scholarly journals Nanoluciferase-based Method for Detecting Gene Expression inC. elegans

2019 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
C Elegans ◽  
Highly Sensitive ◽  
Monitoring Gene Expression ◽  
Green Fluorescent ◽  
Limited Sensitivity ◽  
Inducible Gene

AbstractGenetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, GFP and other fluorophores have limited sensitivity, particularly in tissues that autofluoresce like the intestine of the nematodeCaenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in multi-well format to detect constitutive and inducible gene expression inC. elegans. We optimize detection of bioluminescent signal from NanoLuc inC. elegansand show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by thevha-6promoter. We can reliably detect signal in singlevha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from 1/100 dilution of lysate from a singlevha-6p::Nanoluc-expressing adult and from a singlevha-6p::Nanoluc-expressing adult “hidden” in a pool of 5,000 N2 wild-type animals. We also optimized various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof of concept, we used NanoLuc to monitor promoter activity of thepals-5stress/immune reporter and we were able to measure 300 and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring gene expression inC. elegans.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

scholarly journals Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

2017 ◽  
Vol 199 (6) ◽  
Author(s):  
Erik R. Van Vlack ◽  
Shana Topp ◽  
Jessica C. Seeliger
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Fluorescent Protein ◽  
Lactobacillus Rhamnosus ◽  
Content Type ◽  
Naturally Occurring ◽  
Binding Event ◽  
Steady State Model ◽  
Green Fluorescent ◽  
Inducible Gene

ABSTRACT We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis. Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches. IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.

scholarly journals Dual Labeling with Green Fluorescent Proteins for Confocal Microscopy

2000 ◽  
Vol 66 (1) ◽  
pp. 413-418 ◽  
Author(s):  
Stacie E. Cowan ◽  
Eric Gilbert ◽  
Artem Khlebnikov ◽  
J. D. Keasling
Keyword(s):  
Gene Expression ◽  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Species ◽  
Green Fluorescent Proteins ◽  
Monitoring Gene Expression ◽  
Green Fluorescent ◽  
Multispecies Biofilms

ABSTRACT We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.

scholarly journals CMYB1Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Min Duan ◽  
Peng Huang ◽  
Xi Yuan ◽  
Hui Chen ◽  
Ji Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Transcriptional Activator ◽  
Myb Transcription Factor ◽  
Specific Gene ◽  
Protein Fusion ◽  
Rhythmic Expression ◽  
Green Fluorescent

Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designatedCold induced MYB 1 (CMYB1). Tissue-specific gene expression analysis revealed thatCMYB1was highly expressed in rice stems and nodes. qRT-PCR assay indicated thatCMYB1was dramatically induced by cold stress (>100-folds) and induced by exogenous ABA and osmotic stress. Interestingly,CMYB1showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested thatCMYB1-GFP (green fluorescent protein) fusion protein was localized in the nuclei. Moreover,CMYB1exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together,CMYB1probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

scholarly journals Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0242640
Author(s):  
Jianying Zhang ◽  
Daibang Nie ◽  
Kelly Williamson ◽  
Arthur McDowell ◽  
MaCalus V. Hogan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Treadmill Running ◽  
Whole Body ◽  
Tendon Cells ◽  
Elevated Expression ◽  
Green Fluorescent

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

scholarly journals RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shakil Ahmad ◽  
Momana Jamil ◽  
Muhammad Fahim ◽  
Shujing Zhang ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Growth Factors ◽  
Imaginal Disc ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Small Body ◽  
Functional Characterization ◽  
Oral Feeding ◽  
Developmental Defects ◽  
Melon Fly ◽  
Green Fluorescent

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval–larval lethality, pupal–adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

scholarly journals Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

1999 ◽  
Vol 10 (2) ◽  
pp. 345-360 ◽  
Author(s):  
Dawn Signor ◽  
Karen P. Wedaman ◽  
Lesilee S. Rose ◽  
Jonathan M. Scholey
Keyword(s):  
Signal Transduction ◽  
Fluorescent Protein ◽  
Partial Purification ◽  
Localization Pattern ◽  
Chemosensory Neurons ◽  
C Elegans ◽  
Microtubule Motors ◽  
Sensory Cilia ◽  
Green Fluorescent ◽  
Kinesin Family

Chemosensation in the nervous system of the nematodeCaenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

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