The Optimization of Transfection Conditions for Rat BHK Cells and Chinese Hamster BRL Cells
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Objective:To optimize the Lipofectamine 2000 transfection conditions for rat BHK cells and Chinese hamster BRL cells using β-Gal vectors.Methods: Lipofectamine 2000 were mixed in varied ratios with plasmid DNA to transfect the rat BHK cells and Chinese hamster BRL cells on a 96-well plate.Successfully transfected cells were stained blue due to expression of β-Gal degradable substrates(X-gal),enabling estimation of the transfection efficacy by counting the blue cells under microscope.ANOVA was performed for statistical test.Results: The maximal efficacy was(43.2±9.3)% in BHK cells when β-Gal vector and Lipofectamine 2000 were mixed in a ratio of 0.27∶0.8(μg∶μL),and was(5.7±1.3)% in BRL cells when β-Gal vector and Lipofectamine 2000 were mixed in a ratio of 0.10∶0.3(μg∶μL).Conclusion: Lipofectamine 2000 appears to be useful for the transfection of mammal cells;however,the transfection conditions should be optimized for individual cell types.Keywords:
Lipofectamine
Chinese hamster
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HEK 293 cells
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Cationic liposome-based reagents provide reliable and simple protocol for in vitro transfection in a broad
range of mammalian cell types. High transfection efficacies and low cytotoxicity of a liposome-based transfection
reagent was required to retain adequate viability and high level of transgene of transfected Chinese hamster ovary
(CHO) cells for subsequent assay. Three commercial liposome-based transfection reagents were utilized and
compared for their aptitude to transfect in-vitro CHO cells with reporter gene expressing the red fluorescent
(pDsRed-N1) and fusion pDsRed-N1 plasmid containing human cDNA Coxsackie and Adenovirus Receptor
(pCAR-DsRed). Xtreme HP Plasmid DNA transfection reagent (Roche) gave the highest transfection efficiencies
compared to other tested reagents. The optimal Xtreme HP Plasmid DNA reagent had shown its higher transfection
efficiency (39.25±1.00% MFI) and stable increment of DsRed-proportion cells with incubation period (24-72
hours), allowing sufficient yield and shortened the selection stably clone of successful transfected CHO-CARDsRed.
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pAcGFP-bFADD plasmid with AcGFP report gene was transfected to Hela cell mediated by lipofectamine 2000, and the transfection condition was optimized by adjusting transfection time and concentrations of plasmid DNA and liposome.Twelve hours after transfected, positive cells count was made under fluorescence microscope, and the cell viability was detected by MTT.Results indicated that perfect cell transfection efficiency(62.3% ) was obtained on the condition of the complex of 2.4 μg pAcGFP-bFADD and 3.0 μL lipofectamine 2000 transfecting Hela cell after 6 h, meanwhile, its cell viability could reach 83%.When the concentration of plasmid DNA and liposome increased to 3.2 μg and 6.0 μL respectively, the complex would bring higher toxicity, the inhibition rate reached 42.98%, the difference was extremely significant(P 0.01) compared with control group.After condition optimization, AcGFP-bFADD fusion gene got the expression peak 72 h after transfection.The green fluorescent positive cells ratio could reach 64%.
Lipofectamine
HeLa
Viability assay
MTT assay
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We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.
Lipofectamine
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Objective To lay the foundation of further studies on LMP1 expression in cells A20 and its role by exploring the optimal method for transfecting murine B lymphoma cell line A20.Methods The murine B lymphoma A20 cells were transfected with recombinant plasmid pCDF-LMP1 containing a green fluorescent protein reporter gene by lipofection,nucleofection,or lentiviral vector-mediated transfection.The intracellular distribution of green fluorescence was observed by fluorescence microscopy.The transfection rate of A20 cells was then compared among the three different methods.Results The transfection rate was the lowest in lipofection,with only a few cells with green fluorescence;approximately 10% in nucleofection;and the highest in high-titer lentiviral transfection,reaching up to 80%.Conclusions High-titer lentiviral vector-mediated transfection is the best method for transfecting the suspended A20 cells.It is worth a popularization and provides references for stable transfection of those cells extremely difficult to be transfected.
Nucleofection
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Objective:To compare the efficiency of three widely used transfection reagents.Methods:The cell line C2C12was used as host cell and plasmid pcDNA3.1+CAT containing CAT reporter gene was used as target DNA.10 4 cells per well were seeded in24-well plate and cultured for24h before transfection,and then1.5μg plasmid DNA was transfected with Ca 3 (PO 4 ) 2 ,lipofectamine2000and chitosan,which are used as vectors.The cells were hyˉdrolyzed48h after transfection by freezing and defrosting repeatedly.Relative CAT content in the cell lysate was determined by ELISA.Results:The transfection efficiency of these three non-viral vectors was evaluated on the basis of CAT content.The highest CAT content was found in the cells transfected by liposome.Conclusion:The commercial liposome has the highest transfection efficiency,chitosan,running next to liposome,is relatively better than Ca 3 (PO 4 ) 2 .
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To construct a highly efficient system of transfected somatic cells mediated by liposome, pCMV-EGFP plasmid coated with Transfast~(TM) reagent, was transferred into the goat fetal fibroblasts at passage from 5 to 13. Such parameters affecting transfection as DNA amount, cell confluency, serum, transfection time, and the ratio of liposome and DNA were analyzed, and transfection efficiency of the cells was assessed by flow cytometer(FCM). It was found that the highest efficiency was achieved with 0.75μg DNA, the ratio of liposome and DNA at 1∶1, a 3 h transfection interval, 50% confluent cells, in the absence of serum, demonstrating by the transfection efficiency of 40.7%±5%. Under the conditions of 4h transfection interval, 80% confluent cells, in the presence of serum led to lower efficiency. The transfection efficiency was increased in a DNA dose-dependent manner, but the viability of the transfected fibroblasts was somewhat reduced significantly. These results suggest that Transfast~(TM) technology can transfer exogenous genes into goat fibroblasts effectively and safely.
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Objective To investigate the optimum transfection conditions of cationic liposome transfected CT26 cells(murine colon cancer cells),thus to provide reference for functional identification of spleen deficiency-related gene RPS20 using RNA interfering methods in mice.Methods We transfected pcDNATM6.2-GW/EmGFP-miR-neg Control plasmid into CT26 cells which planted in six-well plate with cationic liposome,and observed the effect of plasmid and Lipofectamine2000 in various proportions on transfection efficiency by detecting fluorescence expression using fluorescence microscopy and flow cytometry.Results The results of detecting transfection efficiency by fluorescence microscope showed that plasmid 4.0μg and 6.0μg had no significant difference in transfection efficiency.The results of flow cytometry showed that the highest transfect efficiency(average being 25.3%)was obtained in the group of Lipofectamine2000 15.0μL+plasmid 4.0μg.Conclusion The optimum transfection condition is tranfection into CT26 cells with plasmid 4.0μg+Lipofectamine2000 15.0μL in six-well plate.
Cytometry
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