Inhibition of gene expression in plant cells by expression of antisense RNA
01 Aug 1986-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 83, Iss: 15, pp 5372-5376
TL;DR: The observation that antisense RNA inhibits gene expression in bacteria and animal systems has been extended to the plant kingdom with immediate practical applications in both basic research and in plant genetic engineering.
Abstract: Due to the paucity of mutations in biochemical pathways in plants, an alternative approach to classical genetics was tested by expressing antisense RNA in plant cells. A series of plasmids was constructed with the bacterial gene for chloramphenicol acetyltransferase (EC 2.3.1.28) linked in either the sense or antisense orientation to several different plant gene promoters. Various ratios of sense and antisense chloramphenicol acetyltransferase gene plasmids were introduced into plant protoplasts by electric field-mediated DNA transfer (“electroporation”) and the level of expression in each combination was monitored by chloramphenicol acetyltransferase assays. Transcription of antisense RNA was found to effectively block the expression of target genes. Thus, the observation that antisense RNA inhibits gene expression in bacteria and animal systems has been extended to the plant kingdom. Antisense RNA techniques have immediate practical applications in both basic research and in plant genetic engineering.
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TL;DR: A striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA is reported.
Abstract: Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated by transient expression of antisense RNA constructs in mammalian cells5,6, and plant protoplasts7, and by stable expression in transgenic plants8. Here, we report a striking inhibition of expression of the endogenous, developmentally regulated gene for polygalacturonase in stably transformed tomato expressing antisense RNA.
793 citations
TL;DR: This Review discusses key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clinical pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.
Abstract: The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA molecules can efficiently trigger RNAi silencing of specific genes, but their therapeutic use has faced numerous challenges involving safety and potency. However, August 2018 marked a new era for the field, with the US Food and Drug Administration approving patisiran, the first RNAi-based drug. In this Review, we discuss key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clinical pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.
779 citations
TL;DR: Genetic manipulation of genes in the ethylene signal transduction pathway will provide agriculture with new tools to prevent or modify ethylene responses in a variety of plants.
Abstract: Ethylene (C2H4), the chemically simplest plant hormone, is among the best-characterized plant growth regulators. It participates in a variety of stress responses and developmental processes. Genetic studies in Arabidopsis have defined a number of genes in the ethylene signal transduction pathway. Isolation of two of these genes has revealed that plants sense this gas through a combination of proteins that resemble both prokaryotic and eukaryotic signaling proteins. Ethylene signaling components are likely conserved for responses as diverse as cell elongation, cell fate patterning in the root epidermis, and fruit ripening. Genetic manipulation of these genes will provide agriculture with new tools to prevent or modify ethylene responses in a variety of plants.
718 citations
Patent•
07 Apr 1999TL;DR: In this paper, the authors proposed a method to reduce the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acids.
Abstract: Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.
534 citations
TL;DR: It is shown that constitutive expression of an 'anti-sense' chalcone synthase gene in transgenic petunia and tobacco plants results in an altered flower pigmentation due to a reduction in levels of both the messenger RNA for the enzyme and the enzyme itself.
Abstract: In most plants flower pigments derive from the flavonoid biosynthesis pathway. Consistent with this pathway in Petunia hybrida the key enzyme in flavonoid synthesis, chalcone synthase, is synthesized in the flower corolla, tube and anthers1. Here we show that constitutive expression of an 'anti-sense' chalcone synthase gene in transgenic petunia and tobacco plants results, with high frequency, in an altered flower pigmentation due to a reduction in levels of both the messenger RNA for the enzyme and the enzyme itself. The pattern of pigmentation varies among flowers of different transgenic plants, indicating that the activity of the anti-sense gene is influenced by DNA sequences that border its site of insertion in both a quantitative and a qualitative way. Backcrossing experiments show that the different pigmentation phenotypes resulting from the expression of anti-sense chalcone synthese gene(s) are stably inherited. These data establish that secondary metabolism in plants can be manipulated using transgenic plants that constitutively synthesize anti-sense RNA.
484 citations
References
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TL;DR: A series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells.
Abstract: We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.
7,438 citations
TL;DR: A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed and a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM).
5,719 citations
TL;DR: A simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed, according to which the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.
Abstract: Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 12 micrograms DNA Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport
2,496 citations
TL;DR: The results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range.
Abstract: We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.
1,277 citations
TL;DR: Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium, demonstrating that the method is applicable to both monocot and dicot protoplasts.
Abstract: We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.
959 citations