Construction of adenovirus vectors using of Cre-loxPrecombination system

利用Cre-loxPrecombination系统构建腺病毒载体

基本信息

批准号：
12672199
负责人：
TASHIRO Fumi
金额：
$ 1.92万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672199/
关键词：
adenovirus vector construction Cre-loxP 作製法

项目摘要

To analyze the function of genes, various methods for introducing genes to mammalian cells and tissues have been developed. Adenovirus vector is drawing attention, because it has high efficiency of introducing genes and it can be infected to non-dividing cells. However, as the viral genome is relatively long(36kb), in constructing vectors there are some problems of complicated procedures or remaining of replicapable viral particles. To overcom these problems, we developed a new constructing method by using cosmid for long DNA and Cre-loxP site-specific recombination system. In present study, we accomplished to develop more efficient and applicable method for constructing adenovirus vectors by adding improvements.1. To insert longer expression cassettes, the cosmid containing adenoviral genome deleted E1 region(PALC) was deleted E3 region(PALCS). PALCS involving EGFP expression cassette was transfected to 293 cells, then replication of virus and the fluorescence of EGFP were observed. The efficient generation of adenovirus vectors and expression of introduced gene were recognized.2. FLP-FRT, site-specific recombination system derived from yeast, like as Cre-loxP, was introduced to constructing adenovirus vectors. PAFCS generated using FRT sequences instead of loxP in pALC3 was investigated the efficiency of replicating adenovirus vectors. In same efficiency of pALC3, PAFCS could produce adenovirus vectors.3. The expression cassette having DNA sequences interposed by loxP ligated to EGFP reporter gene was inserted to PAFCS. It was cotransfected to 293 cells with FLP and Cre recombinase expressing plasmids, and virus replication and EGFP fluorescence were observed. The results showed that this method could control the expression of introduced gene as required. It will be useful to introduce genes of cytotoxic protein, silently at first.More efficient and applicable method for constructing adenovirus vectors was developed by improvements accomplished in this study.

为了分析基因的功能，已经开发了将基因引入基因和组织的各种方法。腺病毒载体引起人们的注意，因为它具有高效率，可以将其感染到非分散细胞中。但是，由于病毒基因组相对较长（36KB），在构造矢量时，存在一些复杂的过程或可复制的病毒颗粒的问题。为了克服这些问题，我们通过将cosmid用于长DNA和CRE-LOXP特定的重组系统开发了一种新的构造方法。在目前的研究中，我们完成了开发更有效和适用的方法来通过增加改进来构建腺病毒载体的方法1。为了插入较长的表达盒，删除了含有腺病毒基因组的宇宙区域（PALC）的E3区域（PALC）。将涉及EGFP表达盒的PALC转染至293个细胞，然后观察到病毒的复制和EGFP的荧光。识别出有效的腺病毒载体和引入基因的表达。2。引入了从酵母中衍生的flp-frt，特定于酵母菌的位点重组系统，例如Cre-loxp，用于构建腺病毒载体。研究了使用FRT序列而不是PALC3中LOXP生成的PAFC，研究了复制腺病毒载体的效率。在同样的PALC3效率下，PAFC可以产生腺病毒载体3。将带有LOXP连接到EGFP报告基因的LOXP插入的DNA序列的表达盒插入了PAFC。它被共转染到293个细胞，该细胞用FLP和表达质粒的CRE重组酶共转染，并观察到病毒复制和EGFP荧光。结果表明，该方法可以根据需要控制引入基因的表达。引入细胞毒性蛋白的基因将是有用的，首先是默默地。通过这项研究中的改进，开发了更有效且适用于构建腺病毒载体的方法。