Protein trap: a new Swiss army knife for geneticists?

Svetlana A. Fedorova, Natalya V. Dorogova

Результат исследования: Научные публикации в периодических изданияхобзорная статья

Аннотация

The protein trap is a powerful tool for genetic and biochemical studies of gene function in the animal kingdom. Although the original protein trap was developed for flies, it can be easily adapted to other multicellular organisms, both known models and ones with an unsequenced genome. The protein trap has been successfully applied to the fruit fly, crustaceans Parhyale hawaiensis, zebrafish, and insect and animal cell cultures. This approach is based on the integration into genes of an artificial exon that carries DNA encoding a fluorescent marker, standardized immunoepitopes, an integrase docking site, and splice acceptor and donor sites. The protein trap for cell cultures additionally contains an antibiotic resistance gene, which facilitates the selection of trapped clones. Resulting chimeric tagged mRNAs can be interfered by dsRNA against GFP (iGFPi—in vivo GFP interference), or the chimeric proteins can be efficiently knocked down by deGradFP technology. Both RNA and protein knockdowns produce a strong loss of function phenotype in tagged cells. The fluorescent and protein affinity tags can be used for tagged protein localisation within the cell and for identifying their binding partners in their native complexes. Insertion into protein trap integrase docking sites allows the replacement of trap contents by any new constructs, including other markers, cell toxins, stop-codons, and binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS, that reliably reflect endogenous gene expression. A distinctive feature of the protein trap approach is that all manipulations with a gene or its product occur only in the endogenous locus, which cannot be achieved by any other method.

Язык оригиналаанглийский
Страницы (с-по)1445-1458
Число страниц14
ЖурналMolecular Biology Reports
Том47
Номер выпуска2
Ранняя дата в режиме онлайн14 ноя 2019
DOI
СостояниеОпубликовано - фев 2020

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