Transcript The effects of Fndc5 overexpression on characteristics of mouse

The effects of Fndc5 overexpression on characteristics of mouse embryonic stem cells and
the expression of three germ layer markers and mitochondrial genes
Farzaneh Rabiee1,2, Mahboobeh Forouzanfar2, Somayeh Tanhaei2, Majid Motavali-bashi1, Kamran Ghaedi1,2, Mohammad Hossein Nasr Esfahani2
1Department
of Biology, School of Sciences, University of Isfahan, Isfahan, Iran.
2Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Objectives
Fndc5 formerly known as peroxisomal protein, is suggested to
be a PGC1-alpha-dependent myokine and is secreted as Irisin ,
responsible for browning of white fat tissues. In adult mouse,
mRNA expression level of Fndc5 is high in heart, skeletal
muscle and brain. Our previous studies have revealed a
significant increase in Fndc5 mRNA when mouse embryonic
stem cells were differentiated into beating bodies and neural
precursor cells . As a step closer to clarify the function of Fndc5
on embryonic stem cell differentiation we have overexpressed
Fndc5 in embryonic stem cell to analyze the expression of
three germ layers marker and mitochondrial genes.
Results
The results depicted that expression of mesodermal and
endodermal genes such as Brachyury,Dkk1, Isl1 and
Foxa2 was higher in those cells with overexpressing
Fndc5 compare with the control. furthermore there were
not significant difference in the expression levels of
ectodermal and mitochondrial markers in overexpressing
Fndc5 group and control.
Methods
Stably transformed overexpressing Fndc5 mouse embryonic
stem cells (Rb20-Fndc5) after induction by doxycyclin were
cultured for three days. Then, 5 × 105 of treated mESCs
were suspended in a non-adhesive dish by the medium
without LIF,and small molecules SB and PD (for stemness
maintenance) for six days in the absence of
Fndc5
overexpression. Media were changed every 2 days. Total
RNA was extracted from cultured cells . cDNA synthesis was
carried out with cDNA Synthesis Kit . Real-time PCR was
performed to estimate the mRNA expression levels of target
genes.
Fig- 1: (aggregation protocole)
References
Conclusions
In conclusion , Fndc5 may be a master gene , whose
product would modulate the expression of endodermal and
mesodermal genes. However it has not any effect on
mitochondrial and ectodermal genes. Further experiments
are required for clarification the function of Fndc5.