Pcaggs

Manufactured by Addgene

Sourced in United States

PCAGGS is a plasmid vector designed for cloning and expression in mammalian cells. It contains key genetic elements necessary for efficient gene expression, including a CMV promoter, a polylinker region for inserting genes of interest, and a selectable marker for antibiotic selection in mammalian cells.

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Lab products found in correlation

Superscript 3 one step rt pcr system with platinum taq polymerase, by Thermo Fisher Scientific (2 mentions) Prl tk, by Promega (1 mentions) Ni nta, by Qiagen (1 mentions) Pet28a vector, by Merck Group (1 mentions) Q5 site directed mutagenesis, by New England Biolabs (1 mentions) Flag beads, by GenScript (1 mentions) Rnasin, by Promega (1 mentions) Plvx ires zsgreen1, by Addgene (1 mentions) Pcsii cmv rfa ires blast, by RIKEN BioResource Center (1 mentions) Pgex 6p 1, by Addgene (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PCAGGS-N-HA vector PCAGGS expression vector PCAGGS vector PCAGGS-FLAG-hsDicer PCAGGS-NICD PCAGGS Donor mClover-LMNA PCAGGS-ChR2-Venus PCAGGS-FlpE

12 protocols using pcaggs

Lentiviral Expression of TRIM5α Variants

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pEasiLV plasmids expressing luciferase, CD8, MX2 or TRIMCYP have been described6 (link). cDNAs encoding TRIM5α and TRIM5α isoform X7 lacking the SPRY domain were amplified using the SuperScript III One-Step RT–PCR System with Platinum Taq polymerase (Invitrogen) from 20 ng RNA obtained from IFNα-treated (500 U ml^-1) U87-MG CD4⁺ CXCR4⁺ cells and inserted into pEasiLV-MCS6 (link) between the AgeI and XhoI restriction sites. The TRIM5α R332G/R335G and H43Y mutants were obtained by site directed mutagenesis (SDM). CRISPR-resistant TRIM5α constructs (rTRIM5α) were produced by SDM using primers designed to silently mutate the guide RNA target sequence. pFLAG-TRIM5α was generated by subcloning a cDNA encoding TRIM5α with an N-terminal FLAG tag into pCAGGS (Addgene) using the EcoRI and XhoI sites. pCAGGS expressing FLAG-tagged GFP, pFLAG-GFP, was derived from a plasmid encoding HA-tagged GFP36 (link). The vectors encoding HA-tagged ubiquitin and the K48R and K63R mutant derivatives have been described37 (link)
38 (link).

Jimenez-Guardeño J.M., Apolonia L., Betancor G, & Malim M.H. (2019). Immunoproteasome activation enables human TRIM5α restriction of HIV-1. Nature microbiology, 4(6), 933-940.

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In Utero Electroporation and Calcium Imaging

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In utero electroporation was performed as described previously²³ (link). To perform calcium imaging of layer 2/3 pyramidal cells, GCaMP6s was cloned into pCAGGS (Addgene plasmid 40753⁷² (link)) and used in combination with DsRed in pCAGGS for visualization (gift from Christiaan Levelt). Pups were in utero electroporated at embryonic day (E) 16.5 after injection of the GCaMP6s (2 μg/μl) and DsRed (1 μg/μl) vectors into the ventricles. Electrode paddles were positioned to target the subventricular zone and 50 V pulses of 50 ms duration were applied.
For in vivo experiments, surgery for craniotomy was performed as described previously⁷ (link)^,³³ (link). Pups were kept at 36-37°C and anesthetized with 2% isoflurane and lidocaine was applied into the skin before neck muscle removal. A head bar was fixed above the V1/S1 region. Isoflurane was dropped to 0.7% before the imaging session. This lightly anesthetized state, which is characterized by rapid and shallow breathing and a relatively high heart rate, was maintained throughout the imaging session. During experiments in the absence of anesthesia, pups were kept undisturbed in the dark and covered by a custom-made holder that prevents heat loss and mimic the conditions in the nest, for one hour before the imaging session started. The welfare of the pup was monitored to minimize distress during the whole imaging session.

Maldonado P.P., Nuno-Perez A., Kirchner J.H., Hammock E., Gjorgjieva J, & Lohmann C. (2021). Oxytocin Shapes Spontaneous Activity Patterns in the Developing Visual Cortex by Activating Somatostatin Interneurons. Current Biology, 31(2), 322-333.e5.

