Effect of Viral Double-Stranded RNA on Mammalian Cells in Culture: Cytotoxicity Under Conditions Preventing Viral Replication and Protein Synthesis

Noninfectious bovine enterovirus double-stranded RNA induces cytopathic effects when added to mammalian cells in culture. This is demonstrated by (51)Cr release from prelabeled murine lymphoma cells and trypan blue uptake. Also, the induction of cell death by this viral RNA occurs in the presence of inhibitors of protein synthesis (cycloheximide and puromycin). The possible role and mechanism of viral, double-stranded RNA as a cytopathic agent in virally infected cells are discussed.     

A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.     

An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.     

Biosynthesis of Protein Amino Acids in Plant Tissue Culture. III. Studies on the Biosynthesis of Arginine

Evidence from isotope competition studies and enzymic studies indicates that n-acetyl glutamic semialdehyde, alpha-n-acetyl-l-ornithine, l-ornithine and l-citrulline are intermediates between glucose and arginine in cells of Paul's Scarlet Rose. Evidence for the presence of alpha-n-acetyl-ornithine aminotransferase (E. C. 2.6.1.11) in cell-free extracts was obtained.     

Life Cycle Analysis of Mammalian Cells: III. The Inhibition of Division in Chinese Hamster Cells by Puromycin and Actinomycin

Analysis of the effects of actinomycin and puromycin on the G₂ and mitotic parts of the life cycle in Chinese hamster ovary cells grown in suspension and synchronized by thymidine treatment has been carried out. Rates of division of partially synchronized cell populations were measured in the presence and absence of the drugs, and various controls were performed to test for absence of complex side effects. Actinomycin produces a block 1.9 hr before completion of division, while puromycin produces a block almost coinciding with the initiation of mitosis. Evidence is presented that the puromycin block may be a double one, inhibiting one kind of protein synthesis that virtually coincides with the beginning of mitosis and another that occurs about 8 min earlier. The data are interpreted in terms of the time interval between messenger formation and its associated protein synthesis in this region of the life cycle. The various events studied have been provisionally mapped in the G₂ and mitotic periods of the life cycle.     

Comparative Studies of Various Artificial microRNA Expression Vectors for RNAi in Mammalian Cells

Artificial microRNA (amiRNA) has recently become an important RNA interference (RNAi) technology for gene therapy and gene function studies. Here nine expression strategies were employed to construct plasmid vectors expressing amiRNA (amiR-Fluc) against firefly luciferase (Fluc). Our results indicate that all nine vectors can successfully produce mature amiR-Fluc and specifically suppress the expression of Fluc, although the RNAi efficiency in different mammalian cells displays obvious differences. Among these nine vectors, three can efficiently co-express DsRed reporter gene linked with amiR-Fluc cassette. Moreover, the recommended number of concatenated amiRNAs in a multi-amiRNA expression vector should not be more than four, and the relative position of an amiRNA in the multi-amiRNA expression vector has no apparent influence on its RNAi activity. In summary, all these results described here provide valuable information for the rational design and application of amiRNA expression vector.     

Optimization of Electrotransfection Conditions of Mammalian Cells with Different Biological Features

We introduced eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) genes into cells with different biological features through electroporation. The effects of conditions, including voltage, capacitor flow, pulse cycle, DNA dosage and buffer, on transfection efficiency were investigated based on fluorescent microscopy and posttransfection survival rate of cells by staining with trypan blue. Better electrotransfection outcomes were achieved in the following epithelial cells: Vero cells at 300?V/850???F, PK15 cells at 300?V/500???F, MDCK cells at 200?V/600???F, F81 cells at 200?V/500???F, cancer cells MB49 at 300?V/400???F, Hela cells at 200?V/450???F, HF-29 cells at 300?V/800???F and B16F1 cells at 200?V/650???F. Among fibroblast cells, better electrotransfection was achieved in BHK21 cells at 300?V/600???F and ST cells at 200?V/750???F. RPMI-1640 medium without antibiotics and serum demonstrated higher electrotransfection efficiency and cell survival rate than other cell culture media as electroporation buffer. Our findings further prove that electroporation transfection is an effective method for genetic transfection. Cells with different biological features require varying transfection conditions to obtain higher transfection efficiency of target genes.     

An Easy-To-Handle Microfluidic Device Suitable for Immunohistochemical Procedures in Mammalian Cells Grown Under Flow Conditions

Microfluidics, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.     

