Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids
Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids
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DOI：
10.1095/biolreprod.113.116905
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发表时间：
2014-05-01
影响因子：
3.6
通讯作者：
Wu, Zhenfang
中科院分区：
文献类型：
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作者：
Li, Zicong;Zeng, Fang;Wu, Zhenfang
转基因的过程包括将外源基因（转基因）引入动物的基因组中。通过原核显微注射（PNI）进行基因转移是用于产生转基因动物的主要方法。然而，这种技术并不总是产生生殖系转基因后代，并且对牲畜的成功率较低。替代方法，如使用转基因成纤维细胞的体细胞核转移，与PNI相比，没有显示出效率的增加，而基于病毒的转基因则受到转基因大小和生物安全考虑的问题的阻碍。我们最近描述了使用piggyBac转座酶为基础的载体pmhyGENIE-3进行的非常成功的小鼠转基因实验。该构建体是一个单一的自失活质粒，含有成功基因转移所需的所有转座元件。在这一系列实验中，我们的实验室已经实施了pmGENIE-3的细胞质注射（CTI），用于将转基因递送到体内受精的猪受精卵中。超过8.00%的注射胚胎发育成转基因动物，其基因组中含有单基因，通常是单转基因。然而，CTI技术是不成功的，在体外受精猪受精卵的注射过程中。总之，在这里，我们描述了一种方法，不仅易于实施，而且还证明了非病毒家畜转基因的最高效率。
The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.