摘要
目的构建神经元抑制性沉默元件(RE-1/NRSE)双链RNA的慢病毒载体。方法根据RE-1/NRSE序列合成靶序列的寡核苷酸序列,退火形成双链DNA,与经HpaⅠ和XhoⅠ双酶切后的pGC-LV载体连接产生L-smRE-1/NRSE慢病毒载体,采用PCR和测序对阳性克隆进行鉴定。用L-smRE-1/NRSE和包装质粒pHelper 1.0、pHelper2.0共转染293T细胞,包装产生慢病毒颗粒,在倒置荧光显微镜下观察293T细胞中绿色荧光蛋白的表达量,并计算病毒滴度,初步观察其对大鼠间充质干细胞的转染效率。结果PCR和测序证实,构建出了RE-1/NRSE双链RNA的慢病毒载体L-smNRSE/RE-1。包装慢病毒,浓缩病毒悬液的滴度为4×108TU/ml。慢病毒颗粒能稳定转染大鼠间充质干细胞,当感染复数为80时,感染效率达100%。结论成功构建了RE-1/NRSE dsRNA的慢病毒表达载体。
Objective To construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element(RE-1/NRSE) double-stranded RNA(dsRNA).Methods The RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector.The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing.A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1,pHelper 1.0,and pHelper 2.0.The titer ...
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2009年第6期760-764,803,共6页
Acta Academiae Medicinae Sinicae
基金
辽宁省自然科学基金(20062090)
辽宁省教育厅科学技术研究项目(2004d227)~~
关键词
神经元抑制性沉默元件
双链RNA
慢病毒载体
repressor element-1/neuron-restrictive silencer element
double-stranded RNA
lentivirus vector
