| Definition |
A protein trap cassette is designed to tag protein(s) encoded by an endogenous locus upon integration of the cassette into the genome. The basic structure of a protein trap cassette is an artificial exon composed of a splice acceptor site, an open reading frame without initiation and stop codons, and a splice donor site. No promoter sequences are present. Upon integration into a coding intron of an endogenous locus, the open reading frame (ORF) encoded by the artificial exon can be incorporated into ('tag') the protein encoded by the locus. This only occurs if the insertion is in the correct orientation and when the frame of the artificial exon corresponds to that of the preceeding exon. Thus, only one out of six insertions in a coding intron will function as a protein trap. To account for each reading frame, three versions of a given protein trap element, differing only in the splice phase, are typically produced for use in an insertional mutagenesis screen. Depending on the nature of the ORF encoded by the artificial exon, the tag may be used to detect the expression pattern and/or subcellular localization of the protein (e.g. if the tag is an epitope tag or fluorescent protein), to drive expression of a gene of interest (e.g. if the tag is a driver that forms part of a binary expression system), or may modify the properties of the endogenous protein (e.g. if the tag affects protein localization or stability). A protein trap cassette may be inserted into a genome as part of a transgenic construct via transposable-element-mediated transgenesis, or may be inserted directly into a modified endogenous locus via a genome engineering method such as homologous recombination or CRISPR. The presence of the splice acceptor and donor sites mean that a protein trap cassette is not inherently mutagenic, since it is designed to insert in frame into a protein. However, an insertion into an intron that splits a critical functional or localization domain may alter the activity of the encoded protein, and the protein trap tag sequence itself may be designed to alter function (for example targeting the protein to a new location within the cell or altering its stability).[ FlyBase:FBrf0216478 FlyBase:FBrf0236840 ] |
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