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2011 | 71 | 1 |
Tytuł artykułu

Generation of stable HEK293 cell lines expressing cell reprogramming factors

Warianty tytułu
Języki publikacji
EN
Abstrakty
EN
Induced pluripotent stem cells (iPSCs) are the product of somatic cell reprogramming into an undifferentiated embryonic-like state. These pluripotent cells might adopt various phenotypes by means of bioengineering methods and therefore might serve for disease modeling, pharmaceutical screening and cellular replacement therapies. Transcription factors such as Oct4, Sox2, Klf4 and Myc play the crucial role in the cell converting. The aim of our study was to obtain the protein extracts for the purpose of cell reprogramming experiments. Methods The cells of HEK 293 (Human Embryonic Kidney) line (ATTC/CRL15-73) have been transfected by non-viral, HiFect method with the pCMV cDNA9R-myc plasmid, coding one of the selected factors: Oct4, Sox2 or Klf4. After transfections, cells were cultured in low density for 2-3 weeks in the presence of neomycin to select the resistant (i.e.transfected) colonies. The expression of c-myc as a marker of stable transfectants was determined by western blot analysis. The overexpressed reprogramming proteins were gently extracted with non-denaturating CellLytic buffer supplemented with protease inhibitor cocktail and stored for the future application. Results. Isolation and propagation of an individual cells from neomycin-resistant colonies allowed us to obtain about 20-30 clones for each transcription factor. The c-myc positive clones have been selected for further in vitro culturing with the purpose of continual generation of Oct4, Sox2 or Klf4 proteins. Conclusions. The presented study resulted in successful generation of stable HEK293 cell lines that could express each of the three human reprogramming factors fused with the myc tag and with polyarginine (9R) to facilitate intracellular trafficking. The extracted proteins might therefore be used in induction of cell reprogramming experiment with the aim of generating IPSCs for potential neurorestorative therapies. Supported by grant 5978/B/P01/2010/38 and No N N302 597838.
Słowa kluczowe
Wydawca
-
Rocznik
Tom
71
Numer
1
Opis fizyczny
p.172
Twórcy
autor
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
autor
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
autor
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
autor
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
autor
  • Medical Research Centre, NeuroRepair Department, Polish Academy of Sciences, Warsaw, Poland
Bibliografia
Typ dokumentu
Bibliografia
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Identyfikator YADDA
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