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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment.
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Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment.

机译：通过重组改造氨苄青霉素抗性载体，用于通过轰击产生秀丽隐杆线虫转基因动物。

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摘要

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5' and 3' ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.
机译：秀丽隐杆线虫是现代生物学研究的重要模式生物。秀丽隐杆线虫研究的一个重要方面是生产用于研究的转基因动物。这些通常是通过显微注射产生的，但是生物弹轰击变得越来越普遍。然而，由于缺乏方便的标记，许多先前产生的用于显微注射的质粒不容易用于轰击。 unc-119基因通常用作标记，因为可以在低放大倍数下观察到unc-119的抢救，因此可以轻松地将抢救的动物与大量非抢救的动物区分开。在这里，我们报道了在大肠杆菌中使用同源重组作为一种将包含unc-119基因的盒插入氨苄青霉素抗性基因位点常用质粒中的方法，该方法比其他方法（如亚克隆方法）更简单。这些盒的侧翼是与氨苄青霉素抗性基因的5'和3'端同源的区域，并含有unc-119基因和卡那霉素抗性基因或unc-119：mCherry融合基因和卡那霉素抗性基因。所得质粒可用于生物弹轰击，以产生表现出unc-119拯救并且还表达受体质粒转基因的动物。

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