Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs

2012 ◽  
pp. 209-229 ◽  
Author(s):  
Shi-Lung Lin ◽  
Shao-Yao Ying
Keyword(s):  
Gene Silencing

Developmental regulation of heterochromatin-mediated gene silencing in Drosophila

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2223-2234 ◽  
Author(s):  
B.Y. Lu ◽  
J. Ma ◽  
J.C. Eissenberg
Keyword(s):  
Cell Cycle ◽  
Gene Silencing ◽  
Mitotic Activity ◽  
Larval Stage ◽  
Developmental Regulation ◽  
Promoter Strength ◽  
Lacz Gene ◽  
High Degree

The roles of differentiation, mitotic activity and intrinsic promoter strength in the maintenance of heterochromatic silencing were investigated during development using an inducible lacZ gene as an in vivo probe. Heterochromatic silencing is initiated at the onset of gastrulation, approximately 1 hour after heterochromatin is first visible cytologically. A high degree of silencing is maintained in the mitotically active imaginal cells from mid-embryogenesis until early third instar larval stage, and extensive relaxation of silencing is tightly associated with the onset of differentiation. Relaxation of silencing can be triggered in vitro by ecdysone. In contrast, timing and extent of silencing at both the initiation and relaxation stages are insensitive to changes in cell cycle activity, and intrinsic promoter strength also does not influence the extent of silencing by heterochromatin. These data suggest that the silencing activity of heterochromatin is developmentally programmed.

scholarly journals Efficient method of prion gene silencing in vitro and in vivo by antisense-oligodeoxynucleotides conjugated with new dimethylaminoethylmethacrylate oligoelectrolite

2011 ◽  
Vol 5 (3) ◽  
pp. 77-88 ◽  
Author(s):  
L. A. Ivanytska ◽  
◽  
V. V. Stadnyk ◽  
Yu. V. Martyn ◽  
M. R. Kozak ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Efficient Method ◽  
Antisense Oligodeoxynucleotides ◽  
Prion Gene

scholarly journals Modulations of SIR-nucleosome interactions of reconstructed yeast silent pre-heterochromatin by O-acetyl-ADP-ribose and magnesium

2017 ◽  
Vol 28 (3) ◽  
pp. 381-386 ◽  
Author(s):  
Shu-Yun Tung ◽  
Sue-Hong Wang ◽  
Sue-Ping Lee ◽  
Shu-Ping Tsai ◽  
Hsiao-Hsuian Shen ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Chromatin Condensation ◽  
Epigenetic Inheritance ◽  
Assembly System ◽  
Sir Proteins ◽  
Epigenetic Gene Silencing ◽  
In Vitro Assembly ◽  
The Individual

Yeast silent heterochromatin provides an excellent model with which to study epigenetic inheritance. Previously we developed an in vitro assembly system to demonstrate the formation of filament structures with requirements that mirror yeast epigenetic gene silencing in vivo. However, the properties of these filaments were not investigated in detail. Here we show that the assembly system requires Sir2, Sir3, Sir4, nucleosomes, and O-acetyl-ADP-ribose. We also demonstrate that all Sir proteins and nucleosomes are components of these filaments to prove that they are SIR-nucleosome filaments. Furthermore, we show that the individual localization patterns of Sir proteins on the SIR-nucleosome filament reflect those patterns on telomeres in vivo. In addition, we reveal that magnesium exists in the SIR-nucleosome filament, with a role similar to that for chromatin condensation. These results suggest that a small number of proteins and molecules are sufficient to mediate the formation of a minimal yeast silent pre-heterochromatin in vitro.

scholarly journals KAP-1 Corepressor Protein Interacts and Colocalizes with Heterochromatic and Euchromatic HP1 Proteins: a Potential Role for Krüppel-Associated Box–Zinc Finger Proteins in Heterochromatin-Mediated Gene Silencing

1999 ◽  
Vol 19 (6) ◽  
pp. 4366-4378 ◽  
Author(s):  
Robert F. Ryan ◽  
David C. Schultz ◽  
Kasirajan Ayyanathan ◽  
Prim B. Singh ◽  
Josh R. Friedman ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Zinc Finger Proteins ◽  
Centromeric Heterochromatin ◽  
Interphase Nuclei ◽  
Epigenetic Gene Silencing ◽  
Associated Proteins

ABSTRACT Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1α and HP1γ) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1α, which are normally found in centromeric heterochromatin, as well as M32 and hHP1γ, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP–KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

In Vitro and In Vivo Gene Silencing by TransKingdom RNAi (tkRNAi)

2008 ◽  
pp. 1-14 ◽  
Author(s):  
Shuanglin Xiang ◽  
Andrew C. Keates ◽  
Johannes Fruehauf ◽  
Youxin Yang ◽  
Hongnian Guo ◽  
...  
Keyword(s):  
Gene Silencing

scholarly journals Switching the intracellular pathway and enhancing the therapeutic efficacy of small interfering RNA by auroliposome

