Novel suction-based in vivo cutaneous DNA transfection platform
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ABSTRACT:
SUBMITTER: Lallow E
PROVIDER: S-EPMC8570601 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.
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Project description:We describe an original electroporation protocol for in vivo plasmid DNA transfection. The right hind limbs of C57 mice are exposed to a specifically designed train of permeabilizing electric pulses by transcutaneous application of tailored needle electrodes, immediately after the injection of pEGFP-C1 plasmid encoding GFP (Green Fluorescente Protein). The electroporated rodents show a greater GFP expression than the controls at three different time points (4, 10, and 15 days). The electroporated muscles display only mild interstitial myositis, with a significant increase in inflammatory cell infiltrates. Finally, mild gait abnormalities are registered in electroporated mice only in the first 48 h after the treatment. This protocol has proven to be highly efficient in terms of expression levels of the construct, is easy to apply since it does not require surgical exposure of the muscle and is well tolerated by the animals because it does not cause evident morphological and functional damage to the electroporated muscle.
| S-EPMC7555604 | biostudies-literature
Project description:The potential applications of cationic poly(ionic liquids) range from medicine to energy storage, and the development of efficient synthetic strategies to target innovative cationic building blocks is an important goal. A post-polymerization click reaction is reported that provides facile access to trisaminocyclopropenium (TAC) ion-functionalized macromolecules of various architectures, which are the first class of polyelectrolytes that bear a formal charge on carbon. Quantitative conversions of polymers comprising pendant or main-chain secondary amines were observed for an array of TAC derivatives in three hours using near equimolar quantities of cyclopropenium chlorides. The resulting TAC polymers are biocompatible and efficient transfection agents. This robust, efficient, and orthogonal click reaction of an ionic liquid, which we term ClickabIL, allows straightforward screening of polymeric TAC derivatives. This platform provides a modular route to synthesize and study various properties of novel TAC-based polymers.
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Project description:Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high-throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.
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Project description:Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of +36 (+36 GFP), can penetrate a variety of mammalian cell lines. Internalization of +36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When +36 GFP is mixed with siRNA, protein-siRNA complexes approximately 1.7 mum in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by +36 GFP suppresses target gene expression. We show that +36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of +36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.
| S-EPMC2659711 | biostudies-literature
Project description:PurposeTo determine whether (a) stem cells loaded with DNA-carrying microbubbles (MBs) can be transfected in vivo, (b) the cells remain alive to express the gene, and (c) gene expression is sufficiently robust to be detected in vivo.Materials and methodsThe study was approved by the Institutional Animal Care and Use Committee. Cationic MBs were prepared, characterized, and loaded with pLuciferase green fluorescent protein (GFP) plasmid. Loading was confirmed with SYBR Gold staining (Life Technologies, Carlsbad, Calif). C17.2 cells were loaded with the DNA-carrying MBs. Two hundred thousand cells suspended in 20 ?L phosphate-buffered saline were mixed with 200 ?L Matrigel (BD Biosciences, San Jose, Calif) and injected in both flanks of eight nude mice. One of the Matrigel (BD Biosciences) injections contained 50 000 cells pretransfected in vitro by using lipofectamine as a positive control. Nine flanks were exposed to 2.25-MHz ultrasonic pulses at 50% duty cycle for 1 minute at 1 W/cm(2) (n = 3) or 2 W/cm(2) (n = 6), and six flanks served as the negative control. Two days later, bioluminescent images were acquired in each mouse every 3 minutes for 1 hour after the intraperitoneal injection of d-luciferin (Perkin Elmer, Waltham, Mass). Differences between groups were assessed by using the nonparametric Kruskal-Wallis test with Wilcoxon rank sum tests for follow-up comparisons. Mice were then killed, plugs were explanted, and alternate sections were stained with hematoxylin-eosin or stained for GFP expression.ResultsMean DNA-loaded MB diameter ± standard deviation was 2.87 ?m ± 1.69 with the DNA associated with the MB shell. C17.2 cells were associated with 2-4 MBs each, and more than 90% were viable. Peak background subtracted bioluminescent signal was fourfold higher when cells were exposed to 2 W/cm(2) pulses as compared with 1 W/cm(2) pulses (P = .02) and negative controls (P = .002). Histologic examination showed cells within the Matrigel (BD Biosciences) with robust GFP expression only after 2 W/cm(2) ultrasound exposure and lipofectamine transfection.ConclusionStem cells loaded with DNA-carrying MBs can be transfected in vivo with ultrasonic pulses and remain alive to demonstrate robust gene expression.
| S-EPMC4521611 | biostudies-literature
Project description:Background Development of the flexible CO2 fiber has presented new opportunities for the use of precision laser cutting in cranial procedures. The efficacy of the CO2 scalpel is further enhanced by combining it with a fluid removal suction capability. Objectives We report our experience with a novel CO2 laser-suction device. Methods The novel laser-suction device was designed in conjunction with OmniGuide Inc. (Cambridge, Massachusetts, USA). We performed a case review of its use in firm tumors that were resistant to resection by bipolar, suction, and ultrasonic aspirator. Results The laser-suction device was applied in three tumors where resection with ultrasonic aspiration failed. Tumor resection using the laser-suction device was successful in all three cases. There were no complications related to the laser-suction device. There were no instances of intraoperative device malfunction. Discussion The CO2 laser combined with suction is a useful instrument for resection of firm tumors that prove to be resistant to ultrasonic aspiration. We also find it to be useful in settings where precise tissue incisions are desired with minimal manipulation. In our experience, the surgical efficiency of the CO2 laser is improved by the laser-suction device. This device allows the surgeon to utilize a suction device and laser in a single hand and enables concurrent use of bipolar electrocautery without repeated instrument changes.
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Project description:Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.
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Project description:Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a laser, to produce temporary mechanical deformations of the cell membrane and facilitate the delivery of plasmid DNA into cells. We show that high stress gradients, produced when picosecond laser pulses with a fluence of 100 mJ/cm2 are absorbed by piezophotonic materials, enable transfection of a plasmid DNA encoding Green Fluorescent Protein (gWizGFP, 3.74 MDa) in COS-7 monkey fibroblast cells with an efficiency of 5% at 20 °C, in 10 minutes. We did not observe significant cytotoxicity under these conditions. Photoacoustic transfection is scalable, affordable, enables nuclear localization and the dosage is easily controlled by the laser parameters.
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Project description:Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies.
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