PhiC31 整合酶介导的基因电转移后在小鼠乳腺中的基因组整合和稳定基因表达。
PhiC31 integrase-mediated genomic integration and stable gene expression in the mouse mammary gland after gene electrotransfer.
出版信息
J Gene Med. 2013 Oct;15(10):356-65. doi: 10.1002/jgm.2723.
BACKGROUND
PhiC31 integrase is capable of conferring long-term transgene expression in various transfected tissues in vivo. In the present study, we investigated the activity of phiC31 integrase in mouse mammary glands.
METHODS
The normal mouse mammary epithelial cell line HC11 was transfected with FuGENE® HD Transfection Reagent (Roche Diagnostics, Shanghai, China). Transfection of the mouse mammary gland in vivo was performed by electrotransfer. Transgene expression was detected by western blotting and an enzyme-linked immunosorbent assay. Genomic integration and integration at mpsL1 was confirmed by a nested polymerase chain reaction.
RESULTS
An optimal electrotransfer protocol for the lactating mouse mammary gland was attained through investigation of different voltages and pulse durations. PhiC31 integrase mediated site-specific transgene integration in HC11 cells and the mouse mammary gland. In addition, the site-specific integration occurred efficiently at the ‘hot spot’ mpsL1. Co-delivery of PhiC31 integrase enhanced and prolonged transgene expression in the mouse mammary gland.
CONCLUSIONS
The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.
背景
PhiC31 整合酶能够在体内转染的各种组织中赋予长期的转基因表达。本研究旨在研究 phiC31 整合酶在小鼠乳腺中的活性。
方法
使用 FuGENE® HD 转染试剂（罗氏诊断公司，上海，中国）转染正常的小鼠乳腺上皮细胞系 HC11。通过电转移进行体内转染小鼠乳腺。通过 Western blot 和酶联免疫吸附试验检测转基因表达。通过巢式聚合酶链反应确认基因组整合和 mpsL1 处的整合。
结果
通过研究不同的电压和脉冲持续时间，获得了适用于哺乳期小鼠乳腺的最佳电转移方案。PhiC31 整合酶介导 HC11 细胞和小鼠乳腺中的特异性转基因整合。此外，特异性整合在“热点”mpsL1 处高效发生。PhiC31 整合酶的共递送增强并延长了小鼠乳腺中的转基因表达。
结论
本研究结果表明，使用 phiC31 整合酶是在小鼠乳腺中实现高效和稳定的转基因表达的一种可行且有效的方法。
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