Transformation of dictyostelium with plasmid DNA by calcium phosphate precipitation.

CSH protocols Pub Date : 2008-12-01 DOI:10.1101/pdb.prot5102
Pascale Gaudet, Petra Fey, Rex Chisholm
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引用次数: 0

Abstract

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Calcium phosphate precipitation is a commonly used method for DNA-mediated transformation in Dictyostelium. Calcium phosphate precipitation produces high-copy-number transformants and is often used for overexpression experiments in conjunction with the G418 resistance gene, which needs to be present at high levels to produce efficient selection.

磷酸钙沉淀法转化质粒DNA的dictyostium。
盘状盘基骨柱是一种单细胞真核生物,通常被称为社会性变形虫,因为它可以在营养条件有限的情况下形成多细胞结构。在细胞运动过程中,细胞间通讯、细胞内信号传导和细胞骨架组织的一般原理来源于盘基骨菌的细胞和分子研究,并被发现在所有真核生物中都是保守的。完整基因组数据库的可用性以及野生型和突变株的存量使盘状盘状盘虫成为一种可接近和强大的模式生物。Dictyostelium能够适应需要将DNA引入细胞的遗传操作,如基因敲除、过表达、反义RNA表达、RNA干扰(RNAi)介导的基因敲低和限制性酶介导的突变。磷酸钙沉淀是Dictyostelium dna介导转化的常用方法。磷酸钙沉淀产生高拷贝数的转化子,通常与G418抗性基因一起用于过表达实验,G418抗性基因需要高水平存在以产生有效的选择。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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