scholarly journals Quantification of durable CRISPR-based gene silencing activity

2021 ◽  
Author(s):  
Muneaki Nakamura ◽  
Alexis Ivec ◽  
Yuchen Gao ◽  
Lei S Qi
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Gene Repression ◽  
Biological Behavior ◽  
Biological Research ◽  
Stable Gene ◽  
Reporter System ◽  
Engineering Applications ◽  
Domain Composition ◽  
Silence Gene

Development of CRISPR-based technologies for regulating gene expression stands to provide novel methods for the study and engineering of biological behavior. New tools capable of inducing long-lasting changes in gene expression will increase the utility of these techniques, providing durable effects from one-time doses of reagents. We describe here a reporter system for quantifying the ability of CRISPR-based effectors to induce stable gene repression. We observe a continuous gradation of the ability of these effectors to silence gene expression, depending on the domain composition and configuration. We also report the creation of a single CRISPR protein capable of producing durable gene silencing. This assay should allow for the continued development of enhanced gene repression tools which will be useful in a wide array of biological research and engineering applications.

scholarly journals Durable CRISPR-Based Epigenetic Silencing

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Muneaki Nakamura ◽  
Alexis E. Ivec ◽  
Yuchen Gao ◽  
Lei S. Qi
Keyword(s):  
Epigenetic Silencing ◽  
Gene Repression ◽  
Biological Behavior ◽  
Biological Research ◽  
Stable Gene ◽  
Epigenetic Changes ◽  
Reporter System ◽  
Epigenome Editing ◽  
Single Protein ◽  
Epigenetic Editing

Development of CRISPR-based epigenome editing tools is important for the study and engineering of biological behavior. Here, we describe the design of a reporter system for quantifying the ability of CRISPR epigenome editors to produce a stable gene repression. We characterize the dynamics of durable gene silencing and reactivation, as well as the induced epigenetic changes of this system. We report the creation of single-protein CRISPR constructs bearing combinations of three epigenetic editing domains, termed KAL, that can stably repress the gene expression. This system should allow for the development of novel epigenome editing tools which will be useful in a wide array of biological research and engineering applications.

scholarly journals Polyornithine-based polyplexes to boost effective gene silencing in CNS disorders

Nanoscale ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 6285-6299 ◽  
Author(s):  
I. Conejos-Sánchez ◽  
E. Gallon ◽  
A. Niño-Pariente ◽  
J. A. Smith ◽  
A. G. De la Fuente ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Viral Vectors ◽  
Neural Cells ◽  
Cns Disorders ◽  
Silence Gene

Novel biodegradable and biocompatible polyornithine derivatives as non-viral vectors for siRNA exhibit effectively silence gene expression in primary neural cells.

The Protein Methyltransferase Prmt5 Links Histone H4 Methylation to Dnmt3a-Dependent DNA Methylation and Transcriptional Silencing of the Fetal Globin Genes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 361-361
Author(s):  
Stephen Jane ◽  
Quan Zhao ◽  
Gerhard Rank ◽  
Loretta Cerruti ◽  
David J. Curtis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Gene Silencing ◽  
Globin Gene ◽  
K562 Cells ◽  
Gene Repression ◽  
Histone H4 ◽  
Cpg Dinucleotides ◽  
Repressor Complex ◽  
Chip Analysis

