Abstract
Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.
Highlights
Functional genomics has benefited greatly from the ability to introduce tag sequences into desired chromosomal loci by homologous recombination, thereby labeling gene products (RNA or protein) and facilitating high-throughput analyses with standardized assays
We designed a strategy for chromosomal gene tagging that allows generating clones in which only the desired tag sequence is inserted into a specified genomic locus
The module is amplified by polymerase chain reaction (PCR) using primers with short overhangs homologous to the genomic locus of interest
Summary
Functional genomics has benefited greatly from the ability to introduce tag sequences into desired chromosomal loci by homologous recombination, thereby labeling gene products (RNA or protein) and facilitating high-throughput analyses with standardized assays. This strategy is common in yeast and gradually within reach in other model organisms [1]. Seamless tagging can be achieved with the two-step delitto perfetto approach [12] (Figure S1C in Supporting Information S1) or using spontaneous marker excision by homologous recombination [13] (Figure S1D in Supporting Information S1) These methods are incompatible with high-throughput genome manipulation required for systematic studies. As biological research goes quantitative, a simple and efficient method enabling minimally-invasive gene tagging is increasingly required
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
