Pegfp n2 vector

Manufactured by Takara Bio

Sourced in United States

The PEGFP-N2 vector is a plasmid designed for the expression of proteins fused to the green fluorescent protein (GFP) in mammalian cells. The vector contains the immediate early promoter of the cytomegalovirus (CMV) to drive high-level expression of the fusion protein. The PEGFP-N2 vector is commonly used for live-cell imaging and protein localization studies.

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18 protocols using pegfp n2 vector

Cloning and Expressing Axolotl and Human Tig1 Proteins

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For cell localisation studies, A. mexicanum (Am) Tig1 and H. sapiens Tig1 were amplified and cloned into a pEGFP-N2 vector (Clontech) in which the eGFP sequence was removed and a myc tag added in either C- or N- terminal positions, using standard cloning techniques. For expression studies, Am TIG1 or Prod1 were cloned into a pEGFP-N2 vector (Clontech), replacing the EGFP sequence. The p53-N2 vector was previously described (Yun et al., 2013). The Am TIG1^P155A mutant version was generated using a site-directed mutagenesis kit (New England Biolabs). For baculovirus production, Am Tig1^wt and Am Tig1^P155A cDNA sequences were coupled with a T2a element followed by a reporter gene, EGFP or mCherry, and inserted into a pOEMGED baculovirus-rescuing vector (Oliveira et al. 2018). Two fragments corresponding to Tig1 gene and T2a-mCherry sequences were amplified from pN2-TIG1-myc vectors using standard PCR techniques, and integrated into a BmtI and EcoRI-HF-digested pOEMGED plasmid via Gibson assembly method (NEB), with 1:3:3 plasmid to amplicon ratio. The Tig1-T2a-EGFP cassette was then transferred to a small transfection vector derived from a SacI and SphI-HF – digested pGEM-T plasmid and ligated using classic cloning techniques. The same strategy was used to generate pOEMGED and pGMT-derived vectors containing the Tig1^P155A sequence.

Oliveira C.R., Knapp D., Elewa A., Gerber T., Gonzalez Malagon S.G., Gates P.B., Walters H.E., Petzold A., Arce H., Cordoba R.C., Subramanian E., Chara O., Tanaka E.M., Simon A, & Yun M.H. (2022). Tig1 regulates proximo-distal identity during salamander limb regeneration. Nature Communications, 13, 1141.

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Evaluating FANCA Plasmid Transfection

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U2OS cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). U2OS cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin–neomycin (PSN) antibiotic mixture (Gibco) at 37 °C with 5% CO₂. To evaluate the transfection efficiency, FANCA plasmids were co-transfected with pEGFP-N2 vector (Clontech) into U2OS cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. In experiments evaluating cell sensitivity to ICL damage, 2 μM mitomycin C (MMC; Sigma) was added into the culture medium 24 h after transfection.

Yang X., Zhang X., Jiao J., Zhang F., Pan Y., Wang Q., Chen Q., Cai B., Tang S., Zhou Z., Chen S., Yin H., Fu W., Luo Y., Li D., Li G., Shang L., Yang J., Jin L., Shi Q, & Wu Y. (2019). Rare variants in FANCA induce premature ovarian insufficiency. Human Genetics, 138(11), 1227-1236.

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Tagging and Truncation of TDP1 and PRMT5

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Human flag-tagged full-length TDP1 (FLAG-TDP1^WT), His-tagged and green fluorescent protein (GFP)-tagged TDP1 constructs were described previously (10 (link),19 (link)). The FLAG-PRMT5 fusion construct was a kind gift from Dr Shilai Bao (Institute of Genetics and Developmental Biology, CAS, China). The flag-tagged N-terminal (1–293 aa) and C-terminal (294–637 aa) truncated PRMT5 and GFP-tagged N-terminal (1–185 aa) truncated TDP1 constructs were generated by polymerase chain reaction amplification using full-length PRMT5 or full-length TDP1 (FLAG-TDP1^WT) as template and were cloned in the mammalian expression vectors pCMV-Tag2 (Stratagene, La Jolla, CA, USA) or pEGFP-N2 vector (CLONTECH) respectively. The following point mutations: TDP1^R361K, TDP1^R586K, TDP1^{R361K, R586K} in FLAG and GFP tagged TDP1 constructs as well as His-TDP1^R361K,R586K were created using the ‘QuickChange’ protocol (Stratagene, La Jolla, CA, USA). All PCR-generated constructs were confirmed by DNA sequencing.

Rehman I., Basu S.M., Das S.K., Bhattacharjee S., Ghosh A., Pommier Y, & Das B.B. (2018). PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes. Nucleic Acids Research, 46(11), 5601-5617.

