scholarly journals A robust and flexible CRISPR/Cas9 based system for neutrophil-specific gene inactivation in zebrafish

2021 ◽  
pp. jcs.258574
Author(s):  
Yueyang Wang ◽  
Alan Y. Hsu ◽  
Eric M. Walton ◽  
Sung Jun Park ◽  
Ramizah Syahirah ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Cell Motility ◽  
Transgenic Fish ◽  
Gene Inactivation ◽  
Specific Gene ◽  
Tissue Specific ◽  
Gateway System ◽  
Gene Functions ◽  
Specific Manner ◽  
Cell Front

CRISPR/Cas9-based tissue-specific knockout techniques are essential in probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live-imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of sgRNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 gene restored neutrophil motility in the corresponding knockout background. Moreover, active Rac and force bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.

scholarly journals A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

2020 ◽  
Author(s):  
Yueyang Wang ◽  
Alan Y. Hsu ◽  
Eric M. Walton ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Cell Motility ◽  
Neutrophil Migration ◽  
Transgenic Fish ◽  
Subcellular Location ◽  
Vector System ◽  
Specific Gene ◽  
Tissue Specific ◽  
Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

scholarly journals Tissue-specific gene expression and protein abundance patterns are associated with fractionation bias in maize

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Jesse R. Walsh ◽  
Margaret R. Woodhouse ◽  
Carson M. Andorf ◽  
Taner Z. Sen
Keyword(s):  
Gene Expression ◽  
Agronomic Traits ◽  
Functional Divergence ◽  
Duplication Event ◽  
Regulatory Control ◽  
Specific Gene ◽  
Protein Abundance ◽  
Genome Duplication Event ◽  
Tissue Specific ◽  
Gene Functions

Abstract Background Maize experienced a whole-genome duplication event approximately 5 to 12 million years ago. Because this event occurred after speciation from sorghum, the pre-duplication subgenomes can be partially reconstructed by mapping syntenic regions to the sorghum chromosomes. During evolution, maize has had uneven gene loss between each ancient subgenome. Fractionation and divergence between these genomes continue today, constantly changing genetic make-up and phenotypes and influencing agronomic traits. Results Here we regenerate the subgenome reconstructions for the most recent maize reference genome assembly. Based on both expression and abundance data for homeologous gene pairs across multiple tissues, we observed functional divergence of genes across subgenomes. Although the genes in the larger maize subgenome are often expressing more highly than their homeologs in the smaller subgenome, we observed cases where homeolog expression dominance switches in different tissues. We demonstrate for the first time that protein abundances are higher in the larger subgenome, but they also show tissue-specific dominance, a pattern similar to RNA expression dominance. We also find that pollen expression is uniquely decoupled from protein abundance. Conclusion Our study shows that the larger subgenome has a greater range of functional assignments and that there is a relative lack of overlap between the subgenomes in terms of gene functions than would be suggested by similar patterns of gene expression and protein abundance. Our study also revealed that some reactions are catalyzed uniquely by the larger and smaller subgenomes. The tissue-specific, nonequivalent expression-level dominance pattern observed here implies a change in regulatory control which favors differentiated selective pressure on the retained duplicates leading to eventual change in gene functions.

scholarly journals Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9

Genetics ◽  
2017 ◽  
Vol 208 (2) ◽  
pp. 673-686 ◽  
Author(s):  
Bridget D. DeLay ◽  
Mark E. Corkins ◽  
Hannah L. Hanania ◽  
Matthew Salanga ◽  
Jian Min Deng ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Gene Inactivation ◽  
Specific Gene ◽  
Tissue Specific ◽  
Tissue Specific Gene

Hypertension Increases Connexin43 in a Tissue-Specific Manner

Circulation ◽  
1997 ◽  
Vol 95 (4) ◽  
pp. 1007-1014 ◽  
Author(s):  
Jacques-Antoine Haefliger ◽  
Einar Castillo ◽  
Ge´rard Waeber ◽  
Gabriela E. Bergonzelli ◽  
Jean-Franc¸ois Aubert ◽  
...  
Keyword(s):  
Tissue Specific ◽  
Specific Manner

scholarly journals A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

2021 ◽  
Author(s):  
Isabel Regadas ◽  
Olle Dahlberg ◽  
Roshan Vaid ◽  
Oanh Ho ◽  
Sergey Belikov ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Specific Gene ◽  
Chromatin Signature ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Histone 3

scholarly journals Tentacle Morphological Variation Coincides with Differential Expression of Toxins in Sea Anemones

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 452
Author(s):  
Lauren M. Ashwood ◽  
Michela L. Mitchell ◽  
Bruno Madio ◽  
David A. Hurwood ◽  
Glenn F. King ◽  
...  
Keyword(s):  
Differential Expression ◽  
Morphological Variation ◽  
Expression Profiles ◽  
The Body ◽  
Sea Anemones ◽  
Tissue Specific ◽  
Venom Composition ◽  
Specific Manner ◽  
Branched Structures ◽  
Morphological Variants

Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails.

scholarly journals Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the −183 to −137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 146
Author(s):  
Takahiro Nakayama ◽  
Toshiyuki Fukutomi ◽  
Yasuo Terao ◽  
Kimio Akagawa
Keyword(s):  
Transcription Factor ◽  
Gene Silencing ◽  
Protein Complex ◽  
Promoter Region ◽  
Neuronal Cell ◽  
Syntaxin 1A ◽  
Tissue Specific ◽  
Cell Tissue ◽  
Specific Manner ◽  
A Cell

The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC.

scholarly journals Retinoid nutritional status differently affects the expression of Japanese quail retinoic acid receptor-beta isoform transcripts in a tissue-specific manner

2001 ◽  
Vol 169 (2) ◽  
pp. 281-290 ◽  
Author(s):  
ZW Fu ◽  
T Kubo ◽  
K Sugahara ◽  
T Noguchi ◽  
H Kato
Keyword(s):  
Retinoic Acid ◽  
Nutritional Status ◽  
Japanese Quail ◽  
Retinoic Acid Receptor ◽  
Mrna Levels ◽  
Tissue Specific ◽  
Specific Manner ◽  
Lower Magnitude ◽  
Lung And Kidney ◽  
Receptor Beta

We investigated the effects of vitamin A (VA) nutritional status on the levels of expression of retinoic acid (RA) receptor-beta (RARbeta) gene in the various tissues of Japanese quail. VA deficiency caused a significant decrease in the mRNA levels of brain, liver, heart, lung and kidney RARbeta2/beta4, whereas no change was observed in the level of testis RARbeta2 transcript. In contrast, reduction in the RARbeta1 transcript caused by VA depletion was observed only in the lung, remaining unchanged in the other tissues. The administration of RA to the VA-deficient quail rapidly induced the expression of RARbeta2/beta4 mRNAs in all the tissues examined, but RA increased the expression of RARbeta1 transcript in the liver, heart, lung and kidney at a lower magnitude. RA could not change the expression of the brain RARbeta1 transcript, while it induced the expression of the testis RARbeta1 mRNA in a temporal way. These results clearly indicate that VA nutritional status differently regulates the expression of RARbeta1 and RARbeta2/beta4 transcripts in a tissue-specific manner.

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