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Lentiviral Expression of TRIM5α Variants

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Jimenez-Guardeño J.M., Apolonia L., Betancor G, & Malim M.H. (2019). Immunoproteasome activation enables human TRIM5α restriction of HIV-1. Nature microbiology, 4(6), 933-940.

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Plasmid Construction for H9N2 Virus Study

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All H9N2 virus genes were individually cloned into the mammalian expression vector pCAGGS (Addgene, Cambridge, MA, USA, kept in our laboratory). Sequences of primers used for constructing plasmids are listed in Table 1. Full length c-jun was cloned into the pCMV-Tag2B-Flag plasmid (Addgene, Cambridge, MA, USA, kept in our laboratory) using homologous recombination. Luciferase reporter plasmids pGL3 and pRL-TK were purchased from Promega (Madison, WI, USA). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG, mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, and rabbit monoclonal anti-β-actin antibody were purchased from ABclonal (ABclonal, Wuhan, China). Rabbit monoclonal antibodies against phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK1/2/3, and JNK1/2/3 were purchased from HuaAn (Huabio, Hangzhou, China). The FcRY monoclonal antibody was prepared in our laboratory, while the FcRY polyclonal antibody was prepared by Wuhan Daian Biotech, Ltd. (Daian, Wuhan, China).

Sun Z., Zhang W., Li J., Yang K., Zhang Y, & Li Z. (2024). H9N2 Avian Influenza Virus Downregulates FcRY Expression in Chicken Macrophage Cell Line HD11 by Activating the JNK MAPK Pathway. International Journal of Molecular Sciences, 25(5), 2650.

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Cloning and Mutagenesis of Human SLC4A2

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The different isoforms of human SLC4A2 gene cDNAs were acquired from Han Jiahuai lab at Xiamen University and then cloned into the mammalian cells expression vector pCAGGS (Addgene) modified to introduce a Flag tag plus an 8 × His tag followed by a Tobacco Etch Virus (TEV) protease cleavage site at the N-terminal of AE2. The Quick-change site-directed mutagenesis method was used to introduce the mutations. All the mutants and wide-type constructs were confirmed by DNA sequencing before functional and structural studies.

Zhang Q., Jian L., Yao D., Rao B., Xia Y., Hu K., Li S., Shen Y., Cao M., Qin A., Zhao J, & Cao Y. (2023). The structural basis of the pH-homeostasis mediated by the Cl−/HCO3− exchanger, AE2. Nature Communications, 14, 1812.

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Site-directed mutagenesis of MX1 and MX2

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Human 293T cells and U87-MG CD4⁺ CXCR4⁺ (3 (link)) cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (10%), l-glutamine, and penicillin-streptomycin. Site-directed mutagenesis was performed on human MX2 or MX1(N_MX2) (residues 1 to 91 of MX2 appended to residues 44 to 662 of human MX1 [23 (link)]) constructs containing a C-terminal FLAG tag using standard PCR amplification techniques. For the loop 4 (L4) deletion mutants, overlapping PCR was used to delete residues 580 to 608 of MX2, and the sequence corresponding to residues 533 to 561 of native human MX1 (10 (link)) was deleted in the context of the MX1(N_MX2) chimera. Mutant constructs were cloned into EasiLV-MCS (3 (link)) using BamHI and XhoI restriction sites. FLAG-tagged mutants then were subcloned into pCAGGS (Addgene) using BclI and XhoI restriction sites. GFP, MX1, and MX2 were subcloned into pCMV4.HA using Acc65I and XbaI to introduce a triple-hemagglutinin (HA) tag at the C terminus before further subcloning into EasiLV-MCS or pCAGGS. An MX1(N_MX2) construct with a C-terminal triple-HA tag was generated by overlapping PCR using HA-tagged MX1 as a template and cloned into pCAGGS using NotI and XhoI restriction sites.

Dicks M.D., Goujon C., Pollpeter D., Betancor G., Apolonia L., Bergeron J.R, & Malim M.H. (2015). Oligomerization Requirements for MX2-Mediated Suppression of HIV-1 Infection. Journal of Virology, 90(1), 22-32.

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Recombinant Expression and Purification of SARS-CoV-2 Spike and Nucleocapsid Proteins

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The ectodomain of the SARS-CoV-2 spike trimer⁴⁵ was cloned into mammalian expression vector pCAGGS (Addgene), with a fold-on tag followed by 6×His tag and Strep tag II at the C-terminal. This expression vector was transiently transfected into HEK293F cells and the spike trimer secreted in the supernatant was purified 3-5 days post transfection by metal-affinity chromatography using an Ni-NTA (Qiagen) column. SARS-CoV-2 nucleocapsid protein (N) was cloned into pET28a(+) vector (Millipore-Sigma) with an AAALE linker and 6×His tag at the C-terminal. The NP construct was then used to transform into Escherichia coli BL21 (DE3) pLysS cells and the target protein was produced and purified from the bacterial lysate by metal affinity chromatography using an Ni-NTA (Qiagen) column, followed by size-exclusion chromatography on a Superdex 200 10/300 GL column.

