Tag-free expression plasmids of zebrafish LGP2 (DrLGP2) (29 (link)) and human LGP2 (HsLGP2) were made by insertion of corresponding ORFs into pcDNA3.1(+) vector (Invitrogen). The ORF of human LGP2 is amplified from an expression plasmid provided by Professor Jin Zhong from Institute Pasteur of Shanghai, Chinese Academy of Sciences (37 (link)). Similarly, Tag-free plasmids of human MDA5 (HsMDA5) and RIG-I (HsRIG-I) were generated by inserting their ORFs into EcoRV site of pcDNA3.1(+) vector (Invitrogen). HA-tagged plasmids (DrLGP2-HA, HsLGP2-HA, HsIRF3-HA) were generated by inserting the ORFs into EcoRV site of pcDNA3.1(+) vector (Invitrogen) that had preexisted a HA coding sequence into NotI site. DrMDA5, DrRIG-I, DrIRF3-HA were described previously (38 (link), 39 (link)). Crucian carp IFNpro-luc (CaIFNpro-luc) and zebrafish IFNφ1pro-luc (DrIFNφ1pro-luc) were reported previously (23 (link), 24 (link)). Human IFNβpro-luc (HsIFNβpro-luc) was kindly provided by Professor Hongbin Shu from Wuhan University (40 (link)).
Pcdna3.1 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Japan, Canada, United Kingdom, Spain
The PcDNA3.1 vector is a plasmid DNA-based expression vector used for the expression of proteins in mammalian cell lines. It contains the human cytomegalovirus (CMV) immediate-early promoter, which drives the expression of the gene of interest. The vector also includes an ampicillin resistance gene for selection in bacteria and a neomycin resistance gene for selection in mammalian cells.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (440 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (182 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (117 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (56 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (45 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (35 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (31 mentions)
Pcdna3, by Thermo Fisher Scientific (24 mentions)
Si nc, by GenePharma (23 mentions)
Opti mem, by Thermo Fisher Scientific (22 mentions)
Sh nc, by GenePharma (20 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (20 mentions)
Dual luciferase reporter assay system, by Promega (20 mentions)
Mir nc, by GenePharma (19 mentions)
Pmirglo vector, by Promega (19 mentions)
Mimic nc, by GenePharma (19 mentions)
Pgl3 basic vector, by Promega (19 mentions)
Si nc, by RiboBio (18 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (18 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (17 mentions)
1 649 protocols using pcdna3.1 vector
1
Plasmid Construction for Immune Genes
2
STXBP5-AS1 Cloning into pcDNA3.1(+) Vector
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pcDNA3.1 (+) vector was purchased from Thermo Fisher Scientific. STXBP5-AS1 DNA was obtained by PCR using cDNA, Pfu DNA polymerase and synthetic oligonucleotide primers incorporating restriction sites. PCR products were ligated into the pcDNA3.1 (+) vector according to the manufacturer’s protocol (Thermo Fisher Scientific) and then sequenced to confirm. Primer sequences were as follows (sequence from 5′ to 3′): STXBP5-AS1 Forward: GGAGTGGGAGTGTG AGGAGAA; Reverse: AAGATCCCTGTGGCAAAATCCC.
3
Modulating Cardiac H/R Injury via miR-142-3p and RUNX1/DRD2
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Lipofectamine® 3000 (Invitrogen) was used in transfection. Negative control (NC) mimic (cat. no. miR1N0000001-1-5) and miR-142-3p mimic (cat. no. miR10000155-1-5) were obtained from Guangzhou RiboBio Co., Ltd. NC mimic or miR mimic at a final concentration of 50 nM were transfected to a H9c2 cell-seeded 6-well plate at 37˚C for 4 h. The complementary DNAs (cDNAs) of RUNX1 and DRD2 were subcloned to pcDNA3.1 vector (Invitrogen: Thermo Fisher Scientific, Inc.), and 2 µg/well of these cDNAs were transfected into a H9c2 cell-seeded 6-well plate at 37˚C for 4 h. The control (without H/R injury) and H/R groups were transfected with pcDNA3.1 vector as the negative control. Subsequent experiments were performed 48 h after transfection.