Abstract Elevated levels of fetal hemoglobin ameliorate the severity of sickle cell disease and β-thalassemia, fuelling interest in the mechanisms underpinning the fetal (γ) to adult (β) switch in β-like globin chain subtype. We have previously identified a tripartite protein complex consisting of p22 NF-E4, CP2 and ALY, collectively known as the stage selector protein (SSP) that binds to the proximal γ-promoters, and fosters the preferential expression of the γ-genes in fetal erythroid cells. We have also identified a 14 kDa isoform of the NF-E4 protein that plays a role in γ-gene repression by binding CP2 and sequestering it away from the γ-promoter, resulting in disassembly of the activator SSP complex. Despite the loss of SSP binding, we showed by chromatin immunoprecipitation (ChIP) analysis that p22 NF-E4 remained bound to the γ-promoter in this context. To determine whether p22 NF-E4 could serve as the cornerstone for assembly of a larger repressor complex in this setting, we analyzed the proteins that were co-immunoprecipitated with p22 NF-E4 from K562 cell extract by mass spectrometry. One protein identified was PRMT5, an arginine methyltransferase that has been linked to gene silencing by establishing repressive arginine methyl marks including symmetrical dimethylation of arginine 3 on histone H4 (H4R3me2s). We confirmed the interaction between the two endogenous proteins by direct co-immunoprecipitation, and co-localized p22 NF-E4 and PRMT5 to the γ-globin gene promoters by ChIP. In vitro methylation studies using PRMT5 co-immunoprecipitated with p22 NF-E4 confirmed that histone H4 was the major substrate of the enzyme complex in K562 cells. In accord with this, we demonstrated a marked increase in H4R3me2s at the γ-promoter by ChIP in the setting of enforced expression of wild type PRMT5, accompanied by silencing of γ-gene expression. To determine whether additional factors cooperated with PRMT5 in γ-gene repression, we interrogated PRMT5 containing immunoprecipitates with antisera to a range of candidate proteins. We isolated a large repressor complex containing members of the NuRD complex and the methyl domain-binding proteins (MBD2 and MDB3). We also isolated the DNA methyltransferase 3a (Dnmt3a), a finding of considerable interest in view of the links between γ-gene silencing and methylation of CpG dinucleotides. Using bisulfite DNA sequencing, we demonstrated in K562 cells in which PRMT5 expression had been enforced, an increase in the density of methylated CpG dinucleotides clustered around the transcriptional start site. In contrast, cells transfected with an expression vector stably expressing hairpin short interfering RNAs, which induced a 90% reduction in PRMT5 protein levels, showed complete abrogation of DNA methylation at these CpGs, coincident with a five-fold induction of γ-gene expression. ChIP analysis of the human β-globin locus in βYAC transgenic mice revealed a marked enhancement of H4R3me2s at the γ-promoters in adult erythropoietic cells, and absence of this repressive mark at the γ-promoter in the E12.5 fetal liver. This data establishes a direct link between the PRMT5-induced repressive histone mark H4R3me2s and DNA methylation in developmental regulation of γ-gene expression. It also provides impetus for new strategies aimed at reactivation of fetal globin gene expression.

scholarly journals Identification of new reference genes with stable expression patterns for gene expression studies using human cancer and normal cell lines

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gergely Attila Rácz ◽  
Nikolett Nagy ◽  
József Tóvári ◽  
Ágota Apáti ◽  
Beáta G. Vértessy
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Reference Genes ◽  
Normal Cell ◽  
Human Cancer ◽  
Expression Patterns ◽  
Biological Research ◽  
Serum Starvation ◽  
Stable Gene ◽  
Human Cancer Cell Lines

AbstractReverse transcription—quantitative real-time PCR (RT-qPCR) is a ubiquitously used method in biological research, however, finding appropriate reference genes for normalization is challenging. We aimed to identify genes characterized with low expression variability among human cancer and normal cell lines. For this purpose, we investigated the expression of 12 candidate reference genes in 13 widely used human cancer cell lines (HeLa, MCF-7, A-549, K-562, HL-60(TB), HT-29, MDA-MB-231, HCT 116, U-937, SH-SY5Y, U-251MG, MOLT-4 and RPMI-8226) and, in addition, 7 normal cell lines (HEK293, MRC-5, HUVEC/TERT2, HMEC, HFF-1, HUES 9, XCL-1). In our set of genes, we included SNW1 and CNOT4 as novel candidate reference genes based on the RNA HPA cell line gene data from The Human Protein Atlas. HNRNPL and PCBP1 were also included along with the „classical” reference genes ACTB, GAPDH, IPO8, PPIA, PUM1, RPL30, TBP and UBC. Results were evaluated using GeNorm, NormFiner, BestKeeper and the Comparative ΔCt methods. In conclusion, we propose IPO8, PUM1, HNRNPL, SNW1 and CNOT4 as stable reference genes for comparing gene expression between different cell lines. CNOT4 was also the most stable gene upon serum starvation.

