Double-inducible gene activation system for caspase 3 and 9 in epidermis

CRISPR activation system is part of the function of CRSPR/Cas9 which is used to manipulate certain genes by activating these genes. CRISPR activation utilizes dCas9 (dead-Cas9) or Cas9 which is deactivated as DNA scissors, so that the use of Cas9 for gene activation does not cause targeted DNA chain termination. This research that uses this CRISPR activation is applied to the zebrafish gene (Danio rerio) aims to determine the mRNA level of the targeted gene. In this study, the ASCL1a and BCL6a genes from zebrafish were targeted as the object of study. The results showed that genes from zebrafish that were targeted had an increase in mRNA levels after being activated using CRISPR activation system. Keywords: CRISPR activation system (CRISPRa), zebrafish (Danio rerio), dCas9, ASCL1a gene, BCL6a gene

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Mitogen-Inducible Gene 6 Triggers Apoptosis and Exacerbates ER Stress-Induced β-Cell Death

Molecular Endocrinology ◽

10.1210/me.2012-1174 ◽

2013 ◽

Vol 27 (1) ◽

pp. 162-171 ◽

Cited By ~ 15

Author(s):

Yi-Chun Chen ◽

E. Scott Colvin ◽

Bernhard F. Maier ◽

Raghavendra G. Mirmira ◽

Patrick T. Fueger

Keyword(s):

Cell Death ◽

Er Stress ◽

Caspase 3 ◽

Cell Mass ◽

Growth Factor Receptor ◽

Protein Translation ◽

Β Cells ◽

Β Cell ◽

Inducible Gene

The increased insulin secretory burden placed on pancreatic β-cells during obesity and insulin resistance can ultimately lead to β-cell dysfunction and death and the development of type 2 diabetes. Mitogen-inducible gene 6 (Mig6) is a cellular stress-responsive protein that can negatively regulate the duration and intensity of epidermal growth factor receptor signaling and has been classically viewed as a molecular brake for proliferation. In this study, we used Mig6 heterozygous knockout mice (Mig6+/−) to study the role of Mig6 in regulating β-cell proliferation and survival. Surprisingly, the proliferation rate of Mig6+/− pancreatic islets was lower than wild-type islets despite having comparable β-cell mass and glucose tolerance. We thus speculated that Mig6 regulates cellular death. Using adenoviral vectors to overexpress or knockdown Mig6, we found that caspase 3 activation during apoptosis was dependent on the level of Mig6. Interestingly, Mig6 expression was induced during endoplasmic reticulum (ER) stress, and its protein levels were maintained throughout ER stress. Using polyribosomal profiling, we identified that Mig6 protein translation was maintained, whereas the global protein translation was inhibited during ER stress. In addition, Mig6 overexpression exacerbated ER stress-induced caspase 3 activation in vitro. In conclusion, Mig6 is transcriptionally up-regulated and resistant to global translational inhibition during stressed conditions in β-cells and mediates apoptosis in the form of caspase 3 activation. The sustained production of Mig6 protein exacerbates ER stress-induced β-cell death. Thus, preventing the induction, translation, and/or function of Mig6 is warranted for increasing β-cell survival.

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Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters

10.1101/2021.05.17.444476 ◽

2021 ◽

Author(s):

Samantha Carrera ◽

Amanda O'Donnell ◽

Yaoyong Li ◽

Karol Nowicki-Osuch ◽

Syed Murtuza Baker ◽

...

Keyword(s):

Histone Acetylation ◽

Gene Activation ◽

Transcriptional Response ◽

Activation Parameters ◽

Regulatory Elements ◽

Lysine Acetylation ◽

Efficient Gene ◽

Inducible Genes ◽

Target Gene Induction ◽

Inducible Gene

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. However, several studies have shown that histone acetylation dynamics rather than hyperacetylation per se are important determinants in gene activation, particularly at inducible genes. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous DUSP1 activation kinetics and that acetylation levels influence allele activation frequencies. Our data therefore point to a complex interplay between acetylation dynamics and target gene induction, which cannot simply be explained by a unified response to acetylation activity. Instead, acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

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Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

Plant Cell Reports ◽

10.1007/s00299-021-02789-z ◽

2021 ◽

Author(s):

Zhifen Zhang ◽

Yinping Guo ◽

Kathleen Monfero Marasigan ◽

Joann A. Conner ◽

Peggy Ozias-Akins

Keyword(s):

Developmental Stage ◽

Transgene Expression ◽

Gene Activation ◽

Early Embryo ◽

Complete Excision ◽

Globular Stage ◽

Activation System ◽

Transgenic Lines ◽

Embryonic Developmental Stage ◽

Spatiotemporal Control

Abstract Key message Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Abstract Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

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Use of the CRISPR/Cas9-based epigenetic gene activation system In Vivo : A new potential therapeutic modality

Hepatology ◽

10.1002/hep.29860 ◽

2018 ◽

Vol 68 (3) ◽

pp. 1191-1193 ◽

Cited By ~ 1

Author(s):

Samar H. Ibrahim ◽

Keith D. Robertson

Keyword(s):

Gene Activation ◽

Therapeutic Modality ◽

Activation System

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Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase

Gene ◽

10.1016/s0378-1119(96)00516-1 ◽

1996 ◽

Vol 181 (1-2) ◽

pp. 207-212 ◽

Cited By ~ 86

Author(s):

Yumi Kanegae ◽

Koichi Takamori ◽

Yumi Sato ◽

Gwang Lee ◽

Michio Nakai ◽

...

Keyword(s):

Mammalian Cell ◽

Recombinant Adenovirus ◽

Gene Activation ◽

Cre Recombinase ◽

Efficient Gene ◽

Activation System

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Essential Role of STAT3 in the Control of the Acute-Phase Response as Revealed by Inducible Gene Activation in the Liver

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1621-1632.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1621-1632 ◽

Cited By ~ 229

Author(s):

Tonino Alonzi ◽

Diego Maritano ◽

Barbara Gorgoni ◽

Gabriella Rizzuto ◽

Claude Libert ◽

...

Keyword(s):

Acute Phase ◽

Gene Activation ◽

Type I ◽

Group Iii ◽

Group I ◽

Conditional Mutant ◽

Bone Marrow Analysis ◽

Good Agreement ◽

Inducible Gene

ABSTRACT We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them with Mx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon. Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly less efficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response to bacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. The different degrees of defectiveness displayed by the various genes allowed us to differentiate them into three separate groups according to their degree of dependence on STAT3. Induction was totally defective for group I genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defective for group III genes. This division was in good agreement with the known structures of the respective promoters. We also found that the overall induction of the transcription factors C/EBPβ and -δ was only minimally defective in the absence of STAT3. Finally, even though corticosterone levels and action were found to be normal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokines was increased and prolonged, probably as a result of STAT3 deletion in macrophages.

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