transfection

This study investigates the role of genes related to breast cancer in apoptosis control. A melittin nucleic acid sequence was synthesized and introduced into a pcDNA3.1(+) Mammalian Expression Plasmid. The cloning accuracy was assessed using PCR testing and enzyme digestion techniques. The vectors were transfected into cells using LipofectamineTM2000. The transfection efficacy of MCF-7 and 4T1 cells was evaluated using fluorescence and bright-field imaging. Pure melittin produced from bee venom had a notable hemolytic impact, with lower hemolytic activity levels than the positive control, Triton X-100...

Within the eukaryotic cell, the actin cytoskeleton is a crucial structural framework that maintains cellular form, regulates cell movement and division, and facilitates the internal transportation of proteins and organelles. External cues induce alterations in the actin cytoskeleton primarily through the activation of Rho GTPases, which then bind to a diverse array of effector proteins to promote the local assembly or disassembly of actin. We have harnessed the extensively studied functions of RhoA in the dynamics of the actin cytoskeleton to craft a practical series for Stage 2 Biology students...

The anti-tumor capacity of natural killer (NK) cells heavily relies on their ability to migrate towards their target cells. This process is based on dynamic actinrearrangement, so-called actin treadmilling, andis tightly regulated by proteins such as cofilin-1. The aim of the present study was to identify the role of cofilin-1 (CFL-1) in the migratory behavior of NK cells and to investigate a possible impact of an obesity-associated micromilieu on these cells, as it is known that obesity correlates with various impaired NK cell functions...

AIM: To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells (LECs) under hyperosmotic stress. METHODS: LECs were treated with hyperosmotic stress at the concentration of 270, 300, 400, 500, or 600 mOsm for 6, 12, 18, 24h in vitro . Polymerase chain reaction (PCR) was employed for the mRNA expression of autophagy-related genes, while Western blotting detected the targeted protein expression. The transfection of stub-RFP-sens-GFP-LC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux...

OBJECTIVES: This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein β (C/EBPβ) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1)...

PURPOSE: Prostate cancer (PCa) is a major urological disease that is associated with significant morbidity and mortality in men. LLGL2 is the mammalian homolog of Lgl. It acts as a tumor suppressor in breast and hepatic cancer. However, the role of LLGL2 and the underlying mechanisms in PCa have not yet been elucidated. Here, we investigate the role of LLGL2 in the regulation of epithelial-mesenchymal transition (EMT) in PCa through autophagy in vitro and in vivo. METHODS: PC3 cells were transfected with siLLGL2 or plasmid LLGL2 and autophagy was examined...

Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes...

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-β1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-β1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-β1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo...

To investigate the role of microRNA-195-3p (miR-195-3p) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. AC16 human cardiomyocyte cells were cultured and pretreated with different concentrations of isoflurane (ISO) (1%, 2%, and 3%), followed by 6 h each of hypoxia and reoxygenation to construct H/R cell models. The optimum ISO concentration was assessed based on the cell viability. miR-195-3p expression was regulated by in vitro cell transfection. Cell viability was determined by MTT assay, and apoptosis was evaluated by flow cytometry...

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields...

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4...

Although fibroblast growth factor 7 (FGF7) is known to promote wound healing, its mass production poses several challenges and very few studies have assessed the feasibility of producing FGF7 in cell lines such as Chinese hamster ovary (CHO) cells. Therefore, this study sought to produce recombinant FGF7 in large quantities and evaluate its wound healing effect. To this end, the FGF7 gene was transfected into CHO cells and FGF7 production was optimized. The wound healing efficacy of N-glycosylated FGF7 was evaluated in animals on days 7 and 14 post-treatment using collagen patches (CPs), FGF7-only, and CP with FGF7 (CP+FGF7), whereas an untreated group was used as the control...

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods...

INTRODUCTION: Spontaneous miscarriage is a common complication of early pregnancy. Previous studies have shown that mitochondrial function plays an important role in establishment of a successful pregnancy. Cytochrome c oxidase subunit 4 isoform 1 (COX4I1), a component of electron transport chain complex Ⅳ, is required for coupling the rate of ATP production to energetic requirements. However, there is very limited research on its role in trophoblast biology and how its dysfunction may contribute to spontaneous miscarriage...

Acute myocardial infarction (AMI) is the high incidence rate and mortality of common cardiovascular disease. Herein, we explored the critical role of TRIM11 in AMI and its underlying mechanism. Serum from patients with AMI were collected from our hospital. Mice of model group received angiotensin II. Mice of model + TRIM11 group received with Ang II and TRIM11 vectors. Mice of sham group received normal saline. H9c2 cells were performed transfections using Lipofectamine 2000 (Thermo Fisher Scientific Inc, Shanghai, China), and treated with Ang II...

Gastric cancer is a highly immunogenic malignancy. Immune tolerance facilitated by myeloid-derived suppressor cells (MDSCs) has been implicated in gastric cancer resistance mechanisms. The potential role of APE1 in regulating gastric cancer metastasis by targeting MDSCs remains uncertain. In this study, the plasmid Plxpsp-mGM-CSF was used to induce high expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in GES-1 cells. For tumor transplantation experiments, AGS, AGS+GM-CSF and AGS+GM-CSF-siAPE1 cell lines were established by transfection, followed by subcutaneous implantation of tumor cells...

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex-vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients However, assessing genome-targeting interventions in a clinically-relevant manner prior to clinical trials remains a major challenge due to the limited modalities available...

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method...

Cationic liposome-mediated delivery of drugs, DNA, or RNA plays a pivotal role in small molecule therapy, gene editing, and immunization. However, our current knowledge regarding the cellular structures that facilitate this process remains limited. Here, we used human pluripotent stem cells (hPSCs), which form compact colonies consisting of dynamically active cells at the periphery and epithelial-like cells at the core. We discovered that cells at the colony edges selectively got transfected by cationic liposomes through actin-related protein 2/3 (Arp2/3) dependent dynamic lamellipodia, which is augmented by myosin II inhibition...

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate...