Generation of transgenic C. elegans by biolistic transformation.
Generation of transgenic C. elegans by biolistic transformation.
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DOI：
10.3791/2090
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发表时间：
2010-08-23
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影响因子：
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通讯作者：
Fisher, Alfred L
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文献类型：
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作者：
Hochbaum, Daniel;Ferguson, Annabel A;Fisher, Alfred L
使用自由活线虫C.秀丽隐杆线虫的实验室数量正在迅速增长。该生物学模型的普及归因于迅速的生成时间和寿命短，容易且廉价的维护，完全测序的基因组以及一系列RNAi资源和突变动物。此外，对秀丽隐杆线虫基因组的分析显示，蠕虫和较高的脊椎动物之间的相似性很大，这表明蠕虫的研究可能是整个小鼠或培养细胞进行的研究的重要辅助。蠕虫研究的强大而重要的部分是能够使用转基因动物研究基因定位和功能。可以通过对蠕虫种系的微注射或使用生物轰击来创建转基因动物。轰炸是一种较新的技术，对许多实验室不太熟悉。在这里，我们描述了一种简单的方案，可以使用Bio-Rad PDS-1000系统用金颗粒产生转基因蠕虫。与DNA对雌雄同体种系的DNA显微注射相比，该方案的优点是不需要操作员的特殊技能，以识别蠕虫解剖结构或进行显微注射。通常从单个轰炸中获得更多多个转基因线。与显微注射相反，生物学轰击会产生带有外染色体阵列和综合转基因的转基因动物。获得综合转基因线的能力可以避免使用诱变方案整合外源DNA。总之，生物轰炸可能是产生转基因动物的一种有吸引力的方法，尤其是对于不感兴趣的调查人员而言，有兴趣投入熟练的时间和精力来熟练地进行显微注射。
The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.