Browse > Article

Transduction of eGFP Gene to Human Embryonic Stem Cells and Their Characterization  

Kim, Yoon-Young (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Ku, Seung-Yup (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Park, Yong-Bin (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Oh, Sun-Kyung (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Moon, Shin-Yong (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Choi, Young-Min (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University)
Publication Information
Clinical and Experimental Reproductive Medicine / v.36, no.4, 2009 , pp. 283-292 More about this Journal
Abstract
Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.
Keywords
Human embryonic stem cells; Enhanced green fluorescent protein (eGFP); Lentivirus;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-7   DOI   PUBMED   ScienceOn
2 Xiong C, Tang DQ, Xie CQ, Zhang L, Xu KF, Thompson WE, et al. Genetic engineering of human embryonic stem cells with lentiviral vectors. Stem Cells Dev 2005; 14: 367-77   DOI   ScienceOn
3 Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 2001; 11: 514-8   DOI   ScienceOn
4 Gropp M, Itsykson P, Singer O, Ben-Hur T, Reinhartz E, Galun E, et al. Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 2003; 7: 281-7   DOI   ScienceOn
5 Niwano K, Arai M, Koitabashi N, Watanabe A, Ikeda Y, Miyoshi H, et al. Lentiviral vector-mediated SERCA2 gene transfer protects against heart failure and left ventricular remodeling after myocardial infarction in rats. Mol Ther 2008; 16: 1026-32   DOI   ScienceOn
6 Pfeifer A, Ikawa M, Dayn Y, Verma IM. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 2002; 99: 2140-5   DOI   ScienceOn
7 Chung S, Andersson T, Sonntag KC, Bjorklund L, Isacson O, Kim KS. Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 2002; 20: 139-45   DOI   ScienceOn
8 Smith-Arica JR, Thomson AJ, Ansell R, Chiorini J, Davidson B, McWhir J. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells 2003; 5: 51-62   DOI   ScienceOn
9 Oh SK, Kim HS, Ahn HJ, Seol HW, Kim YY, Park YB, et al. Derivation and characterization of new human embryonic stem cell lines: SNUhES1, SNUhES2, and SNUhES3. Stem Cells 2005; 23: 211-9   DOI   ScienceOn
10 Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 2003; 21: 111-7   DOI   ScienceOn
11 Lavon N, Yanuka O, Benvenisty N. The effect of overexpression of Pdx1 and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells. Stem Cells 2002; 24: 1923-30   DOI   ScienceOn
12 Hashimoto D, Nagata T, Uchijima M, Seto S, Suda T, Chida K, et al. Intratracheal administration of third-generation lentivirus vector encoding MPT51 from Mycobacterium tuberculosis induces specific CD8+ T-cell responses in the lung. Vaccine 2008; 26: 5095-100   DOI   ScienceOn
13 Suter DM, Cartier L, Bettiol E, Tirefort D, Jaconi ME, Dubois-Dauphin M, Krause KH. Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors. Stem Cells 2006; 24: 615-23   DOI   ScienceOn
14 Gallo P, Grimaldi S, Latronico MV, Bonci D, Pagliuca A, Ausoni S, et al. A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells. Gene Ther 2008; 15: 161-70   DOI   ScienceOn
15 Kwon YD, Oh SK, Kim HS, Ku SY, Kim SH, Choi YM, et al. Cellular manipulation of human embryonic stem cells by TAT-PDX1 protein transduction. Mol Ther 2005; 12: 28-32   DOI   ScienceOn
16 Oh SK, Kim HS, Park YB, Seol HW, Kim YY, Cho MS, et al. Methods for expansion of human embryonic stem cells. Stem Cells 2005; 23: 605-9   DOI   ScienceOn
17 Asano T, Hanazono Y, Ueda Y, Muramatsu S, Kume A, Suemori H, Suzuki Y, Kondo Y, Harii K, Hasegawa M, Nakatsuji N, Ozawa K. Highly efficient gene transfer into primate embryonic stem cells with a simian lentivirus vector. Mol Ther 2002; 6: 162-168   DOI   ScienceOn
18 Kim S, Kim GJ, Miyoshi H, Moon SH, Ahn SE, Lee JH, et al. Efficiency of the elongation factor-1alpha promoter in mammalian embryonic stem cells using lentiviral gene delivery systems. Stem Cells Dev 2007; 16: 537-45   DOI   ScienceOn
19 Laker C, Meyer J, Schopen A, Friel J, Heberlein C, Ostertag W, et al. Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation. J Virol 1998; 72: 339-48   PUBMED   ScienceOn
20 Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003; 21: 319-21   DOI   ScienceOn