Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.
Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.
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DOI：
10.1016/j.jbiotec.2014.07.012
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发表时间：
2014-09
影响因子：
4.1
通讯作者：
Hideyuki Nakanishi;Y. Higuchi;F. Yamashita;M. Hashida
中科院分区：
文献类型：
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作者：
Hideyuki Nakanishi;Y. Higuchi;F. Yamashita;M. Hashida
基于PhiC31整合酶的载体可以选择性地将治疗性基因整合到基因组中的attpor伪attpsite中，但人类基因组中相当数量的伪attpsite存在于内源性基因编码区。为了避免内源性基因中断，我们旨在使用含有Gal4和LexA dna结合基序的序列特异性dna结合蛋白来增强基于phiC31整合酶的载体的整合位点特异性。双dna结合蛋白被设计用于将含有uas的供体载体连接到目标序列，即LexA操作符，并将整合限制在LexA操作符附近的位点。为了分析染色体整合的位点特异性，建立了基因组上有LexA操作符的人细胞系，并用表达dna结合蛋白的供体载体和phiC31整合酶表达载体（辅助载体）转染细胞系。定量PCR结果显示，含有uas的供体载体在LexA操作器周围的整合度比对照高26倍。序列分析证实，集成发生在LexA操作符周围。本文开发的基于双dna结合蛋白的靶向整合策略将为各种应用（包括基因和细胞治疗）提供更安全、更可靠的遗传操作。
PhiC31 integrase-based vectors can integrate therapeutic genes selectively intoattPor pseudo-attPsites in genomes, but considerable numbers of pseudo-attPsites in human genomes exist inside endogenous gene-coding regions. To avoid endogenous gene disruptions, we aimed to enhance the integration site-specificity of the phiC31 integrase-based vector using a sequence-specific DNA-binding protein containing Gal4 and LexA DNA-binding motifs. The dual DNA-binding protein was designed to tether the UAS-containing donor vector to the target sequence, the LexA operator, and restrict integration to sites close to the LexA operator. To analyze the site-specificity in chromosomal integration, a human cell line having LexA operators on the genome was established, and the cell line was transfected with donor vectors expressing the DNA-binding protein and the phiC31 integrase expression vector (helper vector). Quantitative PCR indicated that integration around the LexA operator was 26-fold higher with the UAS-containing donor vector than with the control. Sequence analysis confirmed that the integration occurred around the LexA operator. The dual DNA-binding protein-based targeted integration strategy developed herein would allow safer and more reliable genetic manipulations for various applications, including gene and cell therapies.