Ying Wang, Fei Hao, Jun Deng, Xi-chuan Yang, Bai-yu Zhong, Qing-yi Ye
Dermatology Department of Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Exp Dermatol. 2007 May;16(5):437-44. doi: 10.1111/j.1600-0625.2007.00546.x.
To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/loxP site-specific recombination system.
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.
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.
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.
研究猿猴病毒40大T抗原（SV40LTAg）和Cre/loxP位点特异性重组系统介导的人黑素细胞可逆转染。
用Sofast™转染试剂将重组的SV40LTAg-EGFP-neo-loxP载体转染至原代培养的人黑素细胞，并用G418筛选阳性细胞。对这些阳性细胞克隆进行扩大培养后，通过聚合酶链反应（PCR）、逆转录-聚合酶链反应（RT-PCR）和免疫荧光法检测SV40LTAg的表达。之后，用Cre-ER(T2)逆转录病毒载体的病毒上清感染这些阳性细胞，并用他莫昔芬诱导Cre重组酶发挥作用。在Cre重组酶作用后的第6天和第10天，用上述相同方法检测SV40LTAg的表达，并通过软琼脂试验、无胸腺小鼠研究和核型分析研究细胞致瘤性。在他莫昔芬处理后的第10天，用免疫荧光染色和透射电子显微镜鉴定细胞生物学特性。然后将这些细胞移植到白癜风动物模型中，观察其体内黑素生成能力。
从经SV40LTAg转染的阳性细胞（命名为MCT）中分离出基因组DNA和总RNA，用PCR和RT-PCR方法扩增出特异性的288 bp片段。免疫荧光结果证实SV40LTAg在细胞核中表达。在用Cre-ER(T2)逆转录病毒载体感染的细胞（命名为MCT-Cre）中，他莫昔芬处理后的第6天可检测到SV40LTAg表达，但在第10天，细胞中未检测到SV40LTAg表达。这些结果表明，Cre重组酶的切除效率随时间延长而增加，并可获得完全重组效率。MCT-Cre细胞生物学特性鉴定表明，这些细胞具有正常的亲代细胞样细胞表型，在体外无致瘤性。移植后4周开始色素沉着，3个月形成黑色斑片。病理结果显示，移植区域的表皮和一些毛囊中有大量黑素细胞和黑色素积聚，证实MCT-Cre在体内具有黑素生成功能。
SV�0LTAg和Cre/loxP位点特异性重组系统可介导人黑素细胞可逆转染，这些细胞具有稳定的亲代细胞样表型特征，在体外无致瘤性；此外，这些细胞在体内仍具有黑素生成功能。
