scholarly journals Generation of viral vectors specific to neuronal subtypes of targeted brain regions by Enhancer-Driven Gene Expression (EDGE)

2019 ◽  
Author(s):  
Rajeevkumar Raveendran Nair ◽  
Stefan Blankvoort ◽  
Maria Jose Lagartos ◽  
Cliff Kentros
Keyword(s):  
Gene Expression ◽  
Brain Function ◽  
Viral Vectors ◽  
Neural Circuits ◽  
Tissue Sample ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Brain Regions ◽  
Transgenic Lines ◽  
The Right

SummaryUnderstanding brain function requires understanding neural circuits at the level of specificity at which they operate. While recent years have seen the development of a variety of remarkable molecular tools for the study of neural circuits, their utility is currently limited by the inability to deploy them in specific elements of native neural circuits, i.e. particular neuronal subtypes. One can obtain a degree of specificity with neuron-specific promoters, but native promoters are almost never sufficiently specific restricting this approach to transgenic animals. We recently showed that one can obtain transgenic mice with augmented anatomical specificity in targeted brain regions by identifyingcis-regulatory elements (i.e. enhancers) uniquely active in those brain regions and combining them with a heterologous promoter, an approach we call EDGE (Enhancer-Driven Gene Expression). Here we extend this strategy to the generation of viral (rAAV) vectors, showing that when combined with the right minimal promoter they largely recapitulate the specificity seen in the corresponding transgenic lines in wildtype animals, even of another species. Because active enhancers can be identified in any tissue sample, this approach promises to enable the kind of circuit-specific manipulations in any species. This should not only greatly enhance our understanding of brain function, but may one day even provide novel therapeutic avenues to correct the imbalances in neural circuits underlying many disorders of the brain.

scholarly journals The Association between DRD3 Ser9Gly Polymorphism and Depression Severity in Parkinson’s Disease

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yan Zhi ◽  
Yongsheng Yuan ◽  
Qianqian Si ◽  
Min Wang ◽  
Yuting Shen ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Resting State ◽  
Brain Function ◽  
Clinical Manifestations ◽  
Brain Regions ◽  
Potential Effect ◽  
Analysis Of Covariance ◽  
Significant Difference ◽  
The Right

More and more evidence suggests that dopamine receptor D3 gene (DRD3) plays an important role in the clinical manifestations and the treatment of Parkinson’s disease (PD). DRD3 Ser9Gly polymorphism is the most frequently studied variant point. Our aim was to investigate the potential effect of DRD3 Ser9Gly polymorphism on modulating resting-state brain function and associative clinical manifestations in PD patients. We consecutively recruited 61 idiopathic PD patients and 47 healthy controls (HC) who were evaluated by clinical scales, genotyped for variant Ser9Gly in DRD3, and underwent resting-state functional magnetic resonance imaging. Based on DRD3 Ser9Gly polymorphism, PD patients and HCs were divided into four subgroups. Then, two-way analysis of covariance (ANCOVA) was applied to investigate main effects and interactions of PD and DRD3 Ser9Gly polymorphism on the brain function via amplitude of low-frequency fluctuations (ALFF) approach. The association between DRD3 Ser9Gly-modulated significantly different brain regions, and clinical manifestations were detected by Spearman’s correlations. PD patients exhibited decreased ALFF values in the right inferior occipital gyrus, lingual gyrus, and fusiform gyrus. A significant difference in the interaction of “groups × genotypes” was observed in the right medial frontal gyrus. The ALFF value of the cluster showing significant interactions was positively correlated with HAMD-17 scores (r=0.489, p=0.011) and anhedonia scores (r=0.512, p=0.008) in PD patients with the Ser/Gly or Gly/Gly genotypes. Therefore, D3 gene Ser9Gly polymorphism might be associated with the severity of depression characterized by anhedonia in PD patients.

