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Adeno-Associated Virus-Based Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
When Russell et al. (138) infected stationary and dividing primary human fibroblast cultures with AAV vectors encoding alkaline phosphatase and neomycin resistance genes, they found that the transduction frequency of S-phase cells is about 200 times greater that non-S-phase cells. Neor transduction was equivalent in dividing and nondividing cells, whereas alkaline phosphatase expression was over 20-fold higher in dividing cultures. The authors argued that G418 resistance requires that cells go through division after transduction, wherease the alkaline phosphatase expression does not. Southern blotting of genomic and Hirt extract DNA supported the hypothesis that AAV genomes remain as single-stranded episomes long enough in nondividing cells to then be recruited and expressed once the cell is allowed to enter S-phase. They were able to confirm subsequent random integration in G418-resistant colonies transduced with the alkaline phosphatase vector at 700 particles per cell.
Interleukin-6 Receptor
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Stefan Rose-John, Peter C. Heinrich
Cells were transfected using the calcium phosphate method, as described.20 Stably transfected cell clones were selected in the presence of 100 or 160 μg/ml G 418 for Hep G2 or NIH/3T3 cells, respectively.
Current and future CFTR therapeutics
Published in Anthony J. Hickey, Heidi M. Mansour, Inhalation Aerosols, 2019
Marne C. Hagemeijer, Gimano D. Amatngalim, Jeffrey M. Beekman
Preclinical studies have demonstrated that PTI-428 and/or PTI-CH increased CFTR function in CFTR model systems when combined with other CFTR modulators (67,74). These studies also demonstrated that amplifiers work across CFTR mutation classes (69). Recently, a proof-of-concept study demonstrated that PTI-CH was able to enhance ORKAMBI® (see “Potentiating corrected CFTR proteins”) effectiveness in primary nasal cells for the p.Ile1234_Arg1239del (ΔI1234_R1239‐CFTR) mutation, for which this drug was initially not registered (75). Read-through of p.Gly542X (c.1624G>T, G542X) in primary cell lines by co-treatment of the aminoglycoside G418 with PTI-428 resulted in enhanced read-through efficacy (73). Whether such treatment combinations are feasible with respect to the known toxicity of aminoglycosides needs to be investigated.
Anti-phospholipase A2 receptor antibodies directly induced podocyte damage in vitro
Published in Renal Failure, 2022
Yanfen Li, Juntao Yu, Miao Wang, Zhao Cui, Ming-hui Zhao
The extracellular portion of PLA2R1 was cloned into the HA tag-CMV-14 expression vector and transfected to HEK293 cells. After 48 h, the selection antibiotic G418 (A1720, Sigma, St Louis, USA) 800 μg/mL was added to establish a stable cell line. To generate the recombinant protein, HEK293 cells, growing in DMEM containing 800 μg/mL G418 and 10% fetal bovine serum (FBS, GIBCO, CA, USA), were collected and lyzed using FBS-free DMEM containing 50 μg/mL ascorbic acid (1043003, Sigma). The cell lysate was precipitated overnight with 3 mol/L (NH4)2SO4 and centrifuged for 20 min at 14,000 g. The PLA2R protein was redissolved in PBS and incubated with Pierce Anti-HA Magnetic Beads (88837, Thermo Fisher Scientific, MS, USA) for 30 min at room temperature. The bound, HA-tagged proteins were dissociated from the beads using HA peptide (26184, Thermo).
A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction
Published in Xenobiotica, 2021
Han Li, Yu-Guang Wang, Zeng-Chun Ma, Gao Yun-Hang, Song Ling, Chen Teng-Fei, Zhang Guang-Ping, Yue Gao
HepG2 cells were seeded in 6-well culture plates at a density of 5 × 105 cells per well 24 h prior to transfection. The cells were then transfected with a mixture containing 500 ng of the appropriate reporter gene plasmid and 1 μg of the corresponding expression plasmid or with 500 ng of the pGLuc-basic2 empty vector and 1 μg of pcDNA3.1 myc/His as an internal control, using TransIT-X2 and following the manufacturer’s instructions. Twenty-four hours after transfection, the cells were trypsinised and sub-cultured into 24-well culture plates at a density of 1 × 105 cells per well and allowed to reach 60-80% confluency prior to removing medium and replacing with fresh medium containing 10% FBS and the antibiotic G418 (1000 μg/mL). The G418 selection concentration was previously determined by a cell death experiment. Medium was renewed every three days for one month until small colonies appeared. Ten clones from each transfection were further sub-cloned into 96-well culture plates to obtain monoclonal cells. All clones were chosen for induction testing by treating the appropriate cells with inducers (TCDD, CITCO, RIF) and assessing luciferase assay followed by quantitative PT-PCR for the appropriate CYP mRNA and western blotting for the appropriate CYP protein.
Silence of MEG3 intensifies lipopolysaccharide-stimulated damage of human lung cells through modulating miR-4262
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Xiaoliang Li, Qianqian Zhang, Zhigang Yang
Short-hairpin RNA directed against human MEG3 or Krüppel-like factor (KLF) 4 was sub-cloned into the pGPU6/Neo plasmid (GenePharma, Shanghai, China) to generate sh-MEG3 or sh-KLF4. The plasmid carrying a non-targeting sequence was used as a negative control (NC) of sh-MEG3 or sh-KLF4, termed sh-NC. For overexpression of KLF4, the full-length KLF4 sequences were ligated into pEX-2 plasmid (GenePharma, Shanghai, China) to generate pEX-KLF4, control of which was the empty pEX-2 plasmid, termed pEX. The Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) was utilized for cell transfection in line with the manufacturer’s instructions. G418 (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO) was added to the culture medium for the selection of stably transfected cells. G418-resistant cell clones were established after approximately 4 weeks. MiR-4262 mimic, scramble miRNAs, miR-4262 inhibitor and its NC were purchased from Life Technologies Corporation (Gaithersburg, MD). Cell transfection with miRNAs was also performed with Lipofectamine 3000 reagent, and cells were harvested at 72 h post-transfection in the subsequent experiments.