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Background
DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The “10-23 DNA enzyme” is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs.Methodology
In this study, we inserted a nonpairing bulge at the 5′ end of the “10–23 DNA enzyme” to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5′ end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme.Conclusions
Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12–15 bp away from the core catalytic domain. 相似文献2.
Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes 总被引:7,自引:0,他引:7
Breitler JC Labeyrie A Meynard D Legavre T Guiderdoni E 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(4):709-719
This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in
microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use
influences the integration and expression of the co-transferred gene of interest. Two non-linked marker genes (yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments. Three different
DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid
DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins. Our results demonstrate that microprojectile bombardment-mediated transformation
of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use
of whole-plasmid DNA. Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among
plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization
of molecules prior to integration was rarely observed in gene cassette transformants.
Received: 4 April 2001 / Accepted: 13 August 2001 相似文献
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Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins 总被引:18,自引:3,他引:15 下载免费PDF全文
Boden D Pusch O Silbermann R Lee F Tucker L Ramratnam B 《Nucleic acids research》2004,32(3):1154-1158
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MOTIVATION: A promising and reliable approach to annotate gene function is clustering genes not only by using gene expression data but also literature information, especially gene networks. RESULTS: We present a systematic method for gene clustering by combining these totally different two types of data, particularly focusing on network modularity, a global feature of gene networks. Our method is based on learning a probabilistic model, which we call a hidden modular random field in which the relation between hidden variables directly represents a given gene network. Our learning algorithm which minimizes an energy function considering the network modularity is practically time-efficient, regardless of using the global network property. We evaluated our method by using a metabolic network and microarray expression data, changing with microarray datasets, parameters of our model and gold standard clusters. Experimental results showed that our method outperformed other four competing methods, including k-means and existing graph partitioning methods, being statistically significant in all cases. Further detailed analysis showed that our method could group a set of genes into a cluster which corresponds to the folate metabolic pathway while other methods could not. From these results, we can say that our method is highly effective for gene clustering and annotating gene function. 相似文献
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Bruno Lefebvre Brigitte Vandewalle Justine Longue Ericka Moerman Bruno Lukowiak Valery Gmyr Kathrin Maedler Julie Kerr-conte François Pattou 《BMC biotechnology》2010,10(1):28
Background
In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. 相似文献13.
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Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression
In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular. 相似文献
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