Pollock Steve V, Mukherjee Bratati, Bajsa-Hirschel Joanna, Machingura Marylou C, Mukherjee Ananya, Grossman Arthur R, Moroney James V
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA.
Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305 USA.
Plant Methods. 2017 Apr 3;13:22. doi: 10.1186/s13007-017-0170-x. eCollection 2017.
Random insertional mutagenesis of using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome.
Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants.
The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.
利用耐药盒进行随机插入诱变已在全球数十个实验室中产生了数以万计的转化体。在许多情况下，这些插入突变体有助于阐明这种模式生物中各种生理过程的遗传基础。不幸的是，由于在衣藻基因组中确定插入盒位置存在实验困难，许多有趣突变体的插入位点从未被确定。通常需要数月甚至数年的工作却毫无结果。在此，我们描述一种在衣藻基因组中鉴定插入DNA盒位置的可靠方法。
使用赋予巴龙霉素抗性的DNA盒产生插入突变体。该方案确定了超过80%转化体的盒插入位点。在大多数情况下，插入事件是简单插入，侧翼基因组DNA没有大的缺失。在不到10%的回收转化体中观察到多个插入。
该方法快速、相对便宜，除了电穿孔仪外不需要任何特殊设备。该方案经过调整，以确保在高比例的转化体中始终获得随机插入侧翼的衣藻基因组DNA序列。本文提供了详细方案，以帮助进行衣藻突变体筛选的实验设计和实施。
