Reversible transfection of human melanocytes mediated by Cre/loxP site-specific recombination system and SV40 large T antigen.
OBJECTIVE
To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/loxP site-specific recombination system.


METHODS
The reconstructed SV40LTAg-EGFP-neo-loxP vector was transfected into primary cultured human melanocytes with Sofast(TM) transfection reagent and the positive cells were selected using G418. After expanding culture of these positive cell clones, the expression of SV40LTAg was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent method. After that, these positive cells were infected by virus supernatant of Cre-ER(T2) retrovirus vector and Cre recombinase was induced to act by tamoxifen. On the 6th and 10th day after Cre recombinase acting, the expression of SV40LTAg was detected using the same methods as above, and cell tumorigenicity was studied using soft agar assay, athymic mouse study and karyotype analysis. On 10th day after tamoxifen treatment, cell biological characters were identified with immunofluorescent staining and transmission electron microscopy. Then these cells were transplanted into vitiligo animal model to observe their melanogenesis ability in vivo.


RESULTS
The genome DNA and total RNA were isolated from the positive cells transfected by SV40LTAg (designated as MCT) and specific 288 bp fragment was amplificated using PCR and RT-PCR methods. The results of immunofluorescence confirmed the expression of SV40LTAg in cell nucleus. On the 6th day after tamoxifen treatment in infected cells by Cre-ER(T2) retrovirus vector (designated as MCT-Cre), there could be detected SV40LTAg expression, but on 10th day, there could not be detected SV40LTAg expression in cells. These results showed that the excised efficiency of Cre recombinase increased along with time prolongation, and would obtain complete recombination efficiency. The identification of MCT-Cre cell biological characters showed that these cells had normal parent-cell-like cell phenotype and no tumorigenicity in vitro. The pigmentation started in 4 weeks and formed black macula in 3 months after grafting. The pathological results showed that there had been significant melanocytes and melanin accumulation in epidermis and some hair follicle in transplanted area, which confirmed that MCT-Cre had melanogenesis function in vivo.


CONCLUSIONS
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.
Ying
Wang
W
Dermatology Department of Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Fei
Hao
H
Jun
Deng
D
Xi-chuan
Yang
Y
Bai-yu
Zhong
Z
Qing-yi
Ye
Y
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D008545:Melanoma / Q000209:etiology / Q000235:genetics / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014410:Tumor Stem Cell Assay
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers