Betapudi Venkaiah, Shoebotham Karen, Egelhoff Thomas T
Case Western Reserve University, Cleveland, OH, USA.
Biotechniques. 2004 Jan;36(1):106-12. doi: 10.2144/04361RR01.
Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.
基因打靶是一种强大的分子遗传学技术，已被广泛用于在体内了解特定基因的功能。该技术通过引入的DNA片段与同源靶基因之间的重组来消除内源基因。然而，当需要进行多个基因敲除时，仅有限数量的标记基因的可用性就成为一个难题。在这里，我们描述了一种在盘基网柄菌中进行双基因敲除的新方法，即同时转染两个基因打靶盒，然后仅针对一个标记基因进行克隆选择。随后对杀稻瘟菌素抗性克隆进行基于PCR的筛选，结果显示所选和未选的打靶盒均整合到其各自原来的位点，从而产生完全的基因敲除。对于我们在这些研究中测试的基因（肌球蛋白重链激酶B和C），在转染步骤后，双基因打靶事件的效率在所有杀稻瘟菌素抗性菌落的2%-5%范围内。这种同时破坏多个基因的方法对于其他有兴趣在盘基网柄菌或其他可选择标记数量有限的生物体中创建多个基因破坏体的实验室来说，应该是一种有价值的工具。
