Pcdna3

Manufactured by Promega

Sourced in United States, China, Portugal, United Kingdom

pCDNA3.1 is a plasmid vector designed for high-level expression of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) immediate-early promoter for constitutive expression and a multiple cloning site for inserting the gene of interest.

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Lab products found in correlation

Pcdna3, by Thermo Fisher Scientific (14 mentions) Pgl3 basic, by Promega (7 mentions) Prl tk, by Promega (6 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Dual luciferase reporter assay system, by Promega (5 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (4 mentions) Fugene hd, by Promega (4 mentions) Giga prep kit, by Qiagen (4 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) Pcdna3.1 nfat2, by Promega (3 mentions) Click it plus 5 ethynyl 2 deoxyuridine edu flow cytometry assay kit, by Thermo Fisher Scientific (3 mentions) Pluronic f 127, by GeneCopoeia (3 mentions) Hrp conjugated secondary antibody, by Bioss Antibodies (3 mentions) C myc, by Cell Signaling Technology (3 mentions) Fitc annexin 5 apoptosis detection kit, by BD (3 mentions) Sirna, by RiboBio (3 mentions) Prl sv40, by Promega (2 mentions) Pgl3 basic vector, by Promega (2 mentions) Pcdna3.1 luc vector, by Promega (2 mentions) Reagents for cell culture, by Thermo Fisher Scientific (2 mentions)

65 protocols using pcdna3

Development of Dual-Luciferase Reporter Plasmids

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psiCHECK-2, a dual-luciferase reporter plasmid harboring firefly (F-Luc) and renilla (R-Luc) luciferase genes, was purchased
from Promega (Madison, WI, USA). psiCHECK-2 carrying R-Luc with Prm2 3’-UTR (R-Luc-Prm2 3’-UTR/psiCHECK-2), 6 × YH elements
(R-Luc-6 × YH/psiCHECK-2), and 6 × BoxB sequence (R-Luc-6 × BoxB/psiCHECK-2) were constructed as described in “Supplementary Materials and Methods.” To
facilitate the detection of the reporter mRNAs on Northern blots, F-Luc/pcDNA3 was produced by cloning F-Luc derived from pGL3-basic (Promega)
into pcDNA3 (Thermo Fisher Scientific, Waltham, MA, USA). Similarly, R-Luc from pGL4.73[hRluc/SV40] (Promega) was inserted into pcDNA3,
together with 6 × BoxB sequence, to generate R-Luc-6 × BoxB/pcDNA3. By the use of pcDNA3 vector, expression of the reporter mRNAs is driven by a strong
cytomegalovirus promoter.

OYAMA K., BABA T, & KASHIWABARA S.I. (2020). Functional characterization of testis-brain RNA-binding protein, TB-RBP/Translin, in translational regulation. The Journal of Reproduction and Development, 67(1), 35-42.

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Overexpression and Silencing of SEMA3B and SEMA3B-AS1 in Gastric Cancer

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For overexpression of SEMA3B and SEMA3B-AS1, the sequence of SEMA3B or SEMA3B-AS1 was synthesized and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). The BGC-823 and MGC-803 cells were transfected with SEMA3B or SEMA3B-AS1 expression plasmid (pcDNA3.1-SEMA3B or pcDNA3.1-SEMA3B-AS1) or the empty vector (pcDNA3.1-EV) as control at a final concentration of 2 ug/uL using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). After transfection, cells were incubated in antibiotic-free medium for 5 h, followed by replacement with fresh normal growth medium for a further incubation of 43 h by adding 800 µg/mL of G418 (Life Technologies, Carlsbad, CA, USA) to the supplemented culture medium. Resistant cell clones were isolated and expanded for further characterization. For inhibition of SEMA3B and SEMA3B-AS1, HGC-27 cells were respectively transfected with SEMA3B-or SEMA3B-AS1-specific shRNA plasmid using FuGENE HD Transfection Reagent, and a scrambled shRNA was used as a negative control. For overexpression of miR-6872-5p, BGC-823 and MGC-803 cells were transfected with miR-6872-5p mimic or mimic control (Ambion, Austin, TX, USA) at a final concentration of 25 nmol/L, and for downregulation of miR-6872-5p, HGC-27 cells were transfected with miR-6872-5p inhibitor or inhibitor control (Ambion, Austin, TX, USA).

