Establishment of human embryonic stem cell line stably expressing Epstein-Barr virus-encoded nuclear antigen 1.
Human embryonic stem (hES) cells have the capability of unlimited undifferentiated proliferation, yet maintain the potential to form perhaps any cell type in the body. Based on the high efficiency of the Epstein-Barr virus-based episomal vector in introducing exogenous genes of interest into mammalian cells, we applied this system to hES cells, expecting that this would resolve the problem of poor transfection efficiency existing in current hES cell research. Therefore, the first step was to establish EBNA1-positive hES cells. Using the Fugene 6 transfection reagent, we transfected hES cells with the EBNA1 expression vector and subsequently generated hES cell clones that stably expressed EBNA1 under drug selection. These clones were confirmed to express EBNA1 mRNA by RT-PCR and to express EBNA1 protein by Western blotting. Furthermore, luciferase reporter gene analysis was performed on the EBNA1 clones and revealed that the expressed EBNA1 protein was functional. When the EBNA1-positive cells were injected into severe combined immunodeficient (SCID) mice, they formed teratoma tissues containing all three embryonic germ layers and EBNA1 protein was detected in these teratoma tissues by Western blotting. All the results show that we have successfully created stable EBNA1-hES cells, thus laying a good foundation for further research.
Ren
Cai-Ping
CP
Cancer Research Institute, Central South University, Changsha 410078, China. rencaiping@xysm.net
Zhao
Ming
M
Shan
Wen-Jiao
WJ
Yang
Xu-Yu
XY
Yin
Zhi-Hua
ZH
Jiang
Xing-Jun
XJ
Zhang
Hong-Bo
HB
Yao
Kai-Tai
KT
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004338:Drug Combinations; D004622:Embryo, Mammalian / Q000166:cytology*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D014162:Transfection
D018076:DNA, Complementary; D004338:Drug Combinations; D019309:Epstein-Barr Virus Nuclear Antigens; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D008156:Luciferases; C417029:EBV-encoded nuclear antigen 1