Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.     

Transgenic plant-derived siRNAs can suppress propagation of influenza virus in mammalian cells

We used synthetic DNA oligos to investigate the nucleic acid chaperone properties of the N terminal domain of hepatitis delta antigen (NdAg). We found that NdAg possessed a bona fide chaperone activity. NdAg could distinguish subtle differences in the thermal stability of the base pairing region, and enabled DNA oligos to form a more stable duplex among competing sequences through facilitating strand annealing selectively, stimulating duplex conversion selectively, and stabilizing the more stable duplex. The property of NdAg identified in this study could be applied to improve the efficiency and specificity of dot blot hybridization under conditions of low stringency.     

An M13 vector library for cloning DNA with four nucleotide 3' overhangs

A flexible M13 vector library incorporating the BstXI site has been developed. DNA cut by any currently commercially available restriction endonuclease that generates a 4-nucleotide (nt) 3' overhang can be ligated into a specific clone of the library. The BstXI enzyme recognizes a 6-bp bipartite palindromic sequence. The central nucleotides are not specified, and form a 4-base, 3' overhang when cut by BstXI. 5' CCANNNNN NTGG GGTN NNNNNACC 5' Since the 4-base overhang formed is not part of the BstXI recognition sequence, it is possible to generate a library of 256 different clones by introducing the BstXI site, 151 of the possible 256-member library have been isolated, including all 13 M13BF clones in which the overhang formed by BstXI digestion is complementary to those formed by currently available restriction endonucleases. Of these 13 vectors, BstXI digestion of six clones results in nonpalindromic cohesive ends and should facilitate in vitro tandem gene amplification. The BstXI site is adjacent to the four codons corresponding to the factor Xa recognition sequence. Hence the vector library could facilitate the expression of a fusion protein that could be proteolytically cleaved by factor Xa.     

Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules

RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.     

Mechanisms involved in targeted gene replacement in mammalian cells

The "ends-out" or omega (Omega)-form gene replacement vector is used routinely to perform targeted genome modification in a variety of species and has the potential to be an effective vehicle for gene therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown. Understanding molecular features associated with gene replacement is important and may lead to an increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal mu-delta locus in a murine hybridoma cell line. The results showed that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.     

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single- base 3' overhangs, and one using Pfu polymerase, which produces blunt- ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double- strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.     

Reaction parameters of targeted gene repair in mammalian cells

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.     

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short‐hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.     

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.     

Silencing of HIV-1 gene expression by siRNAs in transduced cells

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.     

Retrovirus-mediated gene expression in mammalian cells.

Significant advances have been made in precisely defining the elements in the Moloney murine leukemia virus genome responsible for tissue-restricted expression. This knowledge should lead to improved expression vectors for gene transfer in mammalian cells. In the past year, retrovirus-mediated gene expression in a diverse range of cell types has been reported. These cells have been used to study gene transfer relevant to a range of inherited diseases.     

Activating gene expression in mammalian cells with promoter-targeted duplex RNAs

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.     

RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues

siRNAs modified with morpholino nucleoside analogues were synthesized and their biological properties were examined in details. The gene silence abilities of modified siRNAs were correlated to the positions of the modifications, some of which appeared to be more potent than the native siRNA. The 3′-end modification improved the stability of siRNAs in serum significantly. Furthermore, the dose–response and time-course experiments demonstrated that the siRNAs with 3′-end modification have potent gene silence activity at lower concentration and prolonged action time. These favorable properties make the morpholino modified siRNA a potentially useful tool in therapeutic applications.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.