Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti
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PROVIDER: S-EPMC10557486 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1), even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.
| S-EPMC3443236 | biostudies-literature
Project description:Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.
| S-EPMC5339718 | biostudies-literature
Project description:We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick beta-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in. Cre activity is detectable in the developing pituitary after initiation of Gh transcription and persists through adulthood with high penetrance in Gh expressing cells and lower penetrance in lactotropes, a cell type that shares a common origin with somatotropes. This Gh-cre transgenic line is suitable for efficient, cell-specific deletion of floxed regions of genomic DNA in differentiated somatotropes and a subset of lactotrope cells of the anterior pituitary gland.
| S-EPMC2696317 | biostudies-literature
Project description:The Streptomyces phage phiC31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5' ends. Rotation of one pair of half sites by 180 degrees relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by phiC31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.
| S-EPMC419440 | biostudies-literature
Project description:BackgroundThe Asian tiger mosquito, Aedes albopictus (Skuse), is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately.Methodology/principal findingsGermline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2-3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2-6%.Conclusions/significanceBoth piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.
| S-EPMC2923142 | biostudies-literature
Project description:RNA-Seq was used for transcriptome sequencing of 0-12 hour embryos.
2011-12-16 | GSE34480 | GEO
Project description:Caenorhabditis elegans benefits from a large set of tools for genome manipulation. Yet, the precise single-copy insertion of very large DNA constructs (>10 kb) and the generation of inversions are still challenging. Here, we adapted the phiC31 integrase system for C. elegans. We generated an integrated phiC31 integrase expressing strain flanked by attP sites that serves as a landing pad for integration of transgenes by recombination-mediated cassette exchange (RCME). This strain is unc-119(-) so RMCE integrants can be produced simply by injection of a plasmid carrying attB sites flanking unc-119(+) and the gene(s) of interest. Additionally, phiC31 integrase is removed concomitantly with integration, eliminating the need to outcross away the integrase. Integrations were obtained for insert sizes up to ∼33.4 kb. Taking advantage of this integration method we establish a dual-color fluorescent operon reporter system able to study post-transcriptional regulation of mRNA. Last, we show that large chromosomal segments can be inverted using phiC31 integrase. Thus, the phiC31 integrase system should be a useful addition to the C. elegans toolkit.
| S-EPMC9208643 | biostudies-literature
Project description:The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.
| S-EPMC3424569 | biostudies-literature
Project description:The genus totivirus in the family Totiviridae contains double-stranded RNA viruses. Their genome has two open reading frames (ORFs) that encode capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). The toti-like viruses recently identified in Anopheles sp. and Aedes aegypti mosquitoes (AaTV) share the same genome organization as other totiviruses. The AaTVs that have been described in distinct geographical regions are monophyletic. In this study, we show that AaTV sequences can be grouped into at least three phylogenetic clades (named A, B, and C). Clades A and B are composed of AaTV sequences from mosquitoes collected in the Caribbean region (Guadeloupe), and clade C contains sequences from the USA. These clades may represent AaTV lineages that are locally adapted to their host populations. We also identified three recombinant AaTV strains circulating in mosquitoes in Guadeloupe. Although these strains have different chimeric patterns, the position of the recombination breakpoint was identical in all strains. Interestingly, this breakpoint is located in a hairpin-like structure in the intergenic region of the AaTV genome. This RNA structure may stall RNA polymerase processivity and consequently induce template switching. In vitro studies should be conducted to further investigate the biological significance of AaTV's intergenic region as a recombination hotspot.
| S-EPMC9699231 | biostudies-literature
Project description:The Cre-LoxP system has been commonly used for cell-specific genetic manipulation. However, many Cre strains exhibit excision activity in unexpected cell types or tissues. Therefore, it is important to identify the cell types in which recombination takes place. Fibroblasts are a cell type that is inadequately defined due to a lack of specific markers to detect the entire cell lineage. Here, we investigated the Cre recombination induced by Col1α2-iCre, one of the most common fibroblast-mesenchymal Cre driver lines, by using a double-fluorescent Cre reporter line in which GFP is expressed when recombination occurs. Our results indicated that Col1α2-iCre activity was more extensive across cell types than previously reported: Col1α2-iCre-mediated recombination was found in not only cells of mesenchymal origin but also those of other lineages, including haematopoietic cells, myocardial cells, lung and intestinal epithelial cells, and neural cells. In addition, study of embryos revealed that recombination by Col1α2-iCre was observed in the early developmental stage before gastrulation in epiblasts, which would account for the recombination across various cell types in adult mice. These results offer more insights into the activity of Col1α2-iCre and suggest that experimental results obtained using Col1α2-iCre should be carefully interpreted.
| S-EPMC10728165 | biostudies-literature