Establishing Standard Protocols for Bacterial Culture in Biological Research in Canisters (BRIC) Hardware

2020 ◽  
Vol 4 (2) ◽  
pp. 58-69 ◽  
Author(s):  
Patricia Fajardo-Cavazos ◽  
Wayne L. Nicholson
Keyword(s):  
Gene Expression ◽  
Bacterial Culture ◽  
Culture Conditions ◽  
Media Culture ◽  
Ground Control ◽  
Biological Research ◽  
Confounding Factors ◽  
Cross Experiment ◽  
Spaceflight Experiments ◽  
Control Samples

AbstractThe NASA GeneLab Data System (GLDS) was recently developed to facilitate cross-experiment comparisons in order to understand the response of microorganisms to the human spaceflight environment. However, prior spaceflight experiments have been conducted using a wide variety of different hardware, media, culture conditions, and procedures. Such confounding factors could potentially mask true differences in gene expression between spaceflight and ground control samples. In an attempt to mitigate such confounding factors, we describe here the development of a standardized set of hardware, media, and protocols for liquid cultivation of microbes in Biological Research in Canisters (BRIC) spaceflight hardware, using the model bacteria Bacillus subtilis strain 168 and Staphylococcus aureus strain UAMS-1 as examples.

scholarly journals Cruciate Ligament Cell Sheets Can Be Rapidly Produced on Thermoresponsive poly(glycidyl ether) Coating and Successfully Used for Colonization of Embroidered Scaffolds

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 877
Author(s):  
Ingrid Zahn ◽  
Daniel David Stöbener ◽  
Marie Weinhart ◽  
Clemens Gögele ◽  
Annette Breier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Cruciate Ligament ◽  
Glycidyl Ether ◽  
Stable Gene ◽  
Type I ◽  
Cell Sheets ◽  
Thermoresponsive Polymers ◽  
Related Phenotype ◽  
Anterior Cruciate

Anterior cruciate ligament (ACL) cell sheets combined with biomechanically competent scaffolds might facilitate ACL tissue engineering. Since thermoresponsive polymers allow a rapid enzyme-free detachment of cell sheets, we evaluated the applicability of a thermoresponsive poly(glycidyl ether) (PGE) coating for cruciate ligamentocyte sheet formation and its influence on ligamentocyte phenotype during sheet-mediated colonization of embroidered scaffolds. Ligamentocytes were seeded on surfaces either coated with PGE or without coating. Detached ligamentocyte sheets were cultured separately or wrapped around an embroidered scaffold made of polylactide acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads functionalized by gas-phase fluorination and with collagen foam. Ligamentocyte viability, protein and gene expression were determined in sheets detached from surfaces with or without PGE coating, scaffolds seeded with sheets from PGE-coated plates and the respective monolayers. Stable and vital ligamentocyte sheets could be produced within 24 h with both surfaces, but more rapidly with PGE coating. PGE did not affect ligamentocyte phenotype. Scaffolds could be colonized with sheets associated with high cell survival, stable gene expression of ligament-related type I collagen, decorin, tenascin C and Mohawk after 14 d and extracellular matrix (ECM) deposition. PGE coating facilitates ligamentocyte sheet formation, and sheets colonizing the scaffolds displayed a ligament-related phenotype.

scholarly journals Recent Advances in the Development of Exogenous dsRNA for the Induction of RNA Interference in Cancer Therapy

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 701
Author(s):  
Tatiana S. Golubeva ◽  
Viktoria A. Cherenko ◽  
Konstantin E. Orishchenko
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Molecular Biology ◽  
Gene Silencing ◽  
Regulation Of Gene Expression ◽  
Research Area ◽  
Disease Treatment ◽  
Practical Applications ◽  
Agricultural Plants ◽  
Insect Reproduction

Selective regulation of gene expression by means of RNA interference has revolutionized molecular biology. This approach is not only used in fundamental studies on the roles of particular genes in the functioning of various organisms, but also possesses practical applications. A variety of methods are being developed based on gene silencing using dsRNA—for protecting agricultural plants from various pathogens, controlling insect reproduction, and therapeutic techniques related to the oncological disease treatment. One of the main problems in this research area is the successful delivery of exogenous dsRNA into cells, as this can be greatly affected by the localization or origin of tumor. This overview is dedicated to describing the latest advances in the development of various transport agents for the delivery of dsRNA fragments for gene silencing, with an emphasis on cancer treatment.

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