Institute For General Microbiology, Christian-Albrechts-University, Kiel, Germany.
Department of Microbiology, University of Illinois, Urbana, IL, USA.
Methods Mol Biol. 2022;2522:105-117. doi: 10.1007/978-1-0716-2445-6_6.
Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.
遗传操作通过无标记交换实现了几个基因组区域的修饰，而不会在基因组中留下选择标记。这里，描述了一种使用编码次黄嘌呤磷酸核糖基转移酶的 hpt 作为反选择标记的方法。对于 Methanosarcina 物种，首先生成 hpt 基因的染色体缺失，该缺失赋予对嘌呤类似物 8-氮杂-2,6-二氨基嘌呤 (8-ADP) 的抗性。在第二步，hpt 基因在质粒上的重新引入导致同源重组事件 (pop-in) 后可选择失去 8-ADP 抗性。随后的 pop-out 事件恢复了 8-ADP 抗性，并可产生约 50%的染色体突变体频率。
