Effectene method

Manufactured by Qiagen

The Effectene method is a transfection reagent used for the introduction of nucleic acids into eukaryotic cells. It facilitates the delivery of DNA, RNA, or other molecules into the target cells.

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Lab products found in correlation

Gateway technology, by Thermo Fisher Scientific (1 mentions) Hiperfect, by Qiagen (1 mentions) Dual luciferase assay kit, by Promega (1 mentions) Palter in vitro mutagenesis system, by Promega (1 mentions) Norepinephrine, by Merck Group (1 mentions) Claycomb medium, by Merck Group (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

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Effectene (695 citations)

5 protocols using effectene method

Generating Stable DAT Mutant Cell Lines

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Porcine aortic endothelial (PAE) cells were grown in F12 medium with 10%
fetal bovine serum (FBS). Effectene method (Qiagen, Valencia, CA) was used for
DNA transfection of PAE cells. Clonal pools of PAE cells stably expressing DAT
mutants were generated by G418 selection for 2–3 weeks. These pools of
G418-resistant cells contained cells not expressing YFP-HA-DAT and 10–20%
cells expressing various levels of YFP-HA-DAT mutants. The cells were grown on
glass coverslips for direct imaging of YFP in fixed cells, or on 12-well tissue
culture plates for biochemical experiments.

Cheng M.H., Ponzoni L., Sorkina T., Lee J.Y., Zhang S., Sorkin A, & Bahar I. (2019). Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations. Neuropharmacology, 161, 107676.

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Functional Analysis of SCN5A Mutation

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The full-length wild-type (WT) human SCN5A cDNA (GenBank ID: NM198056) was subcloned into pcDNA3.1 vector for mammalian expression in the Pathophysiology Laboratory of Fuwai Hospital (Invitrogen; Thermo Fisher Scientific, Inc.). The mutation (C280Sfs*61) was constructed using a QuikChange site-directed mutagenesis kit (Stratagene; Agilent Sumitomo Dainippon Pharma Co., Ltd.) on the WT-SCN5A background, and verified by direct sequencing. 293 cells were transfected with 0.6 µg cDNA of WT or mutant channels using the Effectene method (Qiagen GmbH) (19 (link),20 (link)). 293 cells were cultured in DMEM containing l00 ml/l fetal bovine serum. Cells were spread in 6-well plates 24 h before transfection and the density of cells was 70-80% at the time of transfection. The cells were transfected into pEGFP-SCN5A, pEGFPI.100lQSCN5A and pEGFP-SCN5A/pEGFP-L100lQ SCN5A (1:1) plasmids respectively according to the instructions of I. ipofectaIIIi- neTM2000 and then incubated at 37˚C with 50 ml/l CO₂ for 48 h. Then, subsequent experiments were performed. In the coexpression experiments, WT and mutant were transfected at a 1:1 molar ratio. Enhanced green fluorescent protein gene (0.2 µg) was co-transfected and served as an indicator. All experiments were performed 24-48 h after transfection. Over three independent experiments were conducted to confirm the reproducibility of the results.

Zhou X., Ren L., Huang J., Zhang Y., Cai Y, & Pu J. (2023). Novel SCN5A frame‑shift mutation underlying in patient with idiopathic ventricular fibrillation manifested with J wave in inferior lead and prolonged S‑wave in precordial lead. Experimental and Therapeutic Medicine, 25(6), 287.

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Knockdown and Overexpression Experiments

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For knockdown experiments, cells were transiently transfected with 25 nM siRNA (Supplementary Table S1) using the HiPerFect method (Qiagen, Hilden, Germany) according to the manufacturer's directions. For plasmid transfection, the Effectene method (Qiagen) was used according to the manufacturer's manual. Applying the GATEWAY technology (Invitrogen, Carlsbad, CA, USA), the RACGAP1 complementary DNA (cDNA) sequence (EU176264) was cloned from the pENTR221 vector into the destination vector pT-REx-DEST30. The empty pT-REx-DEST30 vector and the LacZ expression vector, pT-REx/GW30/LacZ (Invitrogen), served as controls. For the expression of Myc-tagged HDAC11, the MYC tag sequence (MEQKLISEEDL) was N-terminally inserted into the expression vector pcDNA3.1 carrying the wild-type HDAC11 sequence.^{19 (link)} The correct sequences of all inserts were verified by DNA sequencing (GATC, Konstanz, Germany).

Thole T.M., Lodrini M., Fabian J., Wuenschel J., Pfeil S., Hielscher T., Kopp-Schneider A., Heinicke U., Fulda S., Witt O., Eggert A., Fischer M, & Deubzer H.E. (2017). Neuroblastoma cells depend on HDAC11 for mitotic cell cycle progression and survival. Cell Death & Disease, 8(3), e2635-.

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Generation of KCNH2 Minigene Luciferase Reporter

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The minigene luciferase reporter construct was generated by subcloning the Renilla luciferase gene downstream of a splicing competent KCNH2 minigene composed of KCNH2 genomic DNA from exon 8 to exon 11. The construction of the KCNH2 minigene has been previously described [11 (link)]. The N-terminus of the minigene was tagged by the Myc epitope, which was inserted in-frame with the KCNH2 and luciferase translation sequence. Expression of the minigene luciferase reporter is driven by a CMV promoter. The vector also contains the firefly luciferase gene driven by the SV40 promoter, which was used as a control for transfection efficiency. The deletion and mutations of U/GU-rich elements downstream of the KCNH2 intron 9 poly(A) signal were performed using the pAlter in vitro mutagenesis system (Promega, Madison, WI). HEK293 cells were transiently transfected with the minigene luciferase reporter construct using the Effectene method (Qiagen, Valencia, CA). After 24 h, cells were harvested and assayed for both firefly and Renilla luciferase activity using the Dual-Luciferase assay kit (Promega). Data were analyzed by normalizing Renilla luciferase activity to firefly luciferase activity and presented as mean ± SEM.

Gong Q., Stump M.R, & Zhou Z. (2014). Upregulation of Functional Kv11.1 Isoform Expression by Inhibition of Intronic Polyadenylation with Antisense Morpholino Oligonucleotides. Journal of molecular and cellular cardiology, 0, 26-32.

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Stable Expression of KCNH2 Gene Constructs

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The full-length and short KCNH2 gene constructs were stably expressed in Flp-In HEK293 cells by co-transfection of the constructs (0.1 µg) with the Flp recombinase expression vector pOG44 (0.9 µg) using the Effectene method (Qiagen) and selected with 100 µg/ml hygromycin. The Flp-In HEK293 cells contain the FRT site at a single genomic locus, allowing stable integration of a single copy of the KCNH2 gene via Flp recombinase-mediated DNA recombination at a specific genomic location in all cell clones. HA epitope-tagged short KCNH2 gene constructs were transiently transfected into HEK293 cells or HL-1 cardiomyocytes by Lipofectamine 2000 (Invitrogen). Flp-In HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. HL-1 murine cardiomyocytes were cultured in Claycomb medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum and 0.1 mmol/L norepinephrine (Sigma-Aldrich).^{15 (link)}

Gong Q., Stump M.R., Deng V., Zhang L, & Zhou Z. (2014). Identification of Kv11.1 Isoform Switch as a Novel Pathogenic Mechanism of Long QT Syndrome. Circulation. Cardiovascular genetics, 7(4), 482-490.

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