Guo W., Liang X., Liu L., Guo Y., Shen S., Liang J, & Dong Z. (2019). MiR-6872 host gene SEMA3B and its antisense lncRNA SEMA3B-AS1 function synergistically to suppress gastric cardia adenocarcinoma progression. Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association, 22(4).

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Validating Zebrafish Gnrh3 Antibody Specificity

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The specificity of the zebrafish Gnrh3 Gap antibody was verified by confirming the recognition and specificity of the antibody to Gnrh3 expressed in a heterologous cell line and by co-localizing Gnrh3 immunoreactivity in gnrh3 transgenic adult brain sections (as described in next section), expressing tdTomato in gnrh3 neurons [13 (link)]. The entire zebrafish gnrh3 coding region (from 28 bp to 312 bp of NCBI Reference Sequence NM_182887.2) was cloned into the pcDNA3.1 (Life Technologies) mammalian expression vector under the control of the CMV promoter. The zebrafish gnrh3-pcDNA3.1 plasmid and the control pcDNA3.1 plasmid were transfected into COS7 cells with FuGENE 6.0 (Promega). As an additional control to check for cross-reactivity, we also transfected cells with the zebrafish gnrh2-pcDNA3.1 plasmid. The cells were grown in 25 cm² sterile cell culture flasks in DMEM supplemented with 10% FBS, 1% glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Biological Industries) at 37°C and 5% CO₂. After 48 hours of incubation, cells were transferred to chamber slides. After 24 hours, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 1 hour at room temperature and were washed with PBS. Blocking and immunostaining for zebrafish Gnrh3 Gap was then conducted as described in the next section.

Spicer O.S., Wong T.T., Zmora N, & Zohar Y. (2016). Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio) Reveals No Effects on Reproductive Performance. PLoS ONE, 11(6), e0158141.

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Klf4 Binding to miR-31 Promoter

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For the binding of Klf4 to the miR-31 promoter, the 1.5 kb region directly upstream of miR-31 transcription binding site was amplified by PCR and inserted into the pGL3 vector (Promega, San Luis Obispo, CA, USA). The Hep3B and Sk-hep-1 cells were co-transfected with either pcDNA3.1 (Promega) or the pcDNA3.1-Klf4 and miR-31 promoter. At 48 h after transfection, luciferase activity was analyzed according to the Dual-Luciferase reporter assay system (Promega), using a GloMax fluorescence reader (Promega).

Tian C., Yao S., Liu L., Ding Y., Ye Q., Dong X., Gao Y., Yang N, & Li Q. (2016). Klf4 inhibits tumor growth and metastasis by targeting microRNA-31 in human hepatocellular carcinoma. International Journal of Molecular Medicine, 39(1), 47-56.

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Cloning mutp53 into pcDNA3 vector

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The full-length coding sequences of mutp53 were PCR-amplified from pLLS89 yeast vectors (kindly provided by Dr Gilberto Fronza, from IST Istituto Nazionale per la Ricerca sul Cancro, Italy) with Vent DNA polymerase (New England Biolabs, Werfen, Porto, Portugal), using the primers pair 5′ GGG GTA CCA TGG AGG AGC CGC AGT CAG 3′ and 5′ CCG CTC GAG TCA GTC TGA GTC AGG CCC TTC 3′, where the restriction sites for KpnI/XhoI (in bold) were included, respectively. The PCR products and the mammalian expression vector pcDNA3 (Invitrogen, Alfagene, Lisbon, Portugal) were digested with KpnI/XhoI (New England Biolabs, Werfen, Carnaxide, Portugal), purified from agarose gel, and ligated with T4 DNA ligase (Promega, VWR, Carnaxide, Portugal), originating the expression vectors pcDNA3-mutp53. These constructs were propagated in NZY5α Escherichia coli cells (NZYTech, Lisbon, Portugal). The sequence of each mutp53 in the constructed vectors was confirmed by sequencing (Eurofins GATC Biotech, Konstanz, Germany) with specific pcDNA3 primers. pcDNA3-mutp53 vectors were extracted using the PureYield™ Plasmid Miniprep System kit (Promega, VWR, Carnaxide, Portugal).