2020 ◽  
Vol 6 (30) ◽  
pp. eaba5379 ◽  
Author(s):  
Md. Nazir Hossen ◽  
Lin Wang ◽  
Harisha R. Chinthalapally ◽  
Joe D. Robertson ◽  
Kar-Ming Fung ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Gold Nanoparticles ◽  
Gene Silencing ◽  
Small Interfering Rna ◽  
Sirna Delivery ◽  
Delivery Systems ◽  
Ovarian Cancer Cells ◽  
Interfering Rna

Gene silencing using small-interfering RNA (siRNA) is a viable therapeutic approach; however, the lack of effective delivery systems limits its clinical translation. Herein, we doped conventional siRNA-liposomal formulations with gold nanoparticles to create “auroliposomes,” which significantly enhanced gene silencing. We targeted MICU1, a novel glycolytic switch in ovarian cancer, and delivered MICU1-siRNA using three delivery systems—commercial transfection agents, conventional liposomes, and auroliposomes. Low-dose siRNA via transfection or conventional liposomes was ineffective for MICU1 silencing; however, in auroliposomes, the same dose gave >85% gene silencing. Efficacy was evident from both in vitro growth assays of ovarian cancer cells and in vivo tumor growth in human ovarian cell line—and patient-derived xenograft models. Incorporation of gold nanoparticles shifted intracellular uptake pathways such that liposomes avoided degradation within lysosomes. Auroliposomes were nontoxic to vital organs. Therefore, auroliposomes represent a novel siRNA delivery system with superior efficacy for multiple therapeutic applications.

scholarly journals RNA interference-mediated gene silencing in murine T cells: in vitro and in vivo validation of proinflammatory target genes

2008 ◽  
Vol 6 (1) ◽  
pp. 3 ◽  
Author(s):  
Tatjana C Gust ◽  
Luisa Neubrandt ◽  
Claudia Merz ◽  
Khusru Asadullah ◽  
Ulrich Zügel ◽  
...  
Keyword(s):  
T Cells ◽  
Rna Interference ◽  
Gene Silencing ◽  
Target Genes ◽  
In Vivo Validation

Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA–DNA heteroduplex substrates

2010 ◽  
Vol 432 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Jason P. Ross ◽  
Isao Suetake ◽  
Shoji Tajima ◽  
Peter L. Molloy
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Double Helix ◽  
In Vitro Assays ◽  
Enzyme Analysis ◽  
Transcriptional Gene Silencing ◽  
Restriction Enzyme Analysis

The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA–DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.

Effect of silencing thymosin beta 4 gene expression on stemness and invasiveness in glioblastoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 2081-2081
Author(s):  
Ghazaleh Tabatabai ◽  
Shanmugarajan Krishnan ◽  
Ana-Maria Florea ◽  
Karl Frei ◽  
Kathy Hasenbach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Cell Line ◽  
Glioma Cells ◽  
Glioma Stem Cell ◽  
Stem Cell Line ◽  
Self Renewal ◽  
Lentiviral Transduction

2081 Background: Thymosin β4 (TB4) is a pleiotropic actin-sequestering polypeptide that is involved in wound healing and developmental processes. TB4 gene silencing promotes differentiation of neural progenitor cells whereas TB4 overexpression initiates cortical folding of developing brain hemispheres. However, a role of TB4 in malignant gliomas has not yet been investigated. Methods: We first analyzed TB4 expression on tissue microarrays and performed REMBRANDT and TCGA database interrogations. We analyzed TB4 expression in a panel of 8 long-term glioma cell lines and 7 glioma-initiating cell lines. Using lentiviral transduction, we modulated TB4 expression in LNT-229, U87MG and the glioma-initiating cell line GS-2. We studied clonogenic survival, migration, invasion, self-renewal, differentiation capacity of TB4-depleted or TB4-overexpressing glioma cells in vitro and tumorigenicity upon orthotopic implantation in vivo. Finally, we performed an Affymetrix gene chip analysis to unravel the molecular network of TB4 signaling effects. Results: TB4 expression increased with the grade of malignancy in gliomas and correlated with patient survival. In vitro, TB4 gene silencing by lentiviral transduction decreased migration, invasion, growth and self-renewal, and promoted differentiation and the susceptibility to undergo apoptotic cell death upon nutrient depletion in LNT-229, U87MG and the glioma stem-cell line GS2, respectively. In vivo, survival of nude mice bearing tumors derived from TB4-depleted glioma cells was improved and the tumorigenicity of the GS2 glioma stem-cell line was decreased. The gene expression pattern was shifted from the mesenchymal towards the pro-neural gene signature upon TB4 gene silencing. The clustering of differentially regulated genes involved TGF-β and p53 signaling networks. Conclusions: TB4 may be a key regulator of malignancy in glioblastoma and therefore a novel candidate molecular target for anti-glioma therapies.

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