BAC TransgeneOmics

By I. Poser, M. Sarov, James R. A. Hutchins, Jean-Karim Hériché, Y. Toyoda, Andrei Pozniakovsky, Daniela Weigl, A. Nitzsche, Björn Hegemann, A. Bird and 17 more. Nature Methods, 2008

631 citations recorded by the source corpus.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.NOTE: In the version of this article initially published online, the name of one individual was misspelled in the Acknowledgments. The second sentence of the Acknowledgments paragraph should read, “We thank I. Cheesman for helpful discussions.” The error has been corrected for all versions of the article.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/245d5f4584f7a9153b3eb699c58c66a4ef17a8a5

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Citations in the corpus

A User's Guide to the Encyclopedia of DNA Elements (ENCODE) (2011, background): “Alternative technologies, such as epitope tagging of TFs in their native genomic context using recom bineering [63,64], are also being explored.”
ENCODE whole-genome data in the UCSC Genome Browser: update 2012 (2011, background): “The scope of DNA-binding site identification has been expanded by the introduction of epitope tagging of proteins (18) where antibodies suitable for chromatin immunoprecipitation are not available.”
A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins (2018, neutral): “BAC constructs and cell lines Stable HeLa cell line expressing C-terminal mCherry tagged human G3BP1 were generated by random integration into the genome via BAC recombineering technology, as previously reported (Poser et al., 2008).”
ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia (2012, background): “of expression nearly physiological (Poser et al. 2008; Hua et al. 2009).”
A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. (2015, positive): “Stable HeLa cell lines expressing human FUS were generated using BAC recombineering technology (Poser et al., 2008).”
Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. (2010, methodology): “GFP Pulldowns Generation of the BACs with GFP-fusion constructs was done as described (Poser et al., 2008).”
Identification of the switch in early-to-late endosome transition. (2010, methodology): “Cell Culture and RNA Interference HeLa cells expressing GFP-tagged Rab5c under the endogenous promoter (Poser et al., 2008) were cultured at 37 C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml…”
Genetic manipulation of genes and cells in the nervous system of the fruit fly (2011, background): “Moreover, recombineering can be extrapolated towards high-throughput efforts to tag hundreds of genes within a small time frame (Poser et al., 2008).”
OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization (2022, background): “In human cells, earlier work has leveraged alternative strategies including expression from bacterial artificial chromosomes ( 16 ) or central-dogma tagging ( 17 ) because of the difficulty of site-specificgeneediting.”