共查询到20条相似文献,搜索用时 0 毫秒
1.
M Hamada H Kawasaki T Kuwabara M Warashina K Taira 《Nucleic acids symposium series》1999,(42):285-286
Ribozymes are expected to be useful as antiviral agents and powerful tools of functional analysis of unknown gene products in vivo. For use of ribozymes in vivo, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro would be expected to work in vivo, whereas ribozymes selected in the intracellular environment should retain their function in vivo. With the eventual aim of using ribozymes as antiviral agents or biological tools in mammalian cells, we then devised a novel selection system in mammalian cells of active ribozymes by targeting at a gene for the cyclin dependent kinase inhibitor (CDKI), p16INK4a. In this system, we found that p16INK4a-knockdown cells became malignant and they formed foci. In the mammalian system, we confirmed that the selected cells harbored the active ribozyme, indicating that our positive selection systems in vivo were operational. 相似文献
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Tong WP Zhou Y Wang X Yang F Wu KL Wu J Zhang Y 《Biochemical and biophysical research communications》2008,367(4):866-873
A dual fluorescence reporter plasmid expressing EGFP and DsRed-Monomer from separate promoters was constructed for quantitative flow cytometry analysis. Cloning the hepatitis B virus (HBV) X gene into the 3′ UTR region of DsRed-Monomer allowed quantifying the efficacy of ten siRNAs designed according to the accessibility of HBx mRNA measured in vitro. Using EGFP as an internal control, a justified calculation of the changed mean fluorescence intensity of DsRed-Monomer in each transfected cell yielded highly consistent results, and revealed all 10 siRNAs achieved over 50% inhibition among which a super effective siRNA achieved 88% inhibition at a very low concentration (0.33 μg/ml). This provides a quantification method critical for therapeutic application of siRNA. 相似文献
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Schwarz DS Ding H Kennington L Moore JT Schelter J Burchard J Linsley PS Aronin N Xu Z Zamore PD 《PLoS genetics》2006,2(9):e140
Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the “seed” sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3′ to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide. 相似文献
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RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation. 相似文献
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Størvold GL Gjernes E Askautrud HA Børresen-Dale AL Perou CM Frengen E 《Molecular biotechnology》2007,35(3):275-282
Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function.
Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene
expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional
genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green
fluorescent protein (EGFP) and a puromycin selection marker.
The functionality of the elements in the pSiRPG vector was validated. The H1(TetO2) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the
Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced
TetR-expressing cell line. This observation strongly indicates that the H1(TetO2) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression
of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies
in breast cell lines that are hard to transfect with conventional plasmid-based methods. 相似文献
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Spits C Le Caignec C De Rycke M Van Haute L Van Steirteghem A Liebaers I Sermon K 《Nature protocols》2006,1(4):1965-1970
Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization. 相似文献
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Sano M Kuwabara T Warashina M Fukamizu A Taira K 《Antisense & nucleic acid drug development》2002,12(5):341-352
Intracellular stability is a critical determinant of the activity of a ribozyme in vivo. In previous studies, we succeeded in constructing an effective system for the expression of ribozymes using the promoter of a human gene for tRNA(Val). The resultant tRNA(Val)-driven ribozymes (tRNA-ribozymes) had a half-life of approximately 100 minutes. In the present study, we established a novel system for the selection of tRNA-ribozymes that were more stable than a previously generated optimally designed tRNA-ribozyme, and we confirmed that the newly selected tRNA-ribozymes worked well. Selective pressure was applied by treating cells that expressed tRNA-ribozymes with actinomycin D, and the system yielded tRNA-ribozymes with enhanced stability. The sequences isolated after selection exhibited some similarities. Furthermore, some selected tRNA-ribozymes had almost the same activity as or higher activity than that of the optimally designed tRNA-ribozyme despite the fact that the selective pressure was not aimed at enhancing the cleavage activity. Our approach might be very useful for selection not only of ribozymes with enhanced stability but also of other functional nucleic acids in vivo. 相似文献
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The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA. 相似文献
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Antisense therapy involves the use of antisense oligonucleotides for altering targeted gene function. However, the low efficiency of cell delivery of antisense oligonucleotides has limited the efficacy of antisense therapeutic approaches. RNA-based antisense or ribozyme oligonucleotides can be either synthesized endogenously (e.g., by a viral vector) or delivered exogenously. However, there is presently no vector delivery system available for DNA-based oligonucleotides. Recently, a novel ssDNA expression vector that can generate intracellularly any ssDNA molecule, such as antisense oligonucleotide or DNA enzyme, has been developed in our laboratory. Here we describe an improved expression vector based on the first-generation two-vector system. To test this new expression vector, we chose to express a single-stranded "10-23" DNA enzyme targeting c-raf mRNA in the human lung carcinoma A549 cell line. After introduction into cells by transient transfection, c-raf-cleaving DNA enzymes produced by this expression vector can significantly suppress the expression of c-raf mRNA. Furthermore, the expressed c-raf DNA enzymes induced cell apoptosis, as indicated by genomic DNA fragmentation assay. Our study further demonstrates the feasibility of using this novel ssDNA expression technology to produce intracellularly any sequence of interest, including antisense oligonucleotides and DNA enzyme molecules. 相似文献
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H. Takamatsu K. Hamamoto K. Ishimura S. Yokoyama M. Tokashiki 《Applied microbiology and biotechnology》1996,45(4):454-457
A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min–1 at 100g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 × 106 cells ml–1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. 相似文献
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We demonstrate a new method for the simultaneous measurement of the activation of key regulatory enzymes within single cells. To illustrate the capabilities of the technique, the activation of protein kinase C (PKC), protein kinase A (PKA), calcium-calmodulin activated kinase II (CamKII), and cdc2 protein kinase (cdc2K) was measured in response to both pharmacological or physiological stimuli. This assay strategy should be applicable to a broad range of intracellular enzymes, including phosphatases, proteases, nucleases, and other kinases. 相似文献
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《Experimental cell research》1971,64(1):163-169
Previous studies have demonstrated that limb cartilage and limb muscle arise from the mesenchyme cells of the early limb bud without any detectable cell movement. In the present study, binary mixtures of these three cell types have been made in order to determine whether cells gain the ability to segregate as part of the differentiative process. Limb cartilage, limb muscle, and limb mesenchyme can each segregate from the other two cell types. Thus, the ability to segregate is, in this case, a differentiative characteristic. 相似文献
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RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin. 相似文献
20.
P Aller 《Revista Espanola de Fisiología》1987,43(4):415-420
The effect of mitomycin C on the accumulation of specific mRNAs was studied in asynchronously growing Swiss 3T3 cells, as well as in synchronously growing serum stimulated ts13 cells (a temperature-sensitive mutant from the BHK cell line). It was observed that the steady-state level of p53 RNA experienced some increase in 3T3 cells treated for 24 h with the drug. In addition, mitomycin when applied to serum stimulated ts13 cells increased the level of p2F1 RNA. Mitomycin diminished the level of core histone H3 RNA, a finding consistent with the inhibitory action of this compound on DNA replication. 相似文献