MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, United States of America.
PLoS One. 2018 May 30;13(5):e0196809. doi: 10.1371/journal.pone.0196809. eCollection 2018.
Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a 'safe haven' for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.
盘基网柄菌拥有成熟的分子遗传学操作技术，可通过使用几种不同的选择性标记物、标记物再循环、同源重组和插入诱变进行转染，所有这些都得到了注释良好的基因组的支持。然而，这项技术是针对突变体、无菌细胞进行优化的，与非无菌野生型细胞不同，突变体、无菌细胞可以在液体培养基中生长。目前迫切需要操纵野生型细胞和巨胞饮缺陷细胞的方法，而这两种细胞都不能在液体培养基中生长。在这里，我们提出了一系列基于在细菌上而非液体培养基上选择盘基网柄菌转染体的分子遗传技术。这些技术不仅扩展了可操作的菌株范围，而且比传统方法更快，通常在几天内就能获得可用数量的转染细胞。本文所述的方法和质粒允许使用染色体外载体进行有效的转染，以及在“安全港”进行染色体整合，以实现相对均匀的细胞间表达、高效的基因敲入和敲除以及诱导表达系统。因此，我们为盘基网柄菌细胞的遗传操作创建了一个全新的系统，不再需要在液体培养基上进行细胞喂养。