4
Regulation of Ki-67 in Breast Cancer Cells
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The cultivated cells were seeded into 6-well plates of 2×105 cells/well and incubated for 12 h. At 70–80% confluence, the cells were divided into 5 groups (MCF-7: blank, mimic control, miR-519d mimic, inhibitor control and miR-519d inhibitor; MDA-MB-231: blank, mimic control, miR-328-3p mimic, inhibitor control and miR-328-3p inhibitor) and the HiPerFect Transfection Reagent (Qiagen, USA) was selected to complete the transfection process. Briefly, 150 ng miRNA oligonucleotides and 4 μL reagent were added into 100 μL RPMI-1640 and mixed well. The mixture was then transferred into the cell culture medium. 48 h after transfection, cells were collected for the subsequent detections.
The 70 bp Ki-67 cDNA of the 3ʹUTR region with the whole coding sequence was constructed into the pcDNA3.1 vector (ThermoFisher, USA). The breast cancer cell lines were transfected with pcDNA3.1 vector containing Ki-67 or empty vector via Lipofectamine 3000 as purchased from the supplier (ThermoFisher, USA). 48 h after incubation, the cells were harvested and sent to subsequent experiments.
The 70 bp Ki-67 cDNA of the 3ʹUTR region with the whole coding sequence was constructed into the pcDNA3.1 vector (ThermoFisher, USA). The breast cancer cell lines were transfected with pcDNA3.1 vector containing Ki-67 or empty vector via Lipofectamine 3000 as purchased from the supplier (ThermoFisher, USA). 48 h after incubation, the cells were harvested and sent to subsequent experiments.
5
Overexpression and Silencing of SOX4 by miR-129-5p
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MiR-129-5p mimics, SOX4 cDNA, SOX4 siRNA1 and SOX4 siRNA2 were obtained from RiboBio Co., Ltd. (Guangzhou, China). PcDNA3.1 vector (Thermo Fisher Scientific, Waltham, MA) was employed to generate miR-129-5p, SOX4, SOX4 siRNA1 or SOX4 siRNA2 expression plasmid, and the empty PcDNA3.1 vector was as a negative control. Transfection to SW1353 and JJ012 cells was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's protocol.
6
Modulating lncRNA GAS5 and TIMP2 in MG-63 Cells
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The specific short-hairpin RNA (shRNA) against human lncRNA GAS5 was cloned into pENTR TM / U6 plasmid (GenePharma, Shanghai, China), and referred as sh-GAS5. A non-targeting shRNA (shNC, GenePharma) was used as a negative control. The full-length of lncRNA GAS5 sequence was transfected into pcDNA™ 3.1 vector (ThermoFisher, Scientific, China) and referred as pc-GAS5. The empty pcDNA3.1 vector was used as a negative control. miR-203a mimic, inhibitor or their respective scramble controls were synthesized by RiboBio (Guangzhou, China). TIMP2 specific siRNA (si-TIMP2) and siRNA negative control were purchased from Santa Cruz (Santa Cruz Biotechnology, USA). For TIMP2 overexpressing, the full-length of TIMP2 sequence was transfected into pcDNA™ 3.1 vector (ThermoFisher, Scientific, China) and referred as pc-TIMP2. All transfections were performed by using Lipofectamine 3000 reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer's protocol. The stably transfected MG-63 cells were selected by using the culture medium containing 0.5 mg/ml G418 (Sigma-Aldrich, St Louis, MO, USA). The stable transfected cells were selected for the subsequent experiments.