Weisberg S.P., Connors T.J., Zhu Y., Baldwin M.R., Lin W.H., Wontakal S., Szabo P.A., Wells S.B., Dogra P., Gray J., Idzikowski E., Stelitano D., Bovier F.T., Davis-Porada J., Matsumoto R., Poon M.M., Chait M., Mathieu C., Horvat B., Decimo D., Hudson K.E., Zotti F.D., Bitan Z.C., Carpia F.L., Ferrara S.A., Mace E., Milner J., Moscona A., Hod E., Porotto M, & Farber D.L. (2020). Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum. Nature immunology, 22(1), 25-31.

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Glycoprotein Expression Plasmid Construction

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Codon-optimized CVS-G and SAD-G were commercially synthesized (Invitrogen) and cloned into pcDNA3.1. Mutations were introduced via Q5 site-directed mutagenesis (New England Biolabs) and constructs were cloned into pCAGGS (Addgene). pCAGGS-CVS-G accession number is ABV24348.1 and pCAGGS-SAD-G accession number is P16288.1. pCAGGS-DOG-G (accession number CUI02214.1) and EBLV1-G (accession number SMD54588.1) have been described previously [16, 34 (link)]. pCAGGS-CVS_SADT and pCAGGS-EBLV_SADT, comprising the SAD cytoplasmic tail sequence, were generated through PCR mutagenesis.

Almasoud I., Charlton F.W., Finke S., Barr J.N, & Mankouri J. (2023). Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains. The Journal of General Virology, 104(12), 001935.

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Dicer Enzymatic Activity Assay

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Human Flag-Dicer and Flag-mutant-Dicer in pCAGGS (Addgene #41584 and #41585) were transfected into NoDice 293T cells using Lipofectamine 2000. Mutant Dicer construct? NoDice 293T cells were seeded in a 6-well plate at a density of 5 × 10⁵ per well one day before transfection. NoDice 293T cells were transfected with 3 μg plasmids. 48 hours after transfection, hDcr-KO 293T cells were infected by PR8/delNS1 (MOI = 1) or DMEM and the infected cells were lysed in cell lysis buffer (CST) 24 hours after infection. The Flag-Dicer was retrieved using FLAG beads (GenScript) according to the manufacturer’s instructions. For dicing assays, Flag-Dicer was incubated with 50 nM dsRNA in dicing buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2, 5% glycerol, 1 mM DTT, RNAsin (Promega)] for 1 h at 37°C followed by Trizol purification. The RNA was resuspended in formamide sample buffer without xylene blue (47.5% formamide, 0.01% SDS, 0.01% bromophenol blue, 0.5 mM EDTA), loaded onto a 15% TBE-Urea gel and visualised by GelRed staining.

Zhang Y., Xu Y., Dai Y., Li Z., Wang J., Ye Z., Ren Y., Wang H., Li W.X., Lu J., Ding S.W, & Li Y. (2021). Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2. PLoS Pathogens, 17(8), e1009790.

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Overexpression of GPNMB in Porcine Alveolar Macrophages

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The full-length porcine GPNMB protein-coding sequence was amplified from PAMs cDNA using the primers listed in Table 2 and then cloned into the eukaryotic expression vector pCAGGS (Addgene, Cambridge, MA, USA) and the bicistronic lentivirus vector pLVX-IRES-ZsGreen1 (Addgene, Cambridge, MA, USA), respectively. The nucleotide sequences of the plasmids expressing GPNMB were determined to ensure that the correct clones were used in this study. When the cells reached approximately 80% confluence, they were transfected with recombinant plasmids or an empty vector. The packaging and production of lentiviral vectors were performed as previously described [30 (link)]. When immortalized PAMs reached 80% confluence, cell monolayers were transduced with a recombinant lentivirus. At 24 h postincubation, the cells were harvested for further analysis.

Xu Y., Wang M., Zhang L., Pan Y., Zhang W., Ma W., Chen H., Tang L., Xia C, & Wang Y. (2023). Glycoprotein Non-Metastatic Melanoma Protein B Restricts PRRSV Replication by Inhibiting Autophagosome-Lysosome Fusion. Viruses, 15(4), 920.

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