Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum

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（Dictyostelium）拥有一套成熟的分子遗传操作技术体系，该体系以转染为核心，整合了多种不同筛选标记、标记回收、同源重组及插入诱变技术，所有技术均依托于注释完备的基因组。然而该技术体系是针对突变型无菌培养细胞（axenic cells）优化设计的，这类细胞与非无菌野生型细胞不同，可在液体培养基中稳定生长。目前学界迫切需要可用于操作野生型细胞以及巨胞饮作用（macropinocytosis）存在缺陷的细胞的遗传操作方法——这两类细胞均无法在液体培养基中生长。本文报道了一套分子遗传技术体系，其核心是通过在细菌培养基而非液体培养基上培养来筛选盘基网柄菌转染子（transfectants）。该套技术不仅拓展了可进行遗传操作的菌株范围，且相较于传统方法更为高效快捷，通常仅需数天即可获得满足实验需求的转染细胞群体。本文所述的技术与质粒可实现染色体外载体的高效转染，还可在安全港（safe haven）位点进行染色体整合以实现细胞间相对均一的基因表达，同时可实现高效的基因敲入、基因敲除以及诱导型表达系统的构建。综上，我们构建了一套全新的盘基网柄菌遗传操作系统，该系统无需再依赖液体培养基进行细胞培养。