Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system.
Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system.
复制标题
利用粘粒中克隆的环状腺病毒基因组和 Cre-loxP 重组系统构建腺病毒载体。
DOI：
10.1089/10430349950017527
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发表时间：
1999
影响因子：
4.2
通讯作者：
J. Miyazaki
中科院分区：
文献类型：
--
作者：
F. Tashiro;H. Niwa;J. Miyazaki
重组腺病毒载体已经通过体内同源重组方法或通过体外直接连接方法产生。然而，由于重组载体的尺寸较大，通过这些方法构建腺病毒载体的效率较低。为了提高构建腺病毒载体的容易性，我们使用了环状形式的腺病毒DNA，其可以在转染到组成型产生腺病毒E1蛋白的293细胞中后以与病毒体DNA相当的效率产生感染性病毒。我们用侧接loxP位点的粘粒载体替换腺病毒DNA环状形式的E1区，得到41-kb的粘粒，命名为pALC。使用粘粒载体克隆系统，将双顺反子表达IL-5和绿色荧光蛋白（GFP）的表达盒容易地插入loxP侧翼的粘粒骨架和pALC的腺病毒基因组之间。将所得粘粒转染到293细胞中不产生任何感染性腺病毒，因为其大小（46 kb）大于腺病毒颗粒的包装容量。然而，将Cre表达质粒与该粘粒共转染到293细胞中有效地切除了loxP侧翼的粘粒载体骨架，并产生了表达IL-5和GFP的腺病毒载体。为了进一步简化我们的方法，我们已经产生了组成型表达Cre重组酶的293细胞系。将pALC粘粒单独转染到该细胞系中有效地产生腺病毒载体。本文提出的腺病毒载体构建方法简单有效，将进一步促进重组腺病毒载体在体内和体外基因转移中的应用。
Recombinant adenoviral vectors have been generated either by the in vivo homologous recombination method or by the in vitro direct ligation method. However, the efficiency of adenoviral vector construction by these methods is low, because of the large size of the recombinant vectors. To improve the ease of constructing adenoviral vectors, we used the circular form of adenoviral DNA, which can generate infectious viruses with an efficiency comparable to that of virion DNA, after transfection into 293 cells constitutively producing adenovirus E1 protein. We replaced the E1 region of the circular form of adenoviral DNA with a cosmid vector flanked by loxP sites, resulting in a 41-kb cosmid, designated pALC. An expression cassette that bicistronically expresses IL-5 and green fluorescent protein (GFP) was readily inserted between the loxP-flanked cosmid backbone and the adenoviral genome of pALC, using the cosmid vector cloning system. Transfection of the resulting cosmid into 293 cells did not produce any infectious adenoviruses because its size (46 kb) was larger than the packing capacity of the adenoviral particles. However, cotransfection of a Cre-expression plasmid with this cosmid into 293 cells efficiently excised the loxP-flanked cosmid vector backbone, and produced the adenoviral vector expressing IL-5 and GFP. To simplify our method further, we have produced a 293 cell line constitutively expressing Cre recombinase. Transfection of pALC cosmid alone into this cell line efficiently generated adenoviral vector. The adenoviral vector construction method presented here is simple and efficient and should further facilitate the application of recombinant adenoviral vectors for in vivo and in vitro gene transfer.
DOI：
10.1073/pnas.91.13.6186
发表时间：
1994-06-21
影响因子：
11.1
作者：
KETNER, G;SPENCER, F;HIETER, P
通讯作者：
HIETER, P