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Constructing EGFP Fusion Vectors of Bovine Immune Regulators

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In order to construct EGFP fusion expression vectors of bovine CTLA-4, CD80, CD86, and CD28, we designed the primers to amplify the ORF region that did not have a stop codon. The amplified region fragment of bovine CTLA-4, CD80, CD86, and CD28 was inserted into the cloning site of a pEGFP-N2 vector (Clontech, Palo Alto, CA, USA). The complete vectors, pEGFP-N2-CD80, pEGFP-N2-CD86, and pEGFP-N2-CD28 were transfected into Cos-7 cells by Lipofectamine 2000 reagent (Thermo Fisher Scientific) and cultured for 48 h. The complete vector, pEGFP-N2-CTLA-4 was transfected into CHO-DG44 cells (provided by Dr. Suzuki, Hokkaido University, Japan) with the Lipofectamine LTX reagent (Thermo Fisher Scientific). CTLA-4-EGFP expressing CHO-DG44 cells were cloned and established CTLA-4-EGFP highly expressing CHO-DG44 cells. The binding of CTLA-4-Ig to the CD80 or CD86 expressing cells or the binding of CD80-Ig or CD86-Ig to CTLA-4 expressing cells was confirmed using flow cytometry with FACSVerse (BD Biosciences, San Jose, CA, USA) and FCS Express 4 (De Novo Software, Glendale, CA, USA) as previously described with some modifications [22 (link)]. Rabbit IgG (Southern Biotech, Birmingham, AL, USA) was used as a control Ig.

Watari K., Konnai S., Maekawa N., Okagawa T., Suzuki Y., Murata S, & Ohashi K. (2019). Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production. BMC Veterinary Research, 15, 380.

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VIPR2 Expression and Modulation

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The pCMV6-AN-Myc-DDK vector (PS100016) and a negative control siRNA (S10C-0600) were purchased from Cosmo Bio (Tokyo, Japan). The pEGFP-N2 vector and pmCherry-N1 vector were purchased from Takara Clontech (Kyoto, Japan). The pCMV6-VIPR2-Myc-DDK plasmid was constructed from the pCMV6-AN-Myc-DDK vector and VIPR2 cDNA. The VIPR2-Myc region from the resultant plasmid was cloned into the pEGFP-N2 vector, and the VIPR2-Myc-DDK region was cloned into pmCherry-N1 vector. The AKT-PH-EGFP vector was described previously (34 (link)). Human VIPR2-siRNAs (si1, 3024813653-000080 and -000090; si2, 3024813653-000020 and -000030; si3, 3024813653-000050 and -000060) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A VIPR2-selective antagonistic peptide KS-133 was synthesized at SCRUM Inc. (Tokyo, Japan) as previously reported (35 (link)).

Asano S., Yamasaka M., Ozasa K., Sakamoto K., Hayata-Takano A., Nakazawa T., Hashimoto H., Waschek J.A, & Ago Y. (2022). Vasoactive intestinal peptide–VIPR2 signaling regulates tumor cell migration. Frontiers in Oncology, 12, 852358.

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Mouse Spata1 Cloning and Expression

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Mouse Spata1 coding region was amplified by PCR using testis cDNA as a template and the following primers: 5′-GAATTCGACAACATGTCGCTCAATCCAAGT-3′ (forward) and 5′-GGATCCCGGTTGGCACACTTCCAGAAAACAT-3′ (reverse). The correct PCR product was ligated into pEGFP-N2 vector (Clontech, California). To make SPATA1/pcDNA3 construct, the Spata1 coding region was amplified using the following primers: 5′-GAATTCGACAACATGTCGCTCAATCCAAGT-3′ (forward), and 5′-CTCGAGCCTAGGTTGGCACACTTCCAGAAAA-3′ (reverse). The correct PCR product was cloned into pcDNA3 vector (Invitrogen, New York). A plasmid used to express IFT20/Flag fusion protein in mammalian cells was kindly provided by Dr. Gregory J. Pazour at University of Massachusetts Medical School.

Zhang L., Zhen J., Huang Q., Liu H., Li W., Zhang S., Min J., Li Y., Shi L., Woods J., Chen X., Shi Y., Liu Y., Hess R.A., Song S, & Zhang Z. (2020). Mouse spermatogenesis-associated protein 1 (SPATA1), an IFT20 binding partner, is an acrosomal protein. Developmental dynamics : an official publication of the American Association of Anatomists, 249(4), 543-555.

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MCAM:GFP Plasmid Transfection

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WM239A cells were transfected with the MCAM:GFP plasmid as previously described61 (link). Briefly, MCAM:GFP was made with PCR amplification of the MCAM coding region from a pSport vector (ATCC Bioproducts) containing the MCAM cDNA. The primers used were 5′-AAGCTTATGGGGCTTCCCAGGCTGGTCTCGCC and 5′- TTTGGATCCCATGCCTCAGATCGATGTATTTCTCTCC. The resulting vector was digested with BamHI and HindIII and ligated into the pEGFP-N2 vector (Clontech).

Singh S.P., Schwartz M.P., Tokuda E.Y., Luo Y., Rogers R.E., Fujita M., Ahn N.G, & Anseth K.S. (2015). A synthetic modular approach for modeling the role of the 3D microenvironment in tumor progression. Scientific Reports, 5, 17814.

