A trial of somatic gene targeting in vivo with an adenovirus vector.
A trial of somatic gene targeting in vivo with an adenovirus vector.
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DOI：
10.1186/1479-0556-3-8
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发表时间：
2005-10-12
期刊：
影响因子：
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通讯作者：
Kobayashi, Ichizo
中科院分区：
文献类型：
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作者：
Ino, Asami;Naito, Yasuhiro;Kobayashi, Ichizo
背景：体内基因靶向为基因分析和基因治疗提供了一种潜在的强大方法。为了灵敏地检测和准确地测量设计的序列变化，我们使用了一种转基因小鼠系统MutaMouse，该系统已开发用于检测体内突变。它携带有lacZ+基因的噬菌体lambda基因组，在体外包装成噬菌体颗粒后检测其向lacZ阴性等位基因的变化。我们还证明了用复制缺陷腺病毒载体进行基因转移可以在体外实现高效和准确的基因靶向。方法：将含有两个lacz阴性单碱基对替代突变等位基因之一的噬菌体lambda转基因对应的8kb长的DNA插入复制缺陷腺病毒载体中。重组腺病毒经尾静脉注射到转基因小鼠体内。24小时后，从肝组织中提取基因组DNA，体外包装回收lambda::lacZ。用苯基- β - d -半乳糖苷在琼脂上检测到lacz阴性噬菌体为斑块前体。结果：lacz阴性重组腺病毒注射小鼠的突变频率与对照小鼠相同（约为万分之一）。我们进一步的限制性分析没有检测到任何设计的重组。结论：这些重组腺病毒在小鼠肝脏的基因靶向率小于1/20000。然而，这些结果将有助于开发一种敏感、可靠和不依赖于pcr的检测方法，用于由病毒载体和其他手段介导的体内基因靶向。
BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro.METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside.RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (approximately 1/10000). Our further restriction analysis did not detect any designed recombinant.CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means.