7
Modulating SNHG5 and miR-205 in Vascular Smooth Muscle
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Small interfering RNA (siRNA) of SNHG5 (si‐SNHG5), siRNA of NC, mir‐205‐5p mimics and NC were designed by RiboBio. si‐SNHG5 and mir‐205‐5p mimics were cloned into the promoter CMV (Cytomegalovirus) expression vector (Invitrogen) following the manufacturer's instructions, and then transferred to large artery smooth muscle cells. All SNHG5 knockout constructs were acquired from RiboBio. SNHG5 overexpression plasmid (pcdna‐SNHG5) was constructed by cloning the full‐length complementary DNA (cDNA) sequence of SNHG5 into pcDNA3.1 vector (Invitrogen). mir‐205‐5p overexpression plasmid (pcdna‐mir‐205‐5p) was obtained by cloning the full‐length cDNA sequence of mir‐205‐5p into pcDNA3.1 vector (Invitrogen). si‐mir‐205‐5p and SMAD4 mimics were cloned into promoter CMV (Cytomegalovirus) expression vector (Invitrogen) and transfected into arterial smooth muscle cells following the manufacturer's instructions.
8
Linc00511 Silencing and miRNA Modulation in Cellular Experiments
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Linc00511 small interfering RNAs (si-linc00511 #1 and si-linc00511 #2) and the corresponding siRNA control (si-nc), miR-126-5p or miR-218-5p mimics/inhibitors and their corresponding controls (miR-NCs) were synthesized by GenePharma (Shanghai, China). The Linc00511 and COL1A1 sequences were inserted into the pcDNA3.1 vector (Invitrogen) to overexpress Linc00511 and COL1A1 (pcDNA3.1-linc00511, oe-linc00511; pcDNA3.1-COL1A1, COL1A1), and the empty pcDNA3.1 vector was applied as the negative control (oe-nc; vector). Cells were seeded (5 × 104 cells/well) in 24-well plates. Transfection was performed with Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. RT–qPCR analysis was performed to assess the transfection efficiency. Forty-eight hours after transfection, cells were harvested and used in subsequent experiments.
9
ING1b Fragment Analysis in Cells
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All expression constructs for ING1b fragment analysis were generated using pcDNA3.1+ vector (Invitrogen, Waltham, MA, USA). The vector was modified by replacing the original MCS with an IRES:mCherry cassette. A new MCS was introduced upstream of the expression cassette and used for cloning ING1b fragments. Introducing the IRES element allowed bicistronic expression of ING1b fragments and mCherry tracer in the same cell, permitting efficient isolation of transfected cells by flow cytometry. For localization experiments, GFP-fused and FLAG-tagged ING1b fragments were cloned into pcDNA3.1+ vector (Invitrogen). For transfections, cells were seeded in six-well tissue culture plates 16 h before transfection. The next day, HEK-293 cells at 80% confluence were transfected using TransIT-293 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer's protocol. HeLa cells were transfected using Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer's protocol. Depending on the experiment, expression levels of transfected constructs were analyzed 24 and/or 48 h after transfection.
10
Cloning and Tagging of Dm5-ht2b Receptor
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An expression-ready construct of Dm5-ht2b was generated in pcDNA3.1 vector (Invitrogen/ThermoFisher Scientific, Darmstadt, Germany). PCR was performed with specific primers (sense primer: 5′-AATAAGCTT CCACCATGGAAGAGGATGTGTATGCC-3′; antisense primer first-round PCR: 5′-TGGGACGTCGTATGGGTATCTGCTCGGTCGCCAGG-3′; antisense primer second-round PCR: 5′-TTTTCTAGACTC GAG TTAAGCGTAGTCTGGGACGTCGTATGGGTA-3′). PCR products were digested with HindIII and XhoI, and subcloned into pcDNA3.1(+) vector (Invitrogen). Thus, the resulting construct contained the Kozak consensus motif (CCACC, Kozak, 1984 (link)) immediately 5′ to the ATG-codon and a hemagglutinin A (HA) epitope tag (amino acid sequence: YPYDVPDYA) at the 3′ end of the Dm5-ht2b cDNA and was named pcDm5-ht2b-HA. The insert fragment was checked by DNA sequencing.
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