King Jason, Insall Robert H
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
BMC Genet. 2003 Jul 10;4:12. doi: 10.1186/1471-2156-4-12.
The relative ease of targeted gene disruption in the social amoeba Dictyostelium has stimulated its widespread use as an experimental organism for cell and developmental biology. However, the field has been hamstrung by the lack of techniques to recombine disrupted genes.
We describe new techniques for parasexual fusion of strains in liquid medium, selection and maintenance of the resulting stable diploid strains, and segregation to make recombined haploids. We have used these techniques to isolate rasS/gefB double nulls. The phenotypes of these mutants are no more severe than either parent, with movement, phagocytosis and fluid-phase endocytosis affected to the same degree as in rasS or gefB single nulls. In addition, we have produced diploids from one AX2- and one AX3-derived parent, providing an axenic strain with fewer secondary phenotypes than has been previously available.
The phenotype of the rasS/gefB double mutant suggests that the RasS and GefB proteins lie on the same linear pathway. In addition, axenic diploids and the techniques to generate, maintain and segregate them will be productive tools for future work on Dictyostelium. They will particularly facilitate generation of multiple mutants and manipulation of essential genes.
在社会变形虫盘基网柄菌中，靶向基因破坏相对容易，这刺激了其作为细胞和发育生物学实验生物的广泛应用。然而，该领域因缺乏重组被破坏基因的技术而受到阻碍。
我们描述了在液体培养基中进行菌株准性融合、选择和维持所得稳定二倍体菌株以及分离以产生重组单倍体的新技术。我们已使用这些技术分离出rasS/gefB双缺失突变体。这些突变体的表型并不比任何一个亲本更严重，其运动、吞噬作用和液相内吞作用受到的影响程度与rasS或gefB单缺失突变体相同。此外，我们从一个源自AX2和一个源自AX3的亲本产生了二倍体，提供了一种比以前可用的菌株具有更少次要表型的无菌菌株。
rasS/gefB双突变体的表型表明RasS和GefB蛋白位于同一条线性途径上。此外，无菌二倍体以及产生、维持和分离它们的技术将成为未来盘基网柄菌研究的有效工具。它们将特别有助于产生多个突变体以及对必需基因的操作。
