The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm(2) with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2010.05.014 | DOI Listing |
Bio Protoc
January 2025
Department of Cell Biology and Human Anatomy, University of California, Davis, School of Medicine, Davis, CA, USA.
Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult.
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January 2025
Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan.
Primary neuronal culture and transient transfection offer a pair of crucial tools for neuroscience research, providing a controlled environment to study the behavior, function, and interactions of neurons in vitro. These cultures can be used to investigate fundamental aspects of neuronal development and plasticity, as well as disease mechanisms. There are numerous methods of transient transfection, such as electroporation, calcium phosphate precipitation, or cationic lipid transfection.
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January 2025
Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan.
Background: Chitosan nanoparticles (CsNPs) are an effective and inexpensive approach for DNA delivery into live cells. However, most CsNP synthesis protocols are not optimized to allow long-term storage of CsNPs without loss of function. Here, we describe a protocol for CsNP synthesis, lyophilization, and sonication, to store CsNPs and maintain transfection efficiency.
View Article and Find Full Text PDFExtracellular vesicles (EVs) are gaining recognition as promising therapeutic carriers for immune modulation. We investigated the potential of EVs derived from HEK293FT cells to stabilize and deliver interleukin-10 (IL-10), a key anti-inflammatory cytokine. Using minicircle (MC) DNA vectors, we achieved IL-10 overexpression and efficient incorporation into EVs, yielding superior stability compared to free, recombinant IL-10 protein.
View Article and Find Full Text PDFMol Ther
January 2025
Perinatal Institute, Division of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA. Electronic address:
Exemplified by successful use in COVID-19 vaccination, delivery of modified mRNA encapsulated in lipid nanoparticles provides a framework for treating various genetic and acquired disorders. However, lipid nanoparticles that can deliver mRNA into specific lung cell types have not yet been established. Here, we sought whether poly(®-amino ester)s (PBAE) or PEGylated PBAE (PBAE-PEG) in combination with 4A3-SC8/DOPE/cholesterol/DOTAP lipid nanoparticles (LNP) could deliver mRNA into different types of lung cells in vivo.
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