Laboratory of Food Microbiology, Department of Microbial and Molecular Systems (M(2)S), Faculty of Bioscience Engineering, KU Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium. Electronic address:
Published: August 2014
AI Article Synopsis
Article Abstract
Flp mediated site specific recombination of frt-sites is frequently used in genetic engineering to excise, insert or invert DNA-cassettes in the chromosome. While constructs flanked by frt-sites are generally considered to be stable in the absence of the Flp enzyme, we observed that P22 chromosomes exceeding wild-type length tend to lose frt-flanked insertions via Flp independent recombination of frt-sites during phage propagation. This spontaneous recombination should be considered when engineering the chromosome of P22 and perhaps of other phages as well.
In this protocol, we introduce a sparse driver system for cell-type specific single-cell labeling and manipulation in , enabling complete and simultaneous expression of multiple transgenes in the same cells. The system precisely controls expression probability and sparsity via mutant sites with reduced recombination efficiency and tunable FLP levels adjusted by heat-shock durations. We demonstrate that this generalizable toolkit enables tunable sparsity, multi-color staining, single-cell trans-synaptic tracing, single-cell manipulation, and analysis of cell-autonomous gene function.
We describe a simple recombineering-based procedure for generating single-copy gene fusions to superfolder GFP (sfGFP) and monomeric Cherry (mCherry). The open reading frame (orf) for either protein is inserted at the targeted chromosomal location by λ Red recombination using an adjacent drug-resistance cassette ( or ) for selection. The drug-resistance gene is flanked by flippase (Flp) recognition target (FRT) sites in direct orientation, which allows removal of the cassette by Flp-mediated site-specific recombination once the construct is obtained, if desired.
Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou, China.
species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help.
Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNA, and locus-specific ssDNA oligonucleotides.
We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials. A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter.
