Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection

Batard, Pascal; Jordan, Martin; Wurm, Florian Μ.

doi:10.1016/s0378-1119(01)00467-x

Cited by 118 publications

(85 citation statements)

References 16 publications

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“…The exact mechanism by which keratinocytes internalize and transport plasmid DNA remains unknown, but a protein-mediated uptake and transport seems to be involved. Besides calciummediated transfection, 46 lysosomotropic agents such as chloroquine were found to improve the transfection of mammalian cells by interfering with several steps involved in transport and release of plasmid DNA from lysosomes. 47 Recent evidence in mice and pigs showed that other tissues may also take up plasmid DNA upon in vivo injection of a supraphysiological dose.…”

Section: Dna-binding Proteins In Keratinocytesmentioning

confidence: 99%

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

et al. 2004

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The skin is an interesting organ for human gene therapy due to accessibility, immunologic potential and synthesis capabilities. In this study, we attempted to visualize and measure the uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a doseand time-dependent manner. Cycloheximide treatment inhibited the uptake by 490%, suggesting a protein-mediated uptake. The inhibition of different internalization pathways demonstrated that blocking macropinocytosis (by amiloride and N,N-dimethylamylorid) reduced DNA uptake by 485%, while the inhibition of clathrin-coated pits (by chlorpromazine) and caveolae (by nystatin/filipin III) did not limit the uptake. Colocalization studies using confocal laser microscopy revealed a time-dependent accumulation of plasmid DNA in endosomes and lysosomes. When a green fluorescent protein (GFP) expression vector was used, specific GFP-RNA became detectable by reverse transcriptase-PCR, whereas measurable amounts of protein could not be identified in FACS experiments. To detect the potential DNA receptors on the keratinocyte surface, membrane proteins were extracted and subjected to South-Western blotting using digoxigenin-labeled calf thymus and l-phage DNA. Two DNA-binding proteins, ezrin and moesin, known as plasma membrane-actin linkers, were identified by one-and two-dimensional-South-Western blots and matrix-assisted laser desorption and ionizationmass spectrometry. Ezrin and moesin are functionally associated with a number of transmembrane receptors such as the EGF, CD44 or ICAM-1 receptor. Taken together, naked plasmid DNA seems to enter human keratinocytes through different pathways, mainly by macropinocytosis. Two DNA-binding proteins were identified that seemed to be involved in binding/trafficking of internalized DNA.

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Section: Dna-binding Proteins In Keratinocytesmentioning

confidence: 99%

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

et al. 2004

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“…[1][2][3][4][5][6][7][8][9][10][11][12] A gene-transferring system mediated by a particulate DNA-calcium phosphate composite [1][2][3] is safer than viral [4][5][6] and lipid-based [7][8][9] systems, but its transferring efficiency is comparatively low. To enhance the gene-transferring efficiency, a surface-mediated genetransferring system derived from a DNA-apatite composite (D-Ap) layer has been developed.…”

mentioning

confidence: 99%

Novel gene-transferring scaffolds having a cell adhesion molecule–DNA–apatite nanocomposite surface

2007

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A low efficiency has long been the most critical problem of conventional gene-transferring systems using calcium phosphates, and this was successfully improved on by using a laminin-DNA-apatite composite (LD-Ap) layer. The genetransferring efficiency of the LD-Ap surface was 1-2 orders of magnitude higher than that of a DNA-calcium phosphate composite surface. This is because laminin enhances cell adhesion and spreading, and this provides regions of high DNA concentration between a cell and the LD-Ap surface. The efficiency of gene transfer of the LD-Ap surface was equivalent to, or even higher than that mediated using a commercial lipid-based transfection reagent applied using the manufacture's recommended optimum conditions. In addition, the gene-transferring efficiency of our system could be controlled by changing the laminin and DNA content in the LD-Ap layer. Moreover, our system is composed of highly safe reagents: apatite, DNA and laminin, all of which are present in the human body. Hence, the LD-Ap surface, which enhances cell attachment on its surface, and mediates a safe, highly efficient and controllable gene transfer, is highly applicable to tissue engineering and gene therapy applications. Gene Therapy (2007) An efficient and safe gene-transferring system is a key technology in tissue engineering and gene therapy applications. 1-12 A gene-transferring system mediated by a particulate DNA-calcium phosphate composite 1-3 is safer than viral 4-6 and lipid-based 7-9 systems, but its transferring efficiency is comparatively low. To enhance the gene-transferring efficiency, a surface-mediated genetransferring system derived from a DNA-apatite composite (D-Ap) layer has been developed. 12 In this system, regions of high DNA concentration are produced locally around cells, and this enhances the gene-transferring efficiency. In this work, the cell adhesion molecule, laminin, 13,14 was immobilized on a D-Ap layer to enhance further the gene-transferring efficiency by strengthening a cell's attachment and facilitating its spreading on a coating's surface. The gene-transferring efficiency was 1-2 orders of magnitude higher on the resulting laminin-DNA-apatite composite (LD-Ap) layer than the gene-transferring efficiency on a D-Ap layer. In this paper, we present an advanced gene-transferring system that has a high efficiency, is safe and has a great potential for use in tissue engineering and gene therapy applications.An LD-Ap layer was coated on a polymer surface in a metastable supersaturated calcium phosphate solution at neutral pH, and ambient pressure and temperature. [15][16][17][18] As controls, an apatite (Ap) layer, a D-Ap layer and a laminin-apatite composite (L-Ap) layer were also coated on the polymer surface. The coating layers were formed by immersing the polymer sample, that had an amorphous calcium phosphate (ACP) layer deposited on its surface, in four different coating solutions: a calcium phosphate solution (CP solution) [15][16][17][18] (for the Ap layer), a CP solution supplemented with DN...