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Engineered GluN2Bct-CTM Fusion Proteins

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CTM-GFP was constructed by introducing a BamHI fragment containing the CTM coding sequence into the pEGFP-N2 vector (Clontech #6081-1). The CTM coding sequence was prepared by annealing custom design oligonucleotides (Integrated DNA Technologies). mCTM-GFP was constructed by performing single point mutations to CTM-GFP plasmid. FLAG-cDAPK1 was constructed by deleting the autoinhibitory domain from WT-DAPK1 (789-936bp). GluN2Bct (1242-1342aa) was prepared by PCR using GluN2B expression vector. GluN2Bct-CTM fragment was obtained by inserting GluN2Bct fragment into CTM-GFP using EcoRI and BglII restriction sites (EcoRI, Fermentas, FD0274; BglII, Fermentas, FD0084), then PCR, introducing NcoI and EcoRI to the fragment (NcoI, Fermentas, FD0574). His-TAT-GluN2Bct-CTM was constructed by cloning GluN2Bct-CTM into the pTAT/pTAT-HA plasmids (generous gift of S. Dowdy, Washington University, St. Louis, MO^{34 (link)}) using NcoI and EcoRI restriction sites. His-TAT-GluN2Bct was constructed by mutating the first amino acid in CTM sequence into stop codon. HA-GluN2Bct-CTM was constructed by PCR using TAT-GluN2Bct-CTM as template, with BamHI site in both forward and reverse primers, and then inserted into pcDNA3.0 with BamHI (Fermentas, FERFD0054). HA-GluN2Bct-CTMm and α-synuclein (ΔDQ) were constructed by performing point mutation PCR.

Fan X., Jin W.Y., Lu J., Wang J, & Wang Y.T. (2014). Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation. Nature neuroscience, 17(3), 471-480.

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Genetic Constructs of GPR15 and Interacting Proteins

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The human GPR15 gene was cloned in pCDNA3.1(+) vector (Life Technologies), pCMVmyc vector (Okamoto and Shikano, 2011 (link)), and pEGFPN2 vector (Clontech) to generate no-tagged GPR15, N-terminal Myc-tagged GPR15, and C-terminal GFP-tagged GPR15 plasmids, respectively. CXCR4, CXCR7, and β2-adrenergic receptor genes were obtained from cDNA Resource Center (Bloomsburg, PA) and cloned in pCDNA3.1(+) vector with N-terminal myc tag. K44A hemagglutinin (HA)–dynamin-1 was a gift from Sandra Schmidt (Addgene plasmid 34683). GRK2, β-arrestin 2 WT, and β-arrestin 1 (319-418) were provided by Jonathan Benovic (Thomas Jefferson University); EGFP-Rab4 and EGFP-Rab11 by Marino Zerial (Max Planck Institute), and C-AP180 by Julie Donaldson (National Institutes of Health). GRK5 and GRK6 genes were obtained from the DNASU Plasmid Repository (Tempe, AZ) and cloned in pCDNA3.1(+) vector. Site-directed mutagenesis was performed by overlap extension PCR.

Okamoto Y, & Shikano S. (2017). Differential phosphorylation signals control endocytosis of GPR15. Molecular Biology of the Cell, 28(17), 2267-2281.

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Constructing EGFP-PD-1/PD-L1 Fusion Vectors

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To construct the enhanced green fluorescent protein (EGFP) fusion expression vectors, the ORF region of the canine PD-1 and canine PD-L1 cDNA that did not have the stop codons was amplified by PCR using the cDNA obtained from the Beagle PBMCs and gene-specific primers [5′-CCG CTC GAG ATG GGG AGC CGG CGG GGG CC-3′ (PD-1 F, containing an XhoI restriction site), 5′-CGC GGA TCC TGA GGG GCC ACA GGC CGG GTC-3′ (PD-1 R, containing a BamHI restriction site), 5′-GAA GAT CTA TGA GAA TGT TTA GTG TC-3′ (PD-L1 F, containing a BglII restriction site), and 5′-GGA ATT CTG TCT CTT CAA ATT GTA TAT C-3′ (PD-L1 R, containing an EcoRI restriction site)]. The amplicons were then cloned into the multicloning site of the pEGFP-N2 vector (Clontech, Palo Alto, CA, USA). These vectors were named pEGFP-N2–cPD-1 and pEGFP-N2–cPD-L1, respectively. For transient expression, Cos-7 cells (5×10⁴/cm²) were transfected with 0.4 µg/cm² of the expression vector pEGFP-N2–cPD-1 or pEGFP-N2–cPD-L1 or empty pEGFP-N2 vector as the mock, using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. The cells were cultured at 37°C and 5% CO₂ for 48 h and harvested using a cell scraper. To confirm the expression of fusion proteins, the cells were observed under a confocal microscope LSM700 (Carl Zeiss Microscopy, Jena, Germany), and the subcellular distributions of EGFP were determined.

Maekawa N., Konnai S., Ikebuchi R., Okagawa T., Adachi M., Takagi S., Kagawa Y., Nakajima C., Suzuki Y., Murata S, & Ohashi K. (2014). Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-γ Production from Tumor-Infiltrating Cells by PD-L1 Blockade. PLoS ONE, 9(6), e98415.

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