Gomes S., Bosco B., Loureiro J.B., Ramos H., Raimundo L., Soares J., Nazareth N., Barcherini V., Domingues L., Oliveira C., Bisio A., Piazza S., Bauer M.R., Brás J.P., Almeida M.I., Gomes C., Reis F., Fersht A.R., Inga A., Santos M.M, & Saraiva L. (2019). SLMP53-2 Restores Wild-Type-Like Function to Mutant p53 through Hsp70: Promising Activity in Hepatocellular Carcinoma. Cancers, 11(8), 1151.

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Cloning and Sequence Verification of Mutant p53

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The full-length coding sequence of mutp53 was PCR-amplified from the yeast vector with Vent DNA polymerase (New England Biolabs, Werfen, Porto, Portugal), using the primers pair 5′ GGG GTA CCA TGG AGG AGC CGC AGT CAG 3′ and 5′ CCG CTC GAG TCA GTC TGA GTC AGG CCC TTC 3′, where the restriction sites for KpnI/XhoI (in bold) were included, respectively. The PCR products and the mammalian expression vector pcDNA3 (Invitrogen, Alfagene, Lisboa, Portugal) were digested with KpnI/XhoI (New England Biolabs, Werfen), purified from agarose gel, and ligated with T4 DNA ligase (Promega, VWR, Carnaxide, Portugal), originating the expression vectors pcDNA3-mutp53. These constructs were propagated in NZY5α E. coli cells (NZYTech, Lisboa, Portugal) as described [12] (link). The sequence of each mutp53 in the constructed vectors was confirmed by sequencing (Eurofins GATC Biotech, Konstanz, Germany) with specific pcDNA3 primers. pcDNA3-mutp53 vectors were extracted using the Pure-YieldTM Plasmid Miniprep System kit (Promega, VWR).

Gomes A.S., Ramos H., Gomes S., Loureiro J.B., Soares J., Barcherini V., Monti P., Fronza G., Oliveira C., Domingues L., Bastos M., Dourado D.F.A.R., Carvalho A.L., Romão M.J., Pinheiro B., Marcelo F., Carvalho A., Santos M.M.M, & Saraiva L. (2020). SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations. Biochimica et biophysica acta. General subjects, 1864(1).

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PON2 Overexpression in U87 and GBM8401 Cells

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The U87 and GBM8401 cells were transiently transfected with the PON2 cDNA construct in 6-cm cell culture dishes using serum free Opti-MEMi medium and Lipofectamine 2000 reagent following manufacturer's instructions (Invitrogen). After 24 h, the medium of the transfected cells was replaced with regular medium. Subsequently, the expression of PON2 protein in U87 and GBM8401 cells was examined using Western blot analysis. PON2 cDNA was constructed into pcDNA 3.0 expression vector (Promega), hence, pcDNA 3.0 (Promega) was used as vector control.

Tseng J.H., Chen C.Y., Chen P.C., Hsiao S.H., Fan C.C., Liang Y.C, & Chen C.P. (2017). Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression. Oncotarget, 8(9), 14666-14679.

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Validating miR-223 Promoter Binding

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A dual-luciferase reporter assay was performed to further validate the results of the predicted binding site of RORα with the miR-223 promoter. Luciferase reporter plasmids (pGL3-basic; Sangon Biotech Co., Ltd.) were constructed with full-length or truncated promoters of miR-223. In addition, the full-length RORα gene was cloned into the pcDNA3.0 (Sangon Biotech Co., Ltd.) plasmid to overexpress RORα. The pcDNA3.0 plasmid cloned with the RORα gene was co-transfected with the pGL3-basic plasmid cloned with the miR-223 promoter and Renilla luciferase reporter plasmids (Promega Corporation) into 293 cells at 37˚C for 24 h using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). After another 24 h, firefly and Renilla luciferase activities were measured in 293 cells using the Dual-Luciferase Reporter Assay system (Dual-Glo^®; cat. no. E2920; Promega Corporation). The sequences of the miRNA-223 mimics and inhibitor are presented in Table SI.