Radiosensitivity of Mammalian Cells: III. Effect of Suboptimal Growth Temperatures on Recovery from Radiation-Induced Division Delay

We investigated the effect of suboptimal growth temperatures on recovery from radiation-induced division delay in Chinese hamster cells. It was found that no recovery occurred during the time that either log-phase or synchronized populations were incubated at 4°C and that injury sustained at low dose rates was cumulative over a period of 6.2 hr at low temperature. Postirradiation conditions influencing recovery from the induced division delay period are different from those affecting survival, suggesting that biochemical damage leading to division delay may be different from that leading to cell death.     

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, α-ketoglutarate, ferrous ion, and ascorbate. The K_m for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)₅·4H₂O.     

Initiation of Mammalian Viral Protein Synthesis

CULTURED human cells (KB) infected with human adenovirus type 2 (Ad 2) provide a model system for protein synthesis in mammalian cells. Adenovirus messenger RNA molecules are transcribed from nuclear viral DNA and transported to the cytoplasm for translation¹. Late after infection (18 h) 9–10 viral mRNA species with sedimentation values of 7S to 32S are present in polysomes (Parsons, Gardner and Green, in preparation) and specify eight viral structural proteins which account for 80–90% of the polypeptides synthesized in vivo^2–4. We now describe an in vitro cell-free system, derived from KB cells infected with Ad 2, which synthesizes 8–9 viral polypeptides and can initiate protein synthesis with a special class of yeast methionyl-tRNA. In vivo and in vitro experiments suggest that methionine is the initiator amino-acid for most, if not all, adenovirus structural proteins.     

Physiological Studies in the Mucorales: III. THE ZYGOTROPISM OF ZYGOPHORES OF MUCOR MUCEDO BREFELD

Some previous work on zygotropism in Mucor is briefly reviewed,and further observations confirming earlier studies presented. In addition to positive zygotropism between (+) and (–)zygophores, there is claimed to be a corresponding ‘repulsion’between zygophores of the same mating type, (+) from (+) and(–) from (–). The stimulus appears to be transmittedby the diffusion of a volatile substance from one zygophoreto another. A tentative explanation of the phenomena describedis advanced, based on two postulated volatile hormones, eachevolved by zygophores of one mating type only, stimulating growthin zygophores of its own mating type and retarding it in thoseof the opposite type.     

Gene Silencing Mediated by Endogenous MicroRNAs under Heat Stress Conditions in Mammalian Cells

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.     

人白细胞介素2受体(IL-2R)cDNA在哺乳类细胞中表达的初步研究

按DNA-磷酸钙共沉淀法将人白细胞介素2受体(IL-2R)cDNA转染小鼠成纤维细胞L929,经RNA点渍杂交分析、荧光标记IL-2染色和抗Tac(人IL-2受体α链)特异性玫瑰花环试验,均证明转入的IL-2R cDNA在L929细胞中表达,其产物具有结合IL-2和抗Tac抗体的能力。本文还报道了T细胞白血病Jukat细胞和Molt-4等细胞系异常表达IL-2R的结果,并对此作了分析和讨论。     

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein^1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown¹⁰.In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.Download video file.^{(167M, mp4)}     

Mammalian Pol III Promoter H1 can Transcribe shRNA Inducing RNAi in Chicken Cells

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. RNAi based on DNA vector is not sufficiently established in chicken species. The present study was performed to evaluate RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 in the chicken cells by using a dual fluorescence reporter assay, a plasmid encoding GFP and a plasmid encoding RFP. The evaluation of RNAi efficiency was performed in two kinds of chicken cell type: primary CEF cells and chicken DT-40 cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescent microscopy, and their mRNAs content were analyzed by quantitative RT-PCR. The intensity of the green fluorescence generated by GFP was greatly suppressed by human H1 promoter transcribed GFP-shRNA. Quantitative RT-PCR analysis showed that normalized GFP mRNA expression was reduced to 37 and 32 in primary CEF and DT-40 cells, respectively. In contrast to GFP, the intensity of the red fluorescence generated by RFP protein and the RFP mRNA levels remained unchanged. Consequently, it was concluded that the RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 is applicable to suppress the gene expression specifically and efficiently in chicken cells. Jing Yuan and Xiaobo Wang - These authors contributed equally to this work.