly, double stranded RNA is cleaved into 21-25
nucleotide small RNA by the cellular machinery. These small interfering RNA finally initiates
sequence specific degradation of complementary mRNA resulting in gene silencing.
Since its discovery in the 1990’s the technique has been proven to be very useful in
determining the relation of a gene to its functions.
In our project we pursue different strategies for an in vitro knock-down of Sox9:
1. A retroviral system using shRNA
We could already knockdown Sox9 on gene and protein level in a mouse fibroblasts cell
line (NIH3T3) and in rat MSCs by retroviral infection via shRNA.
Alteration in the expression of some genes involved in chondrogenesis was shown in
downstream experiments (qPCR).
2. Gene knock-down of Sox9 via microRNA (miRNA)
miRNAs are encoded by genes and transcribed by RNA-polymerase II (shRNAs are
transcribed by RNA-polymerase III) but not translated into protein.
A primary transcript called pri-miRNA is processed to short stem-loop structures (premiRNA) and finally to functional miRNA.
Mature single-stranded miRNA molecules are partially or complete complementary to one
or more messenger RNA (mRNA) molecules whereas partially complementary leads to
translation repression and complete complementary to mRNA cleavage.
Lee and colleagues first described this phenomenon in 1993, yet the term “microRNA”
was only introduced in 2001 in a set of three articles in Science.
In our experiments, we follow two different ways to introduce the miRNA (compounded
by Invitrogen; completely complementary to a Sox9-Sequence) into the cells of interest:
A. by using a constitutive lentiviral system
B. by using a inducible plasmid-based system
First our goal is to establish a successful Sox9 gene knock-down protocol in human cells.
For this purpose we utilize a human chondrosarcoma cell-line (HTB94).
For further studies we are planning to perform a Sox9 knockdown in human primary
mesenchymal stem cells and analyze in downstream experiments alterations in protein
expression (2D-gelectrophoresis) and related pathways (cDNA microarray).
Furthermore, differentiation studies in hMSCs will take place by using the inducible system.
Investigators:
PD Dr. Susanne Grässel
Sabine Stöckl, Dipl. Biol.
Mandy Vogel, Technician
Claudia Göttl, Technician
Grants:
This work is supported by the Deutsche Forschungsgemeinschaft (grant GR 1301/7-1)