Yi S, & Yang Y. (2021). Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway. Experimental and Therapeutic Medicine, 22(6), 1392.

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Generating miR-23a Expression Vector and Reporter

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The expression vector for miR-23a (pc3-miR-23a) was generated by cloning genomic fragments encompassing the miR-23a precursor and its 5′- and 3′-flanking sequences into pcDNA3.0 (Invitrogen Life Technologies, Carlsbad, CA). The plasmid pc3-gab was produced based on pcDNA3.0 by replacing the neomycin open reading frame with an enhanced green fluorescent protein (EGFP) expression cassette. The PAK6 coding sequence was cloned into pc3-gab to generate an expression vector (pc3-gab-pak6).
To create a luciferase reporter construct, a 3cDNA3.0 by replacing the neomycin open reading frame with an enhanced green fluorescent protein (EGFP) expression cassette. The PAK6 coding sequence was cloned into pc3-gab to generate ahe complementary site for the seed region of miR-23a was generated using the fusion PCR method. 293T cells grown in a 48-well plate were cotransfected with 200 ng of pcDNA3.0 or 200 ng of pc3-miR-23a, 10 ng of firefly luciferase reporter containing the wild-type or mutant 3′-UTR of the target gene, and 2 ng of pRL-TK (Promega, Madison, WI).

Cai S., Chen R., Li X., Cai Y., Ye Z., Li S., Li J., Huang H., Peng S., Wang J., Tao Y., Huang H., Wen X., Mo J., Deng Z., Wang J., Zhang Y., Gao X, & Wen X. (2015). Downregulation of microRNA-23a suppresses prostate cancer metastasis by targeting the PAK6-LIMK1 signaling pathway. Oncotarget, 6(6), 3904-3917.

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Plasmid Constructs and Antibodies

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Plasmid IRF7-Flag was a gift from Niu Junqi (Jilin University). Plasmids IRF3-Flag, IRF7-Flag mutants, EV-D68 VP3-HA, EV-D68 VP3-Myc, EV-D68 VP3-Myc mutants, EV71 VP3-HA, CA16 VP3-HA, TRAF6-Flag, and TRAF6-V5 were constructed in the lab. TRAF6-Flag was bought from Miaoling Biology Company. Wang Jianwei, from the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences, kindly provided pGL3-IFN-α4-luc, ubiquitinating K63, IFN-β-luc, and ISRE-luc. Xiaofang Yu kindly provided VR1012. PcDNA3.0 and pRL-SV40 were purchased from Promega. All variants were confirmed by subsequent sequencing.
Antibodies against HA and Flag were purchased from ABclonal (catalog numbers AE008 and AE005, respectively); antibodies against Myc were purchased from Proteintech (catalog number 16286-1-AP); antibodies against EV-D68 VP3 were purchased from Gene Tex (catalog number GTX132315). Rabbit antibodies against IRF3, IRF7, and p-IRF7 were purchased from Cell Signaling Technology (lot numbers 11904P and 4920S, respectively). Goat anti-mouse IgG, goat anti-rabbit IgG, anti-tubulin monoclonal antibody, and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal antibody were purchased from Tianjin Sungene Biotech Co., Ltd. Anti-HA affinity matrix (catalog number 11815016001) was purchased from Roche, and anti-Flag affinity matrix (catalog number A2220) was purchased from Sigma.

Kang J., Huang M., Li J., Zhang K., Zhu C., Liu S., Zhou Z., Wang T, & Wang Z. (2023). Enterovirus D68 VP3 Targets the Interferon Regulatory Factor 7 To Inhibit Type I Interferon Response. Microbiology Spectrum, 11(3), e04138-22.

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