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“…Despite being one of the oldest transfection methods, the procedure has remained almost unchanged, basically composed of either direct or drop-wise mixing of two solutions, one containing DNA and calcium chloride (CaCl 2 ) and the other possessing inorganic phosphate (Pi) (either NaH 2 PO 4 or Na 2 HPO 4 ) in order to induce "supersaturation" of the final solution with respect to CaCl 2 and Pi, resulting in precipitation of microscopically visible and invisible particles with the DNA being adsorbed through electrostatic interactions [12][13][14]. Although the particles have net negative charge, they are capable of binding anionic DNA presumably through the Ca…”

Section: Mechanism Of Dna Delivery By Classical Capi Particlesmentioning

confidence: 99%

“…X-ray diffraction and FT-IR analysis of the precipitated particles established them as "hydroxyapatite" with the molecular formula of Ca 10 (PO 4 ) 6 (OH) 2 [12][13][14]. The resulting DNA/CaPi co-precipitates are usually dispersed onto the cultured cells from several hours to overnight in the incubator allowing them to be taken up by the cells via endocytosis.…”

Section: +mentioning

confidence: 99%

“…The internalized DNA is thus entrapped into the acidic compartments of endosomes which can subsequently be fused with the lysosomes of relatively lower pH, where the DNA is usually fragmented by hydrolytic enzymes (nucleases). However, treatment with glycerol [12,15] or chloroquine [15,16] can help release functional DNA from the acidic vesicles to the cytosol in return for some extent of cell toxicity. Depending on whether the cell is dividing, like a cancer cell or nondividing like most of the normal cells in our body, cytosolic plasmid DNA can enter the nucleus at the time when the nuclear membrane is disrupted during mitosis or slowly pass through the nuclear pores by passive diffusion, respectively [5].…”

Section: +mentioning

confidence: 99%

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pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

Chowdhury¹

2013

OJGen

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The short half-lives due to the enzymatic degradation in blood, the lack of tissue targetability and the incapability to passively diffuse across the plasma membrane and smoothly traffic across the harsh intracelluar environment are the major shortcomings for nucleic acid-based potential therapeutics, such as recombinant plasmid and antisense oligonucleotides or small interferring RNA (siRNA). Plasmid DNA containing a gene of interest could have immense impact as a promising therapeutic drug for treating genetic as well as acquired human diseases at the molecular level with high level of efficacy and precision. Thus both viral and non-viral synthetic vectors have been developed in the past decades to address the aforementioned challenges of naked DNA. While in the viral particles plasmid DNA is integrated into the viral genome, in most non-viral cases the DNA being anionic in nature is electrostatically associated with a cationic lipid or polymer forming lipoplex or polyplex, respectively, or a cationized inorganic gold, silica or iron oxide particle. Due to the potential immunogenicity and carcinogenicity issues with the viral particles, non-viral vectors have drawn much more attention for the clinical evaluation. However, the main concern of using non-biodegradable particles, specially the inorganic ones, is the adverse effects owing to their long term interactions with body components. We have recently developed biodegradable pH-sensitive inorganic nanoparticles of Mg/CaPi and carbonate apatite for efficient transgene delivery to primary, cancer and embryonic stem cells, by virtue of their high affinity binding with the DNA, ability to contact the cell membrane by ionic or ligand-receptor interactions and fast dissolution kinectis in endosomal acidic pH facilitating release of the DNA from the dissolving particles and also from the endosomes.

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Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection

Cited by 118 publications

References 16 publications

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

Novel gene-transferring scaffolds having a cell adhesion molecule–DNA–apatite nanocomposite surface

pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

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