Hochbaum Daniel, Ferguson Annabel A, Fisher Alfred L
Department of Medicine, University of Pittsburgh, USA.
J Vis Exp. 2010 Aug 23(42):2090. doi: 10.3791/2090.
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.
使用自由生活线虫秀丽隐杆线虫的实验室数量正在迅速增加。这种生物学模型的受欢迎程度归因于其快速的世代时间和短寿命、易于且成本低廉的维护、全基因组测序以及一系列RNAi资源和突变动物。此外，对秀丽隐杆线虫基因组的分析揭示了线虫与高等脊椎动物之间存在很大的相似性，这表明对线虫的研究可能是对全小鼠或培养细胞进行研究的重要补充。线虫研究中一个强大且重要的部分是能够使用转基因动物来研究基因定位和功能。转基因动物可以通过对蠕虫种系进行显微注射或通过使用生物弹道轰击来创建。轰击是一种较新的技术，许多实验室对此不太熟悉。在这里，我们描述了一种使用Bio-Rad PDS-1000系统通过用金颗粒进行生物弹道轰击来生成转基因蠕虫的简单方案。与将DNA显微注射到雌雄同体种系中相比，该方案的优点是在识别蠕虫解剖结构或进行显微注射方面不需要操作员具备特殊技能。此外，通常从单次轰击就能获得多个转基因品系。而且与显微注射不同，生物弹道轰击产生的转基因动物既有染色体外阵列又有整合的转基因。获得整合转基因品系的能力可以避免使用诱变方案来整合外源DNA。总之，生物弹道轰击可能是一种有吸引力的生成转基因动物的方法，特别是对于那些不想投入时间和精力来熟练掌握显微注射技术的研究人员来说。
