Reversible transfection of human melanocytes mediated by Cre/loxP site‐specific recombination system and SV40 large T antigen

Wang, Ying; Fei, Hao; Deng, Jun; Yang, Xichuan; Zhong, Baiyu; Ye, Qingyi

doi:10.1111/j.1600-0625.2007.00546.x

Cited by 4 publications

(6 citation statements)

References 28 publications

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“…On days 1, 3, 5, 7, 10, and 14, the MTT method was used to evaluate the number of surviving cells as previously described (Ying et al, 2007).…”

Section: In Vitro Ganciclovir Sensitivitymentioning

confidence: 99%

“…Cell growth rate analysis was carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheylt-etrazolium bromide (MTT; Sigma, St. Louis, MO) method as previously described (Ying et al, 2007). Population doubling level (PDL) for the immortalized LT-␤1 and LT-hTERT-␤1 cells at passage 20 (n = 5), was calculated by the following formulae: CD = ln(Nf/Ni)/ln 2, PDL = CT/CD, where PDL is the population doubling level, CT the cell culture time, Nf the final number of cells, Ni the initial number of cells (Vidal et al, 2006).…”

Section: Growth Kinetics Studiesmentioning

confidence: 99%

“…Although few cell lines transduced by SV40LTAg alone become truly immortalized, cells at early stages of immortalization may provide sufficient populations for use. SV40LTAg has been proven to be an efficient immortalizing gene for some types of cells (Kobayashi et al, 2000;Noguchi et al, 2002;Ying et al, 2007). However, co-expression of SV40LTAg and human telomerase reverse transcriptase (hTERT) has been utilized to enhance the immortalization potential of other types of cells (Matsumura et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector

Wang

Zhang

et al. 2011

Journal of Biotechnology

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“…On days 1, 3, 5, 7, 10, and 14, the MTT method was used to evaluate the number of surviving cells as previously described (Ying et al, 2007).…”

Section: In Vitro Ganciclovir Sensitivitymentioning

confidence: 99%

Section: Growth Kinetics Studiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector

Wang

Zhang

et al. 2011

Journal of Biotechnology

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“…The use of lentivirus, in particular, resulted in high‐efficiency gene transduction in melanocytes and melanoma cells and was an improvement over adenovirus‐ and retroviral‐based vectors (9). An interesting recent report utilized a retroviral vector which encoded the Cre recombinase to remove an experimentally transfected SV‐40 large T antigen gene from melanocytes (10). This ‘reversible transfection’ of melanocytes with oncogenic SV‐40 large T antigen resulted in the short‐term proliferation of melanocytes to generate a large number of cells for treating experimentally induced vitiligo.…”

Section: Introductionmentioning

confidence: 99%

Nucleofection is a highly effective gene transfer technique for human melanoma cell lines

Han

Gai

Yancovitz

et al. 2008

Experimental Dermatology

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Despite the increasing use of gene transfer strategies in the study of cellular and molecular biology, melanoma cells have remained difficult to transfect in a safe, efficient, and reproducible manner. In the present study, we report the successful use of nucleofector technology to transfect human melanoma cell lines. This technology uses an empirically derived combination of cell line-specific solutions and nucleofector programmes to electroporate nucleic acid substrates directly into the cell nucleus. Using a colorimetric b-galactosidase assay, we optimized nucleofection parameters for 13 melanoma cell lines, leading to maximum transfection efficiency and cell survival. The combinations of cell solutions NHEM or T and nucleofector programmes A-24 or U-20 produced the best results. We compared nucleofection with two commercially available lipidbased gene transfer systems, effectene and lipofectamine 2000 using a green fluorescent protein reporter vector. Nucleofection demonstrated a 3-to 40-fold improvement in transfection efficiency when compared with the lipid-based counterparts. Nucleofection was also superior in transfecting small-interfering RNA (siRNA) as determined by Western blot analysis. Lastly, we applied nucleofection to the simultaneous transfection of a p53-dependent luciferase plasmid and p53-siRNA. Experiments using dual transfection showed knockdown of p53 expression and silencing of the reporter plasmid. In conclusion, nucleofection is highly effective for the transfer of nucleic acid substrates, singly or in combination, into human melanoma cell lines.

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“…Reversibly immortalized human melanocytes created using a retroviral vector expressing SV40Tag-EGFP flanked by a pair of LoxP recombination targets have been reported to provide melanocytes with rapid replicative potential in vitro [60]. Following the transplantation of reverted melanocytes into an established vitiligo animal model, the pigmentation formed black macula within three months without tumorigenicity.…”

Section: Reversible Immortalization Of Other Cell Typesmentioning

confidence: 99%

Controlled Expansion of Mammalian Cell Populations by Reversible Immortalization

Noguchi¹,

Kobayashi²

2013

J Biotechnol Biomaterial

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In 1996, reversible immortalization using SV40 large T antigens and the Cre/LoxP system was successfully achieved with primary human fibroblasts. The concept of reversible immortalization involves introducing an immortalizing agent, SV40 large T antigens, into primary cells, expanding the cells in the culture, and finally, efficiently removing the immortalizing agent using Cre/LoxP site-specific recombination. The resulting cell population is essentially identical to the initial primary cells, but greatly increased in number. Since this report, reversible immortalization has been realized with hepatocytes, pancreatic β-cells, hepatic stellate cells, endothelial cells, renal epithelial cells and myogenic cells. This method facilitates the study of cell transplantation as well as cell differentiation, the cell cycle and senescence, by allowing one to control cell proliferation.

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Reversible transfection of human melanocytes mediated by Cre/loxP site‐specific recombination system and SV40 large T antigen

Abstract: Human melanocytes could be mediated by reversible transfection by SV40LTAg and Cre/loxP site-specific recombination system, which had stable parent-cell-like phenotypic characters and no tumorigenicity in vitro; moreover, these cells still had melanogenesis function in vivo.

Cited by 4 publications

References 28 publications

Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector

Reversible immortalization of rat pancreatic β cells with a novel immortalizing and tamoxifen-mediated self-recombination tricistronic vector

Nucleofection is a highly effective gene transfer technique for human melanoma cell lines

Controlled Expansion of Mammalian Cell Populations by Reversible Immortalization

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