scholarly journals Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Development ◽  
2021 ◽  
Author(s):  
Daniel S. Levic ◽  
Naoya Yamaguchi ◽  
Siyao Wang ◽  
Holger Knaut ◽  
Michel Bagnat
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Quantitative Imaging ◽  
Expression Patterns ◽  
Imaging Studies ◽  
Coding Regions ◽  
Endogenous Expression ◽  
Epithelial Biology

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC, and the ECM receptor Integrin β1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

scholarly journals Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

2021 ◽  
Author(s):  
Daniel S Levic ◽  
Naoya Yamaguchi ◽  
Siyao Wang ◽  
Holger Knaut ◽  
Michel Bagnat
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Quantitative Imaging ◽  
Expression Patterns ◽  
Imaging Studies ◽  
Coding Regions ◽  
Fluorescent Markers ◽  
Endogenous Expression ◽  
Epithelial Biology

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor β1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

scholarly journals Spatiotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain

2015 ◽  
Vol 112 (22) ◽  
pp. 6855-6862 ◽  
Author(s):  
Loyal A. Goff ◽  
Abigail F. Groff ◽  
Martin Sauvageau ◽  
Zachary Trayes-Gibson ◽  
Diana B. Sanchez-Gomez ◽  
...  
Keyword(s):  
Neural Development ◽  
Expression Patterns ◽  
Noncoding Rnas ◽  
Brain Regions ◽  
Long Noncoding Rnas ◽  
Adult Brain ◽  
Mammalian Brain ◽  
Endogenous Expression ◽  
Spatiotemporal Expression

Long noncoding RNAs (lncRNAs) have been implicated in numerous cellular processes including brain development. However, the in vivo expression dynamics and molecular pathways regulated by these loci are not well understood. Here, we leveraged a cohort of 13 lncRNA-null mutant mouse models to investigate the spatiotemporal expression of lncRNAs in the developing and adult brain and the transcriptome alterations resulting from the loss of these lncRNA loci. We show that several lncRNAs are differentially expressed both in time and space, with some presenting highly restricted expression in only selected brain regions. We further demonstrate altered regulation of genes for a large variety of cellular pathways and processes upon deletion of the lncRNA loci. Finally, we found that 4 of the 13 lncRNAs significantly affect the expression of several neighboring protein-coding genes in a cis-like manner. By providing insight into the endogenous expression patterns and the transcriptional perturbations caused by deletion of the lncRNA locus in the developing and postnatal mammalian brain, these data provide a resource to facilitate future examination of the specific functional relevance of these genes in neural development, brain function, and disease.

scholarly journals mRhubarb: Engineering of monomeric, red-shifted, and brighter variants of iRFP using structure-guided multi-site mutagenesis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Oliver C. Rogers ◽  
Dorothy M. Johnson ◽  
Elad Firnberg
Keyword(s):  
Near Infrared ◽  
Fluorescent Proteins ◽  
Visible Spectrum ◽  
Tissue Imaging ◽  
Imaging Studies ◽  
Near Ir ◽  
Site Mutagenesis ◽  
Light Absorbance ◽  
Reduced Light

Abstract Far-red and near-infrared fluorescent proteins (FPs) enable in vivo tissue imaging with greater depth and clarity compared to FPs in the visible spectrum due to reduced light absorbance and scatter by tissues. However current tools are limited by low brightness, limited red-shifting, and a non-ideal dimeric oligomerization state. In this study we developed a monomeric variant of iRFP, termed mRhubarb713, and subsequently used a targeted and expansive multi-site mutagenesis approach to screen for variants with red-shifted spectral activity. Two monomeric variants were discovered, deemed mRhubarb719 and mRhubarb720, with red-shifted spectra and increased quantum yield compared to iRFP. These tools build on previously developed near-IR FPs and should enable improved in vivo imaging studies with a genetically encoded reporter.

scholarly journals Dysfunction in nonsense-mediated decay, protein homeostasis, mitochondrial function, and brain connectivity in ALS-FUS mice with cognitive deficits

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Wan Yun Ho ◽  
Ira Agrawal ◽  
Sheue-Houy Tyan ◽  
Emma Sanford ◽  
Wei-Tang Chang ◽  
...  
Keyword(s):  
Cognitive Deficits ◽  
Brain Connectivity ◽  
Expression Patterns ◽  
Long Term Potentiation ◽  
Protein Homeostasis ◽  
Spine Density ◽  
Nonsense Mediated Decay ◽  
Endogenous Expression ◽  
Mitochondrial Functions

AbstractAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of the same disease spectrum of adult-onset neurodegenerative diseases that affect the motor and cognitive functions, respectively. Multiple common genetic loci such as fused in sarcoma (FUS) have been identified to play a role in ALS and FTD etiology. Current studies indicate that FUS mutations incur gain-of-toxic functions to drive ALS pathogenesis. However, how the disease-linked mutations of FUS affect cognition remains elusive. Using a mouse model expressing an ALS-linked human FUS mutation (R514G-FUS) that mimics endogenous expression patterns, we found that FUS proteins showed an age-dependent accumulation of FUS proteins despite the downregulation of mouse FUS mRNA by the R514G-FUS protein during aging. Furthermore, these mice developed cognitive deficits accompanied by a reduction in spine density and long-term potentiation (LTP) within the hippocampus. At the physiological expression level, mutant FUS is distributed in the nucleus and cytosol without apparent FUS aggregates or nuclear envelope defects. Unbiased transcriptomic analysis revealed a deregulation of genes that cluster in pathways involved in nonsense-mediated decay, protein homeostasis, and mitochondrial functions. Furthermore, the use of in vivo functional imaging demonstrated widespread reduction in cortical volumes but enhanced functional connectivity between hippocampus, basal ganglia and neocortex in R514G-FUS mice. Hence, our findings suggest that disease-linked mutation in FUS may lead to changes in proteostasis and mitochondrial dysfunction that in turn affect brain structure and connectivity resulting in cognitive deficits.

scholarly journals Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR

Biomolecules ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1276
Author(s):  
Salma Saeed Khan ◽  
Yi Shen ◽  
Muhammad Qaiser Fatmi ◽  
Robert E. Campbell ◽  
Habib Bokhari
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Fluorescent Proteins ◽  
Transcriptional Factor ◽  
Thiol Groups ◽  
Protein Protein Interactions ◽  
Protein Thiol ◽  
New Family

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein–protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As3+ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As3+ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As3+ in various biological and environmental systems.

scholarly journals Charcot-Marie-Tooth 2B mutations in rab7 cause dosage-dependent neurodegeneration due to partial loss of function

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Smita Cherry ◽  
Eugene Jennifer Jin ◽  
Mehmet Neset Özel ◽  
Zhiyuan Lu ◽  
Egemen Agi ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Protein Function ◽  
Quantitative Imaging ◽  
Small Gtpase ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Mutant Proteins ◽  
Charcot Marie Tooth ◽  
Function Mechanism

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10–50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function.

scholarly journals In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Li He ◽  
Richard Binari ◽  
Jiuhong Huang ◽  
Julia Falo-Sanjuan ◽  
Norbert Perrimon
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Patterns ◽  
Cell Type ◽  
Dynamic Information ◽  
New Genes ◽  
Dual Color ◽  
Dynamic Expression ◽  
Spatio Temporal

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

scholarly journals Spaciotemporal Association and Bone Morphogenetic Protein Regulation of Sclerostin and Osterix Expression during Embryonic Osteogenesis

Endocrinology ◽  
2004 ◽  
Vol 145 (10) ◽  
pp. 4685-4692 ◽  
Author(s):  
Yoshio Ohyama ◽  
Akira Nifuji ◽  
Yukiko Maeda ◽  
Teruo Amagasa ◽  
Masaki Noda
Keyword(s):  
Bone Morphogenetic Protein ◽  
Bone Growth ◽  
Expression Patterns ◽  
Cranial Bone ◽  
Nodule Formation ◽  
Endogenous Expression ◽  
Sost Gene ◽  
Morphogenetic Protein

Abstract Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.

Studying the Cell Biology of Apicomplexan Parasites Using Fluorescent Proteins

2004 ◽  
Vol 10 (5) ◽  
pp. 568-579 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Boris Striepen
Keyword(s):  
Protein Trafficking ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Biological Processes ◽  
Organelle Biogenesis ◽  
Apicomplexan Parasites ◽  
Experimental Protocols ◽  
Green Fluorescent

The ability to transfect Apicomplexan parasites has revolutionized the study of this important group of pathogens. The function of specific genes can be explored by disruption of the locus or more subtly by introduction of altered or tagged versions. Using the transgenic reporter gene green fluorescent protein (GFP), cell biological processes can now be studied in living parasites and in real time. We review recent advances made using GFP-based experiments in the understanding of protein trafficking, organelle biogenesis, and cell division inToxoplasma gondiiandPlasmodium falciparum. A technical section provides a collection of basic experimental protocols for fluorescent protein expression inT. gondii. The combination of thein vivomarker GFP with an increasingly diverse genetic toolbox forT. gondiiopens many exciting experimental opportunities, and emerging applications of GFP in genetic and pharmacological screens are discussed.

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