Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function
Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function
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DOI：
10.2144/000112993
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发表时间：
2008-12-01
期刊：
影响因子：
2.7
通讯作者：
Wilkinson, Gavin W. G.
中科院分区：
文献类型：
--
作者：
Stanton, Richard J.;McSharry, Brian P.;Wilkinson, Gavin W. G.
随着生物信息学审问大量序列数据的能力增强，需要更有效的技术来测试基因功能预测。复制缺陷重组腺病毒（Ad）载体被广泛用于表达分析，因为它们提供了在广泛的细胞类型中非常有效的转基因表达。为了促进重组病毒的快速、高通量生成，我们重新设计了一种腺病毒载体（指定为AdZ），允许使用重组技术进行单步定向基因插入。重组允许将PCR产物、合成序列或编码shrna的寡核苷酸直接插入到Ad载体中，而不需要转移载体。载体通过将I-SceI归巢内切酶结合到载体中，使其“自我切除”，从而优化了高通量应用，从而消除了在转染到包装细胞之前对载体进行线性化的需要。AdZ载体允许基因以其天然形式或与链球菌，V5或GFP标签表达。将四环素操作符插入人巨细胞病毒主要立即早期（HCMV MIE）启动子下游，可以沉默表达tet抑制因子的辅助细胞中的转基因，从而使载体与毒性基因产物的克隆兼容。AdZ矢量系统稳健、简单，适合零星和高通量应用。
With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.