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[稳定表达bcr-abl融合基因片段的小鼠SP2/0细胞系的建立]

[Establishment of mouse SP2/0 cell line stably expressing bcr-abl fusion gene fragment].

作者信息

Jiang Yang-Wen, Qian Li, Liu Wei, Gong Wei-Juan, Wang Bing, Guan Jun, Ji Ming-Chun

机构信息

Department of Hematology, Affiliated Hospital of Yangzhou University, Yangzhou 225001, China.

出版信息

Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2005 Aug;13(4):601-4.

Abstract

To establish SP2/0 cell line H-2(d) stably expressing bcr-abl fusion gene fragment, the bcr-abl fusion gene was subcloned into retroviral vector pLXSN from pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with the help of lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected to determine viral titer, the viral titer was 2 x 10(7) CFU/ml. The SP2/0 cells were infected with the collected viral supernatant. The results showed that after G418 selection, the bcr-abl fusion gene was integrated into the chromosome of SP2/0 cells infected stably, with recombinant retrovirus and expressed in SP2/0 cells confirmed by PCR and RT-PCR respectively. In conclusion, the mouse tumor cell lines expressing the bcr-abl fusion protein were successfully established and would be used as a experimental cell model for anti-CML immunotherapy.

摘要

为建立稳定表达bcr-abl融合基因片段的SP2/0细胞系H-2(d),将bcr-abl融合基因从pGEMbcr-abl亚克隆到逆转录病毒载体pLXSN中。借助脂质体将重组逆转录病毒载体pLXSNbcr-abl转染到PT67包装细胞中。经G418筛选后选出阳性克隆并进行培养。然后收集病毒上清液测定病毒滴度,病毒滴度为2×10(7) CFU/ml。用收集的病毒上清液感染SP2/0细胞。结果显示,经G418筛选后,bcr-abl融合基因稳定整合到感染的SP2/0细胞染色体中,通过PCR和RT-PCR分别证实重组逆转录病毒在SP2/0细胞中存在并表达。综上所述,成功建立了表达bcr-abl融合蛋白的小鼠肿瘤细胞系,可作为抗慢性粒细胞白血病免疫治疗的实验细胞模型。

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