Pshuttle cmv

Manufactured by Addgene

Sourced in United States

The PShuttle-CMV is a plasmid vector designed for gene expression in mammalian cells. It contains a cytomegalovirus (CMV) promoter, which drives the expression of the inserted gene of interest. The plasmid also includes an ampicillin resistance gene for selection in bacterial hosts.

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Close spelling variants of this product

PShuttle-CMV vector PShuttle-CMV-iRFP plasmid

7 protocols using pshuttle cmv

Visualizing CXCR7-Mediated β-Arrestin Recruitment

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HEK293 cells (ATCC CRL-11268) were used for the β-arrestin recruitment assay, as previously described^{52 (link)}. In brief, HEK293 cells were transfected with human β-arrestin-2-ω and human CXCR7-α. The cells were stimulated with human CXCL12 (R&D Systems, Minneapolis, MN, USA) or TC14012 at 37 °C for 90 min and then, Gal-Screen (Thermo Fisher Scientific) was added and the cells were further incubated at 25 °C for 90 min. Luminescence was measured in an ARVO X3 plate reader (PerkinElmer, Waltham, MA, USA). In addition, HEK293 cells were transfected with a rat CXCR7 expression plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). The CXCR7 expression plasmid was synthesized by amplifying a rat Cxcr7 mRNA sequence (RefSeq: NM_053352.1) with a FLAG tag (forward primer: 5′-TTTTGCGGCCGCGCCACCATGGATTACAAGGACGATGACGACAAGGGAGGAGGCTCCGATGTGCATCTGTTTGAC-3′, reverse primer: 5′-TTTTAAGCTTTCACTTGGTGTTCTGCTC-3′) from cDNA prepared from total RNA isolated from neonatal rat heart and inserting it in pShuttle-CMV (#16403, Addgene, Watertown, MA, USA). The cells were cultured for 36 h. After 12 h of starvation, the cells were stimulated with CXCL12.

Ishizuka M., Harada M., Nomura S., Ko T., Ikeda Y., Guo J., Bujo S., Yanagisawa-Murakami H., Satoh M., Yamada S., Kumagai H., Motozawa Y., Hara H., Fujiwara T., Sato T., Takeda N., Takeda N., Otsu K., Morita H., Toko H, & Komuro I. (2021). CXCR7 ameliorates myocardial infarction as a β-arrestin-biased receptor. Scientific Reports, 11, 3426.

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Construction of Adenoviral Vectors for IL-2 Expression

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Replication-deficient E1/E3-deleted adenovirus vectors Ad5-CMV-oIL-2 (Ad-oIL-2) and Ad5-CMV-Null (Ad-Null) were constructed using the AdEasy XL Adenoviral Vector System (Agilent). Mouse orthogonal IL-2 clone 3A10 cDNA was synthesized (GenScript) with 5′ KpnI and 3′ HindIII restriction sites and subcloned into the multiple cloning site of pShuttle-CMV (Addgene). pAdEasy-1-containing BJ5183-AD-1 Escherichia coli cells were transformed with PmeI-linearized pShuttle-CMV-oIL-2 for homologous recombination. The resulting recombinant Ad plasmid was sequence-verified and expanded in XL10-Gold Ultracompetent cells before PacI linearization and transfection into HEK293T cells. High-titre adenoviruses were purified by caesium chloride (CsCl₂) gradient centrifugation after multiple rounds of amplification. CsCl₂ was exchanged to A195 buffer^{53 (link)} with Amicon Ultra-15 centrifugal filter units (Millipore). Viral titre (VP per ml) was determined spectrophotometrically (Nanodrop, Thermo Fisher Scientific).

Kalbasi A., Siurala M., Su L.L., Tariveranmoshabad M., Picton L.K., Ravikumar P., Li P., Lin J.X., Escuin-Ordinas H., Da T., Kremer S.V., Sun A.L., Castelli S., Agarwal S., Scholler J., Song D., Rommel P.C., Radaelli E., Young R.M., Leonard W.J., Ribas A., June C.H, & Garcia K.C. (2022). Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature, 607(7918), 360-365.

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Generation of Recombinant Adenovirus Expressing hACE2

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Codon-optimized hACE2 sequences were cloned into the shuttle vector (pShuttle-CMV, Addgene 240007) to generate pShuttle-hACE2. pShuttle-hACE2 was linearized with PmeI and subsequently cotransformed with the HuAdv5 backbone plasmid (pAdEasy-1 vector; Addgene 240005) into E. coli strain BJ5183 to generate pAdV5-ACE2 by homologous recombination as described (He et al., 1998 (link)). The pAdEasy-1 plasmid containing the HuAdV5 genome has deletions in E1 and E3 genes. hACE2 is under transcriptional control of a cytomegalovirus promotor and is flanked at its 3′ end by a SV40 polyadenylation signal. The pAd-hACE2 was linearized with PacI restriction enzyme before transfection into T-Rex 293 HEK cells (Invitrogen) to generate HuAdv5-hACE2. Recombinant HuAdv5-hACE2 was produced in 293-HEK cells and purified by CsCl density-gradient ultracentrifugation. The viral titer was determined by plaque assay in 293-HEK cells.

