Hung Chiung-Yu, Wise Hua Zhang, Cole Garry T
Department of Biology and South Texas Center for Emerging Infectious Diseases, University of Texas, San Antonio, TX, USA.
Methods Mol Biol. 2012;845:131-47. doi: 10.1007/978-1-61779-539-8_9.
The following transformation protocol is based on homologous recombination that occurs between a gene disruption or gene replacement construct and a target gene of Coccidioides. The DNA constructs employed contain either the gene that encodes for hygromycin B or phleomycin resistance, which are present in the pAN7.1 or pAN8.1 plasmid vectors, respectively. Hygromycin B or phleomycin are used to select for transformants at concentrations that inhibit growth of the parental strain. Coccidioides protoplasts generated from germinated arthroconidia are used for the transformation experiments. The plasmid DNA constructs are taken up by the protoplasts in the presence of calcium and polyethylene glycol. Twenty to 100 transformants/μg DNA can be obtained in each transformation experiment. Approximately 5-10% of the transformation events are homologous recombinations. Coccidioides cells in all developmental stages, including arthroconidia, are multinucleate. Since all Coccidioides nuclei are haploid, only one run of transformation is sufficient to create a mutant strain. However, the transformed protoplasts develop into heterokaryotic cells that typically contain both the parental and mutated nuclei. To isolate a homokaryotic strain, we perform multiple subcultures of the single colonies which contain heterokaryotic cells on selection plates with hygromycin B or phleomycin to enrich for the mutated nuclei. Homokaryotic mutants can be obtained after three to four subcultures of isolated colonies. In this protocol, we describe the methodology for preparation of Coccidioides protoplasts, transformation and isolation of homokaryotic mutants.
以下转化方案基于在球孢子菌的基因破坏或基因替换构建体与靶基因之间发生的同源重组。所使用的DNA构建体包含编码潮霉素B或博来霉素抗性的基因，它们分别存在于pAN7.1或pAN8.1质粒载体中。潮霉素B或博来霉素用于以抑制亲本菌株生长的浓度选择转化体。从发芽的关节孢子产生的球孢子菌原生质体用于转化实验。质粒DNA构建体在钙和聚乙二醇存在下被原生质体摄取。每次转化实验可获得20至100个转化体/μg DNA。大约5 - 10%的转化事件是同源重组。球孢子菌在所有发育阶段的细胞，包括关节孢子，都是多核的。由于所有球孢子菌核都是单倍体，仅一轮转化就足以创建突变菌株。然而，转化的原生质体发育成异核细胞，通常包含亲本核和突变核。为了分离同核菌株，我们在含有潮霉素B或博来霉素的选择平板上对含有异核细胞的单菌落进行多次传代培养，以富集突变核。分离菌落经过三到四次传代培养后可获得同核突变体。在本方案中，我们描述了球孢子菌原生质体制备、转化和同核突变体分离的方法。
