Targeted transgene insertion into human chromosomes by adeno-associated virus vectors
Targeted transgene insertion into human chromosomes by adeno-associated virus vectors
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DOI：
10.1038/nbt0702-735
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发表时间：
2002-07-01
影响因子：
46.9
通讯作者：
Russell, DW
中科院分区：
文献类型：
--
作者：
Hirata, R;Chamberlain, J;Russell, DW
二倍体人类细胞的精确遗传操作需要有效的方法，其中细胞衰老和低常规基因靶向率限制了实验和治疗选择。我们以前已经表明，基于腺相关病毒（AAV）的线性单链DNA载体可以准确地将小（1 kb）功能性转基因盒引入正常人成纤维细胞中的次黄嘌呤磷酸核糖转移酶（HPRT）和I型胶原（COL1A1）位点。转基因盒以高频率（在最佳条件下总细胞群体的1%）插入，并且没有二次突变。对插入的转基因盒的选择可用于富集靶向事件，使得>70%的存活细胞已经用适当设计的载体进行基因靶向。这种方法应该证明是有用的二倍体人类细胞的功能基因组分析和治疗基因靶向。
Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.