scholarly journals Main constraints for RNAi induced by expressed long dsRNA in mouse cells

2019 ◽  
Vol 2 (1) ◽  
pp. e201800289 ◽  
Author(s):  
Tomas Demeter ◽  
Michaela Vaskovicova ◽  
Radek Malik ◽  
Filip Horvat ◽  
Josef Pasulka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Systematic Analysis ◽  
Positive Effects ◽  
Rnai Efficiency ◽  
Mouse Cells ◽  
Dicer Cleavage ◽  
Microrna Pathway ◽  
Microrna Function

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

scholarly journals Main constraints for RNAi induced by expressed long dsRNA in mouse cells

2018 ◽  
Author(s):  
Tomas Demeter ◽  
Michaela Vaskovicova ◽  
Radek Malik ◽  
Filip Horvat ◽  
Josef Pasulka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Positive Effects ◽  
Dsrna Binding ◽  
Mouse Cells ◽  
Dicer Cleavage ◽  
Microrna Pathway ◽  
Microrna Function ◽  
Specific Mrna

RNA interference (RNAi) is sequence-specific mRNA degradation guided by small RNAs (siRNAs) produced from long double-stranded RNA (dsRNA) by RNase Dicer. Proteins executing RNAi are present in mammalian cells but sustain a gene-regulating microRNA pathway while dsRNA-induced innate immunity relies on a sequence-independent interferon response. While striving to benchmark mammalian RNAi analysis, we report that the main RNAi constraint is siRNA production, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of dsRNA-binding Dicer co-factors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of Protein Kinase R, a key dsRNA sensor for interferon response, had minimal positive effects in fibroblasts. Without increasing Dicer activity, RNAi can occur when the first Dicer cleavage of an abundant dsRNA produces an efficient siRNA. In mammals, efficient RNAi may effectively employ substrates, which have some features of microRNA precursors, hence bringing the two pathways mechanistically even closer. At the same time, Dicer substrate optimization, which viruses would avoid, represents an opportunity for evolving RNAi, yet unlikely as an antiviral system.

scholarly journals Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome.

2021 ◽  
Author(s):  
Miguel Martinez-Ara ◽  
Federico Comoglio ◽  
Joris van Arensbergen ◽  
Bas van Steensel
Keyword(s):  
Mammalian Cells ◽  
Mouse Genome ◽  
Regulatory Elements ◽  
Genomic Context ◽  
Systematic Analysis ◽  
Chromatin Folding ◽  
Reporter Assays ◽  
Mouse Cells ◽  
Genomic Regions ◽  
Single Promoter

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterised in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE - promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity for a single promoter to quantitative differences in activation across a broad set of promoters. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order TF motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.

Participation in WorldSkills Russia championships is an indicator of training specialists

2021 ◽  
Vol 2 (3) ◽  
pp. 278-285
Author(s):  
E. V. Sobina
Keyword(s):  
Professional Education ◽  
Material Base ◽  
Educational Organizations ◽  
Sources Of Information ◽  
Railway Transport ◽  
Systematic Analysis ◽  
Young Professionals ◽  
Positive Effects ◽  
Problems Of Practice ◽  
Training Specialists

The issues of the participation of students of professional educational organizations in the WorldSkills Russia championships are considered through the prism of quality indicators for training specialists.A systematic analysis of theoretical sources of information devoted to the integration of the movement "Young Professionals" (WorldSkills Russia) into the vocational education system, the problems of forming competencies among students in the process of introducing WorldSkills Russia standards has been carried out.The method of direct observation is used, with the help of which empirical material was collected about the experience of participation of the Volgograd Technical School of Railway Transport — branch of the Rostov State Transport University, in the development of new competencies in WorldSkills Russia; polls of teachers; comparison; generalization.The key problems of practice-oriented activities of students are identified, the experience of the participation of students and teachers of the branch in the WorldSkills Russia championship movement is summarized.The effectiveness of the participation of students in the WorldSkills Russia championships for the period 2017-2021 has been analyzed.The positive effects of involvement in the championship movement have been determined: improving the qualifications of teachers, strengthening the material base of the educational organization in accordance with the requirements of the infrastructure competency sheets. It is shown that the involvement of students in the WorldSkills Russia championship movement provides an opportunity to popularize and increase the prestige of the profession, motivate them to professional education and improve their qualifications.

scholarly journals A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

2009 ◽  
Vol 206 (9) ◽  
pp. 1899-1911 ◽  
Author(s):  
Sarah M. McWhirter ◽  
Roman Barbalat ◽  
Kathryn M. Monroe ◽  
Mary F. Fontana ◽  
Mamoru Hyodo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Map Kinases ◽  
Second Messenger ◽  
Innate Immune ◽  
Guanosine Monophosphate ◽  
Host Cells ◽  
Type I Interferons ◽  
Interferon Response ◽  
Type I

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Correction of Genetic Defects in Mammalian Cells by the Input of Small Amounts of Foreign Genetic Material

1973 ◽  
Vol 13 (3) ◽  
pp. 841-861
Author(s):  
YVONNE L. BOYD ◽  
H. HARRIS
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Hybrid Cell ◽  
Genetic Material ◽  
Chinese Hamster ◽  
Heat Sensitivity ◽  
Chinese Hamster Cells ◽  
Inosinic Acid ◽  
Mouse Cells

Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were cultivated in a selective medium in which possession of these enzymes was essential for cell survival and growth. Clones of cells able to grow in this medium were isolated and studied. A detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase activity.

Recombination events after transient infection and stable integration of DNA into mouse cells

1985 ◽  
Vol 5 (4) ◽  
pp. 659-666 ◽  
Author(s):  
S Subramani ◽  
J Rubnitz
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Transcription Unit ◽  
Sister Chromatid Exchanges ◽  
Neomycin Resistance Gene ◽  
Stable Integration ◽  
Mouse Cells ◽  
Chromatid Exchanges ◽  
Neomycin Resistance ◽  
Constructed Plasmids

To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.

scholarly journals Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

2001 ◽  
Vol 21 (22) ◽  
pp. 7807-7816 ◽  
Author(s):  
Shicheng Yang ◽  
Stephen Tutton ◽  
Eric Pierce ◽  
Kyonggeun Yoon
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transient Transfection ◽  
Interferon Response ◽  
Green Fluorescent Protein Gene ◽  
Double Stranded Rna ◽  
Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

The Role of Protein Kinase R in the Interferon Response

2011 ◽  
Vol 31 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Agnieszka Pindel ◽  
Anthony Sadler
Keyword(s):  
Protein Kinase ◽  
Interferon Response ◽  
Protein Kinase R

scholarly journals Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells.

1988 ◽  
Vol 8 (9) ◽  
pp. 3929-3933 ◽  
Author(s):  
K Tokunaga ◽  
K Takeda ◽  
K Kamiyama ◽  
H Kageyama ◽  
K Takenaga ◽  
...  
Keyword(s):  
Differential Expression ◽  
Functional Diversity ◽  
Mammalian Cells ◽  
Cell Types ◽  
Regulatory Mechanisms ◽  
Untranslated Regions ◽  
Cdna Clones ◽  
Expression Levels ◽  
Mouse Tissues ◽  
Mouse Cells

We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.

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