Gene disruption in Coccidioides using hygromycin or phleomycin resistance markers.
Gene disruption in Coccidioides using hygromycin or phleomycin resistance markers.
复制标题
使用潮霉素或腐草霉素抗性标记物破坏球孢子菌中的基因。
DOI：
10.1007/978-1-61779-539-8_9
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Cole,GarryT
中科院分区：
文献类型：
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作者：
Hung,Chiung-Yu;Wise,HuaZhang;Cole,GarryT
以下转化方案是基于在基因破坏或基因置换构建体与球孢子菌属的靶基因之间发生的同源重组。所采用的DNA构建体含有编码潮霉素B或腐草霉素抗性的基因，它们分别存在于pAN 7.1或pAN 8.1质粒载体中。用潮霉素B或腐草霉素筛选转化子，其浓度应能抑制亲本菌株的生长。质粒DNA构建体在钙和聚乙二醇存在下被原生质体吸收。在每个转化实验中可以获得20至100个转化体/μg DNA。在所有发育阶段的球孢子细胞，包括节孢子，都是多核的。由于所有球孢子菌核都是单倍体，因此只需一次转化就足以产生突变株。然而，转化的原生质体发育成通常含有亲本和突变核的异源细胞。为了分离同核菌株，我们在含有潮霉素B或腐草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，以富集突变的细胞核。将分离的菌落继代培养3 ~ 4次后，可获得同核突变体。本文介绍了球孢子菌原生质体的制备、转化和同核突变体的分离方法。
The following transformation protocol is based on homologous recombination that occurs between a gene disruption or gene replacement construct and a target gene ofCoccidioides. 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.Coccidioidesprotoplasts 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.Coccidioidescells in all developmental stages, including arthroconidia, are multinucleate. Since allCoccidioidesnuclei 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 ofCoccidioidesprotoplasts, transformation and isolation of homokaryotic mutants.