Because human embryonic stem (hES) cells can differentiate into virtually any cell type in the human body, these cells hold promise for regenerative medicine. The genetic manipulation of hES cells will enhance our understanding of genes involved in early development and will accelerate their potential use and application for regenerative medicine. The objective of this study was to increase the transfection efficiency of plasmid DNA into hES cells by modifying a standard reverse transfection (RT) protocol of lipofection. We hypothesized that immobilization of plasmid DNA in extracellular matrix would be a more efficient method for plasmid transfer due to the affinity of hES cells for substrates such as Matrigel and to the prolonged exposure of cells to plasmid DNA. Our results demonstrate that this modification doubled the transfection efficiency of hES cells and the generation of clonal cell lines containing a piece of foreign DNA stably inserted in their genomes compared to results obtained with standard forward transfection. In addition, treatment with dimethyl sulfoxide further increased the transfection efficiency of hES cells. In conclusion, modifications to the RT protocol of lipofection result in a significant and robust increase in the transfection efficiency of hES cells.
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http://dx.doi.org/10.1089/scd.2009.0505 | DOI Listing |
Cell Div
January 2025
Center for Clinical Laboratories, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, 550004, China.
Objective: This study aimed to investigate the regulatory effects of long non-coding RNA-ANRIL on CDKN2A in the cell cycle of Kasumi-1 cells and elucidate the underlying molecular mechanisms.
Methods: ANRIL and CDKN2A expression levels were quantified using RT-qPCR in peripheral blood samples from acute myeloid leukemia (AML) patients. CDKN2A knockdown efficiency was validated via RT-qPCR, and cell cycle distribution was analyzed using flow cytometry.
Acta Biomater
January 2025
College of Chemistry, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, PR China. Electronic address:
mRNA-based protein replacement therapy has become one of the most widely applied forms of mRNA therapy, with lipid nanoparticles (LNPs) being extensively studied as efficient delivery platforms for mRNA. However, existing LNPs tend to accumulate in the liver or kidneys after intravenous injection, highlighting the need to develop vectors capable of targeting specific organs. In this study, we synthesized a small library of ionizable lipids and identified PPz-2R as a promising candidate, exhibiting lung-targeting capabilities, high mRNA transfection efficiency, and good stability through structure-activity relationship studies.
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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.
View Article and Find Full Text PDFPeerJ
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.
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