Site-specific transgene integration in the human genome catalyzed by phiBT1 phage integrase.
Site-specific transgene integration in the human genome catalyzed by phiBT1 phage integrase.
复制标题
由 phiBT1 噬菌体整合酶催化的人类基因组中的位点特异性转基因整合。
DOI：
10.1089/hum.2007.110
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发表时间：
2008
影响因子：
4.2
通讯作者：
Woo,SavioLC
中科院分区：
文献类型：
--
作者：
Chen,Li;Woo,SavioLC
链霉菌噬菌体phiBT1整合酶催化噬菌体attP位点和细菌attB位点之间的重组（att，附着），导致噬菌体DNA以单向方式整合到细菌宿主基因组中。多个伪attB和attP位点在哺乳动物基因组中偶然存在，并且可以与野生型attP和attB序列重组。phiBT1系统先前已被用于实现小鼠苯丙氨酸羟化酶cDNA的位点特异性整合到苯丙酮尿小鼠的肝细胞中，从而导致疾病表型的完全和永久纠正，而没有明显的毒性。在这里，我们报道了人类细胞中三个伪attp和两个伪attb位点的鉴定，它们位于五个不同染色体的基因间区域。在插入连接处没有人类基因组序列的微缺失，并且表达了整合的转基因。表达phiBT1整合酶的人细胞核型正常，假attb和attp位点之间没有染色体易位。聚合酶链反应分析了从表达phiBT1整合酶的各种人类细胞类型分离的基因组DNA，使用引物连接来自不匹配的人类染色体的伪attb和attp位点。在正常的人类肝细胞、外周血单核细胞、血管微内皮细胞和其他两种转化的人类细胞系中未检测到染色体易位事件，尽管在人类黑色素瘤细胞系中观察到一个这样的事件。结果表明，染色体易位的发生是人类细胞类型依赖的，并且phiBT1系统可以将转基因特定位点整合到人类基因组中，可以用于选择应用。
The Streptomyces phage phiBT1 integrase catalyzes recombination between phage attP and bacterial attB sites (att, attachment), resulting in phage DNA integration into the bacterial host genome in a unidirectional manner. Multiple pseudo-attB and-attP sites are present serendipitously in mammalian genomes and can recombine with wild-type attP and attB sequences. The phiBT1 system has been used previously to achieve site-specific integration of murine phenylalanine hydroxylase cDNA into hepatocytes of mice with phenylketonuria, which led to the complete and permanent correction of the disease phenotypes without apparent toxicities. Here we report the identification of three pseudo-attP and two pseudo-attB sites in human cells, which are located in intergenic regions of five different chromosomes. There are no microdeletions of human genomic sequences at the insertional junctions and the integrated transgenes are expressed. Human cells expressing phiBT1 integrase showed normal karyotypes without chromosomal translocations between the pseudo-attB and-attP sites. Polymerase chain reaction analyses were performed on genomic DNA isolated from various human cell types expressing phiBT1 integrase, using primers flanking the pseudo-attB and-attP sites from mismatched human chromosomes. No chromosomal translocation events were detected in normal human hepatocytes, peripheral blood mononuclear cells, vascular microendothelial cells, and two other transformed human cell lines, although one such event was observed in a human melanoma cell line. The results suggest that the occurrence of chromosomal translocations is human cell type dependent, and that the phiBT1 system for site-specific integration of transgenes into the human genome can be used in selected applications.