EFFICIENT MANIPULATION OF THE HUMAN ADENOVIRUS GENOME AS AN INFECTIOUS YEAST ARTIFICIAL CHROMOSOME CLONE
EFFICIENT MANIPULATION OF THE HUMAN ADENOVIRUS GENOME AS AN INFECTIOUS YEAST ARTIFICIAL CHROMOSOME CLONE
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DOI：
10.1073/pnas.91.13.6186
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
HIETER, P
中科院分区：
文献类型：
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作者：
KETNER, G;SPENCER, F;HIETER, P
构建了含有完整的人2型腺病毒基因组的酵母人工染色体（YAC），并显示来自YAC的病毒DNA在引入哺乳动物细胞后具有感染性。腺病毒YAC可以在酵母宿主中使用基于同源重组的方法有效地操纵，并且突变病毒，包括表达酿酒酵母CDC 27基因的人类类似物的变体，容易地从YAC的修饰衍生物中回收。将强大的酵母遗传技术应用于感染性腺病毒克隆，有望显著增强腺病毒的遗传分析，并简化用于疫苗或用于基因转移至哺乳动物细胞或整个动物的基于腺病毒的载体的构建。腺病毒YAC是通过腺病毒2型病毒粒子DNA与携带病毒左右基因组末端片段的YAC载体质粒之间的体内同源重组产生的。这种重组克隆策略通常适用于构建含有其他DNA片段的YAC，例如其他病毒的基因组。此外，它是非常有效的，并且可以允许直接从基因组DNA靶向克隆高等生物体的基因组区段。
A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the YAC. The application of powerful yeast genetic techniques to an infectious adenovirus clone promises to significantly enhance the genetic analysis of adenovirus and to simplify the construction of adenovirus-based vectors for vaccines or for gene transfer to mammalian cells or whole animals. The adenovirus YAC was produced by homologous recombination in vivo between adenovirus 2 virion DNA and YAC vector plasmids carrying segments of the viral left and right genomic termini. This recombinational cloning strategy is generally applicable to the construction of YACs containing other DNA segments, such as the genomes of other viruses. Further, it is very efficient and may permit the targeted cloning of segments of the genomes of higher organisms directly from genomic DNA.