ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2014, Vol. 45 ›› Issue (8): 1253-1259.doi: 10.11843/j.issn.0366-6964.2014.08.007

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Research on Establishing Chicken Embryonic Stem Cell Line by pEGFP-hTERT Transfection

HUANG Xiao-mei1，ZHANG Lei1，ZUO Qi-sheng1，SHI Qing-qing1，LI Dong1，TANG Bei-bei1，ZHANG Ya-ni1，SONG Jiu-zhou2，LI Bi-chun1*   

  1. （1.Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province，College of Animal Science and Technology，Yangzhou University，Yangzhou 225009，China；2.Animal and Avian Science Dept.University of Maryland，College Park，MD 20742，America）
  • Received:2014-01-09 Online:2014-08-23 Published:2014-08-23

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Abstract:

This experiment was conducted to transfect chicken embryonic stem cells with recombinant plasmid pEGFP-hTERT，optimize culture system of chicken embryonic stem cells，and establish embryonic stem cell lines.hTERT gene was obtained and then subcloned into pEGFP-N1 to construct eukaryotic expression vector pEGFP-hTERT.cESCs were transfected with pEGFP-hTERT after transfection DF-1 cells，which were cultured with 80% conditioned medium，20% ES decarboxylase basal medium and 10 μmol•L-1 vitamin C.After detection with AKP staining and associated specific surface antigen SSEA-1，SOX2 and OCT4，cESCs were selected with G418 and detected by real-time fluorescent quantitative PCR.The results showed that vitamin C could promote cESCs proliferation，green fluorescence could be detected in DF-1 cells aftert transfected with pEGFP-hTERT，cESCs transfected with pEGFP-hTERT could be passaged to 10th generation and remained undifferentiated state，and stem cell surface specific antigen detection was positive，as well there were no significant differences in expression levels of related genes（P>0.05）.The results indicated that cESCs could be passaged to 10th generation consistently，and remained undifferentiated state，which were transfected with pEGFP-hTERT and cultured with 80% BRL-CM added vitamin C.This would be used in embryonic stem cells immortalization and establishing cell lines.

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