Efficient Introduction of Genetic Elements at Specific Insertion Loci.

While some genetic modifications are conducted at the native loci of the manipulated genes, "neutral" loci for insertion of (heterologous) genetic elements are equally important for a versatile model system. This, e.g., applies for insertion of genetic complementation constructs or for transplantation of heterologous metabolic pathways. Here, the most frequently used insertion loci of Ustilago maydis is presented, namely the so-called ip locus and three loci encoding secreted proteases like the upp3 locus. The strategy of counter selection provides additional benefits in strain generation as it significantly speeds up the identification of correct mutants. Considerations and strategies for construct design and strain generation based on published standard protocols are provided, followed by a detailed example for upp3 locus insertion combined with counter selection. In essence, these versatile strategies for genetic manipulation have the power to simplify and speed up genetic engineering significantly.