The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements or enhancers. Here, we present a rapid and highly efficient system for the large-scale analysis of enhancer elements, site-specifically integrated into the Drosophila genome. This system, which is scalable for either small projects or high-throughput approaches, makes use of the Gateway cloning technology and the PhiC31 site-specific integration system, which allows the insertion of constructs at predetermined genomic locations. Thus, this system allows not only a fast and easy analysis of reporter gene expression in live animals, but also the simultaneous analysis of different regulatory outputs on a cellular resolution by recombining in the same animal distinct enhancer elements fused to different fluorescent proteins.
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http://dx.doi.org/10.1002/dvg.20637 | DOI Listing |
Stem Cells
January 2025
Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom.
To enable robust expression of transgenes in stem cells, recombinase-mediated cassette exchange at safe harbor loci is frequently adopted. The choice of recombinase enzyme is a critical parameter to ensure maximum efficiency and accuracy of the integration event. We have explored the serine recombinase family of site-specific integrases and have directly compared the efficiency of PhiC31, W-beta, and Bxb1 integrase for targeted transgene integration at the Gt(ROSA)26Sor locus in mouse embryonic stem cells.
View Article and Find Full Text PDFJ Agric Food Chem
January 2025
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States.
, an environmental bacterium, holds promise as a biocontrol agent due to its ability to produce bioactive compounds effective against plant pathogens, such as fungi, oomycetes, and Gram-positive bacteria. However, it lacks activity against Gram-negative bacteria. To address this, we applied new genetic tools to manipulate the phenazine biosynthetic gene cluster () from , converting to a robust producer of phenazine antibiotics.
View Article and Find Full Text PDFAppl Environ Microbiol
December 2024
MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
Over the past three decades, the integrase (Int) from phage C31 has become a valuable genome engineering tool across various species. C31 Int was thought to mediate unidirectional site-specific integration ( × to and ) in the absence of the phage-encoded recombination directionality factor (RDF). However, we have shown in this study that Int can also catalyze reverse excision ( × to and ) at low frequencies in and , causing genetic instability in engineered strains.
View Article and Find Full Text PDFSci Rep
November 2024
School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, James Parsons Building, Byrom Street, L3 3AF, Liverpool, UK.
Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL.
View Article and Find Full Text PDFG3 (Bethesda)
October 2024
Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS UMR5239, Université Claude Bernard Lyon 1, 9 rue du Vercors, 69007 Lyon, France.
The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G.
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