Characterization of the mononuclear phagocyte system in zebrafish

Blood ◽  
2011 ◽  
Vol 117 (26) ◽  
pp. 7126-7135 ◽  
Author(s):  
Valerie Wittamer ◽  
Julien Y. Bertrand ◽  
Patrick W. Gutschow ◽  
David Traver
Keyword(s):  
Fluorescent Protein ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Mononuclear Phagocyte ◽  
Mononuclear Phagocytes ◽  
Reporter Line ◽  
Transgenic Lines ◽  
Green Fluorescent ◽  
And Function ◽  
Antigen Presenting

Abstract The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFPhi; cd45:DsRedhi cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

scholarly journals Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration

2021 ◽  
Author(s):  
Yingxi Cao ◽  
Yu Xia ◽  
Joseph B Balowski ◽  
jianhong ou ◽  
Lingyun Song ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Injury ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Heart Regeneration ◽  
Epicardial Cells ◽  
Transgenic Lines ◽  
Functional Features ◽  
Multiple Trees ◽  
Regulatory Controls

The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.

scholarly journals Fear Extinction Recall Modulates Human Frontomedial Theta and Amygdala Activity

2018 ◽  
Vol 29 (2) ◽  
pp. 701-715 ◽  
Author(s):  
Matthias F J Sperl ◽  
Christian Panitz ◽  
Isabelle M Rosso ◽  
Daniel G Dillon ◽  
Poornima Kumar ◽  
...  
Keyword(s):  
Animal Studies ◽  
Neural Circuits ◽  
Conditioned Fear ◽  
Brain Regions ◽  
Amygdala Activity ◽  
The Right ◽  
First Time ◽  
Insight Into ◽  
Analogous Effect ◽  
Theta Range

Abstract Human functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) studies, as well as animal studies, indicate that the amygdala and frontomedial brain regions are critically involved in conditioned fear and that frontomedial oscillations in the theta range (4–8 Hz) may support communication between these brain regions. However, few studies have used a multimodal approach to probe interactions among these key regions in humans. Here, our goal was to bridge the gap between prior human fMRI, EEG, and animal findings. Using simultaneous EEG–fMRI recordings 24 h after fear conditioning and extinction, conditioned stimuli presented (CS+E, CS−E) and not presented during extinction (CS+N, CS−N) were compared to identify effects specific to extinction versus fear recall. Differential (CS+ vs. CS−) electrodermal, frontomedial theta (EEG) and amygdala responses (fMRI) were reduced for extinguished versus nonextinguished stimuli. Importantly, effects on theta power covaried with effects on amygdala activation. Fear and extinction recall as indicated by theta explained 60% of the variance for the analogous effect in the right amygdala. Our findings show for the first time the interplay of amygdala and frontomedial theta activity during fear and extinction recall in humans and provide insight into neural circuits consistently linked with top-down amygdala modulation in rodents.

scholarly journals A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

2021 ◽  
Vol 25 (3) ◽  
pp. 331-336
Author(s):  
A. V. Smirnov ◽  
T. A. Shnaider ◽  
A. N. Korablev ◽  
A. M. Yunusova ◽  
I. A. Serova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Knockout ◽  
Milk Proteins ◽  
Transgenic Animals ◽  
Regulatory Elements ◽  
Milk Composition ◽  
Peptide Sequence ◽  
Start Codon ◽  
Signal Peptide Sequence ◽  
Casein Genes

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

scholarly journals Analysis of Brain Functional Connectivity Neural Circuits in Children With Autism Based on Persistent Homology

2021 ◽  
Vol 15 ◽  
Author(s):  
Di Liang ◽  
Shengxiang Xia ◽  
Xianfu Zhang ◽  
Weiwei Zhang
Keyword(s):  
Functional Connectivity ◽  
Neural Circuits ◽  
Persistent Homology ◽  
Brain Networks ◽  
Brain Regions ◽  
Autism Spectrum ◽  
Unknown Etiology ◽  
Control Group ◽  
Threshold Values ◽  
The Right

