Open reading frames of the mouse GPR43 (Gene ID: 233079) gene were amplified by PCR and inserted in a pCD513B-1 plasmid (Public Protein/Plasmid Library) to construct a pCD513B-1-GPR43 expression vector (CMV promoter). PCR and restriction enzyme digestion were used to confirm a successfully constructed recombinant GPR43 overexpression vector. Transfection was done by a Lipofectamine®3000 RNAiMax reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions.
Lipofectamine 3000 rnaimax reagent
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Lipofectamine® 3000 RNAiMax reagent is a lipid-based transfection reagent designed for efficient delivery of small interfering RNA (siRNA) and other nucleic acids into a variety of mammalian cell types. The reagent is optimized for high transfection efficiency and low cytotoxicity.
Automatically generated - may contain errors
7 protocols using lipofectamine 3000 rnaimax reagent
1
Overexpression of Mouse GPR43 Gene
2
GPR43 Knockdown via siRNA Transfection
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siRNA targeting GPR43 (sense: 5′-CCAGCCTGGATCCATTATT-3′, antisense: 5′-AAUAAUGGAUCCAGGCUGG-3′) or control siRNA (sense: 5′-UUCUCCGAACGUGUCACGU-3′; antisense: 5′-ACGUGACACGUUCGGAGAA-3′) was synthesized by Ribo Biotech (Guangzhou, China). Transfections were performed using the Lipofectamine® 3000 RNAiMax reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. Experiments were performed with these cells at 24 h posttransfection.
3
Silencing TXNIP Gene Expression
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Control siRNA and TXNIP siRNA were synthesized by GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine® 3000 RNAiMax reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. The cells were collected for Western blotting and RT-PCR at 48 h post-transfection.
4
Silencing TXNIP gene expression
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Control siRNA and TXNIP siRNA were synthesized from GenePharma (Shanghai, China). Cell transfection was using Lipofectamine® 3000 RNAi Max reagent, as per manufacturer's instructions (Invitrogen, Karlsruhe, Germany). The cells were collected for western blotting and RT-PCR analysis at 48 h posttransfection.
5
Silencing TXNIP Gene Expression
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Control siRNA and TXNIP siRNA were synthesized by GenePharma (Shanghai, China). Cell transfection was performed using Lipofectamine® 3000 RNAiMax reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. The cells were collected for Western blotting and RT-PCR at 48 h posttransfection.
6
HIBCH mRNA Knockdown in IPEC-J2 Cells
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Three candidate RNA (siRNA) targeting the HIBCH mRNA coding region of and a negative control siRNA were designed and purchased from GenePharma, Shanghai, China, to determine the 3-HIB function. IPEC-J2 cells were seeded at 1 × 105 cells/well in six-well plates containing induction medium and cultured for 24 h. Transfection was conducted using Lipofectamine 3000 reagent RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols. We measured the specificity and effectiveness of IPEC-J2 cell siRNAs by determining the HIBCH protein expression after siRNA transfection for 48 h. siHIBCH-3 was the most expressed protein; thus, was selected for further analysis.
7
Silencing Nrf2 mRNA Expression
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A total of three candidate siRNAs targeting the coding region of Nrf2 mRNA and an NC siRNA (Table S1 ) were synthesized by Genepharma (Shanghai, China). The specificity and effectiveness of the PMEC siRNAs were evaluated by determining Nrf2 protein expression after siRNA transfection for 48 h. The siNrf2‐2 was found to be the most effective and thus selected for further analysis (60% silence, Figure S1 ). Transfection was conducted with Lipofectamine 3000 reagent RNAiMAX (Invitrogen, Carlsbad, CA, USA) by following the manufacturer's protocols.
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