Transfection and expression of plasmid DNA in plant cells by an arginine‐rich intracellular delivery peptide without protoplast preparation

Chen, Chung‐Pin; Chou, Jyh-Ching; Liu, Betty Revon; Chang, Mei‐Chi; Lee, Han-Jung

doi:10.1016/j.febslet.2007.03.076

Cited by 81 publications

(71 citation statements)

References 45 publications

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“…Our supposition is supported by previous findings related to the ability of CPP peptides to transfer their cargos (e.g. DNA, proteins) to various cells of monocot and dicot plants (Chen et al, 2007;Chugh and Eudes, 2007, 2008a, 2008b, Chugh et al, 2010. Further studies need to be performed to verify our assumption.…”

Section: Resultssupporting

confidence: 70%

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

et al. 2012

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Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particlemediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat 2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.Transgenesis, or genetic transformation, finds many applications in plant biology, e.g. in cell biology and gene function studies. Importantly, it also allows for the generation of plants with improved agricultural traits significantly faster than any conventional breeding practice. The technology is based on the delivery of genes of interest from a broad range of sources into a plant genome. Two major transformation techniques include Agrobacterium-mediated DNA delivery and

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Section: Resultssupporting

confidence: 70%

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

et al. 2012

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“…Successful nucleic acid (DNA and dsRNA) transfection has been reported in protoplast, tobacco cell suspension culture, mungbean and soyabean root tips, and permeabilized immature wheat embryos (3,13,64,99,103). Posttranscriptional gene silencing was achieved using sense and antisense constructs of 0.4 and 0.9 kb in dsRNA/R12-treated tobacco cell suspension culture (64).…”

Section: Somatic Cellsmentioning

confidence: 99%

“…Recent investigations show that CPPs, such as Tat, Tat2, arginine-rich intracellular delivery peptides (AID), pVEC, transportan, and penetratin, can be taken up by suspensions of protoplasts from tobacco cells and triticale mesophyl cells (13,32,64,99). We studied uptake of pVEC and transportan in various plants tissues (32).…”

Section: Somatic Cellsmentioning

confidence: 99%

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

2010

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SummaryNovel classes and applications of cell-penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP-mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP-macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP-cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.

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“…Macropinocytosis dependent transduction of fluorescent proteins by arginine-rich intracellular delivery (AID) and Tat-protein transduction domain has been reported in onion and corn root tip cells [28,29]. Cationic oligopeptides such as polyarginine have been shown to deliver dsRNA to induce post-transcriptional gene silencing in tobacco suspension cells [30]. AID has been reported to deliver plasmid DNA in plant root cells [31].…”

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confidence: 99%

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

Chugh¹,

Eudes²

2008

The FEBS Journal

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Cell-penetrating peptides (CPPs) comprise a fast growing class of short length peptides that differ in sequence, size and charge but share a common characteristic ability to translocate across the plasma membrane. It has been demonstrated that CPPs can act efficiently as nonviral delivery vehicles for macromolecules that are much larger in size than their own and lack the self-potential to enter living cells due to the The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat 2 , mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene ⁄ ethanol (1 : 20, v ⁄ v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cellpenetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active b-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat 2 -mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene ⁄ ethanol (1 : 40, v ⁄ v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat 2 -mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat 2 -GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat 2 -plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat 2 -plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent LipofectamineÔ 2000 to the Tat 2 -plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.Abbreviations AID, arginine-rich intracellular delivery; CPP, cell-penetrating peptide; EIPA, 5-(N-ethyl N-isopropyl) amirolide; b-GUS, b-glucuronidase; M-Tat, mutated-Tat; pVEC, peptide vascular endothelial-cadherin.

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Transfection and expression of plasmid DNA in plant cells by an arginine‐rich intracellular delivery peptide without protoplast preparation

Cited by 81 publications

References 45 publications

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

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