Hassan A.O., Kafai N.M., Dmitriev I.P., Fox J.M., Smith B.K., Harvey I.B., Chen R.E., Winkler E.S., Wessel A.W., Case J.B., Kashentseva E., McCune B.T., Bailey A.L., Zhao H., VanBlargan L.A., Dai Y.N., Ma M., Adams L.J., Shrihari S., Danis J.E., Gralinski L.E., Hou Y.J., Schäfer A., Kim A.S., Keeler S.P., Weiskopf D., Baric R.S., Holtzman M.J., Fremont D.H., Curiel D.T, & Diamond M.S. (2020). A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2. Cell, 183(1), 169-184.e13.

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Lentiviral and Adenoviral Vector Construction

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The following constructs were procured for this study: GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); Rab35 (gift from Peter McPherson; Addgene plasmid #47424); Neo DEST (705–1) (gift from Eric Campeau & Paul Kaufman; Addgene plasmid #17392); pMDLg/pRRE (gift from Didier Trono; Addgene plasmid # 12251); pVSVG (gift from Bob Weinberg; Addgene plasmid #8454); psPAX2 (gift from Didier Trono; Addgene plasmid #12260); pShuttle-CMV (gift from Bert Vogelstein; Addgene plasmid #16403); and AdEasier-1 cells (gift from Bert Vogelstein; Addgene, #16399).

Francis C.R, & Kushner E.J. (2021). Capturing membrane trafficking events during 3D angiogenic development in vitro. Microcirculation (New York, N.y. : 1994), 29(6-7), e12726.

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Plasmid Constructs for Nrf1 Overexpression

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The following plasmids were used. pcDNA3.1, pcDNA3.1-2xFLAG-SREBP1c (Addgene plasmid 26802)^{18 (link)} and pShuttle-CMV (Addgene plasmid 16403)^{19 (link)}. A pCMV-Sport6 vector encoding the mouse Nrf1 cDNA (GenBank accession: BC047283.1) was acquired from Open Biosystems (ThermoScientific). The Nrf1 cDNA was PCR amplified with the addition of tag elements incorporated onto its 5′ (his) or 3′ (flag or myc) ends, along with suitable restriction sites, which were then utilized to subclone into the pShuttle-CMV vector.

Zhang Y., Nicholatos J., Dreier J.R., Ricoult S.J., Widenmaier S.B., Hotamisligil G.S., Kwiatkowski D.J, & Manning B.D. (2014). Coordinated regulation of protein synthesis and degradation by mTORC1. Nature, 513(7518), 440-443.

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Construction of Adenoviral Vectors for IL-2 Expression

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Replication-deficient E1/E3 –deleted adenovirus vectors Ad5-CMV-oIL2 (Ad-oIL2) and Ad5-CMV-Null (Ad-Null) were constructed using AdEasy™ XL Adenoviral Vector System (Agilent). Mouse orthogonal IL-2 clone 3A10 cDNA was synthesized (GenScript) with 5’ KpnI and 3’ HindIII restriction sites and subcloned into the multiple cloning site of pShuttle-CMV (Addgene). pAdEasy-1 –containing BJ5183-AD-1 E.coli cells were transformed with PmeI –linearized pShuttle-CMV-oIL2 for homologous recombination. Resulting recombinant Ad plasmid was sequence-verified and expanded in XL10-Gold Ultracompetent cells before PacI –linearization and transfection into HEK293T cells. High-titer adenoviruses were purified by cesium chloride (CsCl₂) gradient centrifugation after multiple rounds of amplification. CsCl₂ was exchanged to A195 buffer⁵³ with Amicon Ultra-15 centrifugal filter units (Millipore). Viral titer (VP/ml) was determined spectrophotometrically (Nanodrop, ThermoFisher).

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Generating Adenoviruses Expressing LacZ, DN Atf2, and C2/Atf2

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We used the modified AdEasy system (Luo et al., 2007 (link)) to generate non-inducible adenoviruses expressing LacZ, DN Atf2, and C2/Atf2. To construct Ad-DN Atf2, we cloned the coding sequence for mouse Atf2 (NM_001025092) and used overlap extension PCR to introduce threonine to alanine mutations (T51A; T53A) corresponding to amino acids T69/71 in human ATF2. To construct Ad-C2/Atf2, we cloned C2/Atf2 from the pCMV-Flag-C2/Atf2 vector (Steinmuller and Thiel, 2003 (link)) generously provided by Dr Gerald Thiel. As previously described, this vector contains the DNA-binding domain of ATF2 fused with the activation domain of ATF4 to generate a constitutively active ATF2 mutant. These sequences, along with the coding sequence for LacZ, were digested with XhoI and KpnI and ligated into pShuttle-CMV (Addgene, Cambridge, MA). These vectors were electroporated into BJ5183-AD-1 cells (Stratagene, La Jolla, CA), which contain AdEasy-1 encoding the adenoviral backbone, for recombination. Potential recombinants were screened and successful recombinants were PacI linearized and transfected in 293 cells by calcium phosphate transfection. Titre was determined by use of Adeno-X Rapid Titer Kit (Clontech). Virus was delivered experimentally as crude lysate.

Mamrosh J.L., Lee J.M., Wagner M., Stambrook P.J., Whitby R.J., Sifers R.N., Wu S.P., Tsai M.J., DeMayo F.J, & Moore D.D. (2014). Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution. eLife, 3, e01694.

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