Active integration: new strategies for transgenesis
Active integration: new strategies for transgenesis
复制标题
DOI：
10.1007/s11248-007-9077-z
复制
发表时间：
2007-06-01
影响因子：
3
通讯作者：
Moisyadi, Stefan
中科院分区：
文献类型：
--
作者：
Shinohara, Eric T.;Kaminski, Joseph M.;Moisyadi, Stefan
本文介绍了生产转基因动物的新方法，并进一步强调这些技术将来如何应用于基因治疗。有几种被动的转基因方法，如原核显微注射（PNI）和胞质内精子注射介导的转基因（ICSI-Tr），它们依赖于宿主的修复机制转基因（tg）插入。ICSI-Tr已被证明是产生转基因动物的有效手段，其转染效率约为出生动物的45%。此外，因为这涉及在受精期间将转基因注射到卵母细胞的细胞质中，所以传统上使用该技术发生有限的嵌合现象。目前的主动转基因技术涉及使用病毒，例如可以将基因插入宿主基因组的解除武装的逆转录病毒。然而，这些方法受到可插入序列的大小、高胚胎死亡率和插入随机性的限制。已经开发了一种新的活性方法，其将ICSI-Tr与重组酶或转座酶组合以增加转染效率。这种技术被称为“主动转基因”，这意味着在tg引入过程中，通过提供给卵母细胞的酶将tg插入宿主基因组中。基于DNA的方法减轻了与纯化酶相关的许多成本和时间。进一步的研究表明，RNA可以作为转座酶的来源。使用RNA可以防止如果DNA转座酶整合到宿主基因组中可能发生的持续转座酶活性的问题。目前，piggyBac是在哺乳动物系统中稳定整合的最有效的转座子，并且随着进一步的研究进行以阐明改善piggyBac的特异性和功效的修饰，产生转基因动物的效率应该进一步提高。随后，这些方法有一天可能用于人类的基因治疗。
This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.