Autism spectrum disorder (ASD) is a complex neuropsychiatric disorder with a complex and unknown etiology. Statistics demonstrate that the number of people diagnosed with ASD is increasing in countries around the world. Currently, although many neuroimaging studies indicate that ASD is characterized by abnormal functional connectivity (FC) patterns within brain networks rather than local functional or structural abnormalities, the FC characteristics of ASD are still poorly understood. In this study, a Vietoris-Rips (VR) complex filtration model of the brain functional network was established by using resting-state functional magnetic resonance imaging (fMRI) data of children aged 6–13 years old [including 54 ASD patients and 52 typical development (TD) controls] from the Autism Brain Imaging Data Exchange (ABIDE) public database. VR complex filtration barcodes are calculated by using persistent homology to describe the changes in the FC neural circuits of brain networks. The number of FC neural circuits with different length ranges at different threshold values is calculated by using the barcodes, the different brain regions participating in FC neural circuits are discussed, and the connectivity characteristics of brain FC neural circuits in the two groups are compared and analyzed. Our results show that the number of FC neural circuits with lengths of 8–12 is significantly decreased in the ASD group compared with the TD control group at threshold values of 0.7, 0.8 and 0.9, and there is no significant difference in the number of FC neural circuits with lengths of 4–7 and 13–16 and lengths 16. When the thresholds are 0.7, 0.8, and 0.9, the number of FC neural circuits in some brain regions, such as the right orbital part of the superior frontal gyrus, the left supplementary motor area, the left hippocampus, and the right caudate nucleus, involved in the study is significantly decreased in the ASD group compared with the TD control group. The results of this study indicate that there are significant differences in the FC neural circuits of brain networks in the ASD group compared with the TD control group.

scholarly journals Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 273 ◽  
Author(s):  
Marian Seda ◽  
Emma Peskett ◽  
Charalambos Demetriou ◽  
Dale Bryant ◽  
Gudrun E. Moore ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Transgenic Animals ◽  
Product Inhibition ◽  
Transgenic Zebrafish ◽  
Incomplete Penetrance ◽  
Missense Mutations ◽  
Wild Type ◽  
Protein Levels ◽  
Transgenic Lines

Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.

scholarly journals Endogenous zebrafish proneural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

2020 ◽  
Author(s):  
Maira P. Almeida ◽  
Jordan M. Welker ◽  
Stephen C. Ekker ◽  
Karl J. Clark ◽  
Jeffrey J. Essner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Cost Effective ◽  
Regulatory Elements ◽  
Cre Recombinase ◽  
Model Systems ◽  
Control Of Gene Expression ◽  
Endogenous Gene ◽  
Native Promoter ◽  
Transgenic Lines

AbstractThe Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous gene expression patterns are needed. Here, we apply CRISPR/Cas9 targeting to integrate a 2A-Cre recombinase transgene with 48bp homology arms into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

scholarly journals The marked diversity of unique cortical enhancers enables neuron-specific tools by Enhancer-Driven Gene Expression

2018 ◽  
Author(s):  
Stefan Blankvoort ◽  
Menno P. Witter ◽  
James Noonan ◽  
Justin Cotney ◽  
Cliff Kentros
Keyword(s):  
Gene Expression ◽  
Neural Circuit ◽  
Neuronal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
List Type ◽  
Enhancer Activity ◽  
Genetic Tools ◽  
Transgenic Lines ◽  
Genetic Mechanisms

SUMMARYUnderstanding neural circuit function requires individually addressing their component parts: specific neuronal cell types. However, not only do the precise genetic mechanisms specifying neuronal cell types remain obscure, access to these neuronal cell types by transgenic techniques also remains elusive. While most genes are expressed in the brain, the vast majority are expressed in many different kinds of neurons, suggesting that promoters alone are not sufficiently specific to distinguish cell types. However, there are orders of magnitude more distal genetic cis-regulatory elements controlling transcription (i.e. enhancers), so we screened for enhancer activity in microdissected samples of mouse cortical subregions. This identified thousands of novel putative enhancers, many unique to particular cortical subregions. Pronuclear injection of expression constructs containing such region-specific enhancers resulted in transgenic lines driving expression in distinct sets of cells specifically in the targeted cortical subregions, even though the parent gene’s promoter was relatively nonspecific. These data showcase the promise of utilizing the genetic mechanisms underlying the specification of diverse neuronal cell types for the development of genetic tools potentially capable of targeting any neuronal circuit of interest, an approach we call Enhancer-Driven Gene Expression (EDGE).HighlightsEnhancer ChIP-seq of cortical subregions reveals 59372 putative enhancers.3740 of these are specific to particular cortical subregions.This reflects the remarkable anatomical diversity of the adult cortex.Unique enhancers provide a means to make targeted cell-type specific genetic tools.

Sign in / Sign up

Export Citation Format

Share Document