Split-QF System for Fine-Tuned Transgene Expression in Drosophila.
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PROVIDER: S-EPMC6499530 | BioStudies |
REPOSITORIES: biostudies
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Project description:We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This "Q system" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional "logic gates" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis.
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Project description:Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.
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Project description:We established a transcription-based binary gene expression system in Caenorhabditis elegans using the recently developed Q system. This system, derived from genes in Neurospora crassa, uses the transcriptional activator QF to induce the expression of target genes. Activation can be efficiently suppressed by the transcriptional repressor QS, and suppression can be relieved by the nontoxic small molecule quinic acid. We used QF, QS and quinic acid to achieve temporal and spatial control of transgene expression in various tissues in C. elegans. We also developed a split Q system, in which we separated QF into two parts encoding its DNA-binding and transcription-activation domains. Each domain showed negligible transcriptional activity when expressed alone, but expression of both reconstituted QF activity, providing additional combinatorial power to control gene expression.
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Project description:Visualization and manipulation of defined motoneurons have provided significant insights into how motor circuits are assembled in <i>Drosophila</i>. A conventional approach for molecular and cellular analyses of subsets of motoneurons involves the expression of a wide range of <i>UAS</i> transgenes using available <i>GAL4</i> drivers (eg, <i>eve</i> promoter-fused <i>GAL4</i>). However, a more powerful toolkit could be one that enables a single-cell characterization of interactions between neurites from neurons of interest. Here we show the development of a <i>UAS > LexA > QF</i> expression system to generate randomly selected neurons expressing one of the 2 binary expression systems. As a demonstration, we apply it to visualize dendrite-dendrite interactions by genetically labeling <i>eve</i> <sup>+</sup> neurons with distinct fluorescent reporters.
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Project description:Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.
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Project description:In Drosophila, the GAL4/UAS/GAL80 repressible binary expression system is widely used to manipulate or mark tissues of interest. However, complex biological systems often require distinct transgenic manipulations of different cell populations. For this purpose, we recently developed the Q system, a second repressible binary expression system. We describe here the basic steps for performing a variety of Q system experiments in vivo. These include how to generate and use Q system reagents to express effector transgenes in tissues of interest, how to use the Q system in conjunction with the GAL4 system to generate intersectional expression patterns that precisely limit which tissues will be experimentally manipulated and how to use the Q system to perform mosaic analysis. The protocol described here can be adapted to a wide range of experimental designs.
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Project description:Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid and reliable method for screening aneuploidies, but in Brazil, it is not used in public services. We investigated the accuracy of QF-PCR for the prenatal recognition of common aneuploidies and compared these results with cytogenetic results in our laboratory.A ChromoQuant QF-PCR kit containing 24 primer pairs targeting loci on chromosomes 21, 13, 18, X and Y was employed to identify aneuploidies of the referred chromosomes.A total of 162 amniotic fluid samples analyzed using multiplex QF-PCR were compared with karyotyping analysis. The QF-PCR results were consistent with the results of cytogenetic analysis in 95.4% of all samples.QF-PCR was demonstrated to be efficient and reliable for prenatal aneuploidy screening. This study suggests that QF-PCR can be used as a rapid diagnostic method. However, rearrangements and some mosaic samples cannot be detected with this test; thus, those exceptions must undergo cytogenetic analysis.
2017-01-01 | S-EPMC5525164 | BioStudies
Project description:The ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2800 transgenic lines for use with the split-GAL4 intersectional method.
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Project description:Quantitative fluorescence-polymerase chain reaction (QF-PCR) is useful for the detection of aneuploidies involving chromosomes 13, 18, 21, X and Y. Due to the rapid turn-around time and reduced cost compared to traditional karyotyping, QF-PCR has been used as an alternative test for both pre- and postnatal aneuploidy detection in Johannesburg, South Africa since 2001. An internal review of 13,396 aneuploidy tests processed using QF-PCR between January 2015 and December 2019 was performed, and the results showed that the majority (~ 88%) of cases were postnatal tests, with prenatal samples accounting for only ~ 12% of cases. The most common aneuploidies detected were Trisomy 21 (20.6%), Trisomy 18 (3.7%) and Trisomy 13 (2.4%), while sex chromosome aneuploidies were only detected in < 1% of cases. The average percentage of positive cases over the 5-year period was 32.1% for postnatal samples and 11.3% for prenatal samples. QF-PCR testing of the common aneuploidies is being used appropriately, and the high percentage of positive cases demonstrates the value of QF-PCR as prenatal and postnatal tests, particularly in limited resource settings. The higher proportion of positive postnatal cases suggests that referrals are clinically appropriate. However, there is under- and uneven utilization of genetic services in many provinces in South Africa, and the state of prenatal genetic services is poor, as reflected by the low number of prenatal referrals. These results demonstrate the need for programs which will improve the genetic knowledge of referring doctors and the general public, thereby improving the broader utilisation of QF-PCR aneuploidy diagnostic testing, so that patients receive appropriate diagnoses and subsequent management.
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Project description:BACKGROUND:Our aim was to conduct a comprehensive genetic evaluation using the combination of QF-PCR (quantitative fluorescence polymerase chain reaction) and aCGH (array comparative genomic hybridization) for the detection of the frequency and type of chromosome aberrations in recurrent miscarriage (RM) in the clinical setting. METHODS:This retrospective study was conducted on 73 first-trimester products of conception (POC) between September 2014 and February 2017. The POCs were collected from 73 women with at least one previous miscarriage and analyzed for chromosomal anomalies using QF-PCR and aCGH as part of the routine clinical evaluation. RESULTS:Chromosome aberrations were detected in 52/73 POCs (71.2%), of which 41 (56.2%) were identified by QF-PCR and an additional 11 (15.1%) by aCGH. Numerical aberrations constituted 92.3% of abnormalities, with trisomies as the most common subtype (72.9%). Causative structural aberrations were found in three samples (5.8%). The frequency of chromosome aberrations was not dependent on the number of previous miscarriages, whereas it significantly increased with advanced maternal age. CONCLUSION:Our results confirm that chromosome aberrations are the most common cause of RM and that QF-PCR and aCGH combination should be included in the routine genetic analysis of POCs of couples with miscarriage.
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