Insertionsmutagenese

Insertion mutagenesis is a genetic technique used to disrupt, modify, or report on gene function by inserting a DNA sequence into the genome. It is employed to generate mutant libraries, study gene roles, map genetic pathways, and tag genes with reporters or selectable markers. The approach can involve random insertions to survey large portions of the genome or targeted insertions to study a specific locus, depending on the organism and method.

Common methods include transposon mutagenesis, in which mobile DNA elements insert at numerous genomic sites to create loss-of-function mutations and enable screening for phenotypes. Transposon libraries are widely used in bacteria, yeast, and plants. Viral or virus-based insertion mutagenesis uses integrating vectors, such as retroviruses or lentiviruses, to insert reporter or disrupting sequences in mammalian cells, enabling high-throughput functional screens and gene discovery. Targeted insertion methods, including homologous recombination and CRISPR-Cas9–mediated donor template insertion, allow precise integration of reporters, tags, or selectable markers at a predefined locus, though this shifts from random insertion mutagenesis toward targeted genome modification. Gene-trap and promoter-trap vectors combine insertion with a reporter to identify actively transcribed genes and regulatory elements.

Applications of insertion mutagenesis span functional genomics screens to identify genes involved in growth, metabolism, or disease-relevant phenotypes; discovery of essential genes; tagging of genes for expression studies; and generation of mutant libraries in diverse organisms. Limitations include insertion bias, multiple insertions complicating interpretation, potential off-target effects, and position effects on reporter expression. Biosafety and organism-specific regulatory considerations are important in its use.