pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin

Chong, Kevin Wai Yin; Lee, Alan Yiu Wah; Koay, Evelyn S C; Seet, Sze Jee; Cheung, Nam Sang

doi:10.1016/j.jneumeth.2006.05.017

Cited by 12 publications

(12 citation statements)

References 14 publications

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“…N1E-115 cells were transfected with the indicated plasmids as previously described (40). For differentiation, N1E-115 cells were starved for 24 hours in serum-free Neurobasal medium (Invitrogen) supplemented with 1% L-glutamine and 1% penicillin/ streptomycin.…”

Section: Practical Notes For Using the Fret Sensor Construction Kitmentioning

confidence: 99%

A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space

et al. 2013

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Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

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Section: Practical Notes For Using the Fret Sensor Construction Kitmentioning

confidence: 99%

A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space

et al. 2013

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“…Cationic Lipofectamine® 2000 and anionic DNA must interact electrostatically in order to form complexes, and the state of protonation in the transfection system is an important rate-limiting step [38]. Furthermore, in gaining entry to the cell, positively charged complexes interact with the relative negative charge of the plasma membrane; entry of negatively charged complexes is prevented [39].…”

Section: Discussionmentioning

confidence: 99%

“…This study has found the optimum pH for transfection of the HaCaT cell line, under the conditions used, to be pH 7. The optimum pH for transfection has been reported by other authors to be pH 9 [22], pH 8 [38], and pH 7 [39]. Optimum pH may, however, vary according to the liposome vector and to the solution, as well as cell line factors.…”

Section: Discussionmentioning

confidence: 99%

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

Teagle¹,

Birchall

Hargest³

2016

Skin Pharmacol Physiol

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Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent.

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“…For differentiation, N1E-115 cells were starved for 24 h in serum-free Neurobasal medium (Invitrogen) supplemented with 1% l-glutamine and 1% penicillin/streptomycin. For the double siR-NA-mediated KD and plasmid transfection, cells were transfected as previously described (Chong et al, 2006), using 400 ng of the plasmid pcDNA-Lifeact-GFP-IRES-NLS-mCherry and 20 pmol Stealth Select siRNAs (Invitrogen). 1 µl of Transfectin (Bio-Rad) was used as transfection reagent.…”

Section: Accession Numbers the Accession Number For The Pcdna Lifeacmentioning

confidence: 99%

“…For differentiation, PC12 Neuroscreen-1 cells were starved for 24 h in DMEM with 1% l-glutamine supplemented with 1% glucose, 1% penicillin/streptomycin, and 1% horse serum; 50 ng/ ml NGF was added to the medium to induce differentiation. For PC12 transfection, cells were transfected as previously described (Chong et al, 2006), using 400 ng of the plasmid pcDNA-Lifeact-GFP-IRES-NLS-mCherry. 0.8 µl of Transfectin (Bio-Rad) was used as transfection reagent.…”

Section: Accession Numbers the Accession Number For The Pcdna Lifeacmentioning

confidence: 99%

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Fusco¹,

Lefort²,

Smith³

et al. 2016

J Exp Med

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Abbreviations used in this paper: CV, computer vision; FRET, Förster resonance energy transfer; GAP, GTPase-activating protein; GEF, guanine nucleotide-exchange factor; HDS, hierarchical data structure; HS, high stringency; KD, knockdown; LS, low stringency; MDS, morphodynamic signature; MLCK, myosin light chain kinase; MP, morphogenetic process; MRCK, myotonin-related dystrophyn kinase myosin light chain kinase; pMLC, myosin light chain phosphorylation.

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pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin

Cited by 12 publications

References 14 publications

A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space

A Versatile Toolkit to Produce Sensitive FRET Biosensors to Visualize Signaling in Time and Space

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

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