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Pentr4 vector

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The PENTR4 vector is a plasmid designed for bacterial transformation and protein expression. It provides a cloning site for inserting genes of interest and contains elements necessary for bacterial replication and selection.

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Lab products found in correlation

Bp clonase, by Thermo Fisher Scientific (1 mentions) Pgwb502, by Thermo Fisher Scientific (1 mentions) Lr clonase, by Thermo Fisher Scientific (1 mentions) Plenti6 v5 dest vector, by Thermo Fisher Scientific (1 mentions) Effectene transfection reagent, by Qiagen (1 mentions) Pegfp, by Takara Bio (1 mentions) Pfuse, by InvivoGen (1 mentions)

5 protocols using pentr4 vector

1

Adenoviral Vector Encoding HBV Genome

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We designed a pENTR4 vector (Thermo Fisher Scientific, Waltham, MA, USA) for cloning into replication-deficient recombinant adenovirus serotype 5 (Thermo Fisher Scientific, USA), which consists of a HBV 1.3-overlength genome [28 (link)] linked to a click beetle green (CBG99) luciferase separated by a P2A linker site derived from the porcine teschovirus. The P2A linker and the CBG99-luciferase were cloned in frame onto the second open reading frame coding for the HBc protein and after the HBV polyA-sequence. Generation and titration of serotype 5 adenoviruses was performed as described before [29 (link)].
Manske K., Schneider A., Ko C., Knolle P.A., Steiger K., Protzer U, & Wohlleber D. (2021). In Vivo Bioluminescence Imaging of HBV Replicating Hepatocytes Allows for the Monitoring of Anti-Viral Immunity. Viruses, 13(11), 2273.
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2

Generation of Transgenic Arabidopsis Lines

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To generate overexpressing lines (p35S:cD27-LIKE1), D27-LIKE1 cDNA was amplified using primers attb.D27-l1.cds.F/attb.D27-l1.cds.stop.R, then cloned into pDONR221 donor vector with BP clonase (Thermo Fisher, Waltham, MA, USA) and recombined into pGWB502 (Nakagawa et al., 2007) (link) with LR clonase (Thermo Fisher, Waltham, MA, USA). The expression of D27-LIKE1 has been assessed in homozygous overexpressing lines (Supplementary Figure S7.)
To generate complementation lines (pD27-LIKE1:cD27-LIKE1 in d27-like1-1), 2.2 kb promoter fragments (with 5'UTR) were amplified with primers attb.D27-like1.prom.F/attb.D27-like1.prom.R and cloned into the BamHI/EcoRI site of pENTR4 vector (Thermo Fisher, Waltham, MA, USA). D27-LIKE1 cDNA has been cloned into the EcoRI/NotI site of pENTR4 harbouring the D27-LIKE1 promoter fragment. The assembled construct has been recombined into pGWB501 (Nakagawa et al., 2007) (link). Vectors were introduced into Agrobacterium tumefaciens GV3101 and used for floral dip transformation (Végh et al., 2017) . T3-T5 homozygous transgenic plants were used in all experiments.
Gulyás Z., Moncsek B., Hamow K.Á., Stráner P., Tolnai Z., Badics E., Incze N., Darkó É., Nagy V., Perczel A., Kovács L, & Soós V. (2022). D27-LIKE1 isomerase has a preference towards trans/cis and cis/cis conversions of carotenoids in Arabidopsis. The Plant journal : for cell and molecular biology, 112(6).
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3

Lentiviral Transduction for Invasion Assay

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Full-length α1-antitrypsin (α1-AT), α1-antitrypsin variant Pittsburgh (α1-PIT), and α1-antitrypsin variant Portland (α1-PDX) constructs were kind gifts from Gary Thomas (Oregon Health Sciences University) [26 (link)]. The inserts were subcloned into the pIEx-5 vector (Novagen) using the Acc65I and HindIII sites, generating a C-terminal S-tag. Positive clones confirmed by sequence analysis were subcloned into the pENTR4 vector (Invitrogen) using the Acc65I and XhoI sites and recombined into the pLenti6/V5 DEST vector (Invitrogen) using the GATEWAY system. Lentiviruses were generated as previously described [24 (link),25 (link)]. ECs were transduced for 3d and selected with blasticidin (1μg/ml) for 8d. Blasticidin was removed for 24hr and invasion assays were conducted.
Kang H., Duran C.L., Abbey C.A., Kaunas R, & Bayless K.J. (2015). Fluid shear stress promotes proprotein convertase-dependent activation of MT1-MMP. Biochemical and biophysical research communications, 460(3), 596-602.
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4

Retroviral Transduction of ARG1 in Cell Lines

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Human ARG1 cDNA was obtained from the IMAGE consortium, cloned into pENTR4 vector (Invitrogen, Carlsbad, CA), then subcloned into a GATEWAY compatible pBABE vector. To generate retrovirus, the packaging cell line HEK 293T was co-transfected with pBABE vector or pBABE-ARG1, along with plasmids expressing gag/pol and VSVg (vesicular stomatitis virus glycoprotein), using Effectene transfection reagent (Qiagen). At 48 hours posttransfection, the viral supernatants were filtered and used to infect cells. LAN-5 and Kelly cells were transduced and selected for two weeks with 0.5μg/ml puromycin. Cells were harvested for analysis after 72h of subsequent treatment.
Hackett C.S., Quigley D.A., Wong R.A., Chen J., Cheng C., Song Y.K., Wei J.S., Pawlikowska L., Bao Y., Goldenberg D.D., Nguyen K., Gustafson W.C., Rallapalli S.K., Cho Y.J., Cook J.M., Kozlov S., Mao J.H., Van Dyke T., Kwok P.Y., Khan J., Balmain A., Fan Q, & Weiss W.A. (2014). eQTL and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma. Cell reports, 9(3), 1034-1046.
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5

Antibody Fragment Expression Protocols

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Artificial gene synthesis (Mr Gene, GmbH, Germany) composed of a 6His-Tag and a triple c-myc Tag was inserted into the pHEN2 phagemid vector (Griffin 1. library) between NotI and BamHI sites. CcdB gene from pENTR4 vector (Invitrogen - ThermoFisher Scientific, France) was inserted into the pHEN2 vector between NcoI and NotI sites. This vector allows to express antibody fragments in fusion, upstream, with the pelB leader to drive secretion in the periplasm and downstream with the PIII protein of M13 phages. An amber stop codon is present between the antibody and the pIII. This stop codon is partially suppressed in SupE E. coli. For expression and purification of dimeric antibodies, hs2dAb were inserted in vectors derived from pFuse (Invivogen, France) as described in Moutel et al. (2009) (link). For intrabody expression in mammalian cells, hs2dAb were digested by NcoI and NotI and ligated into the pIb-mEGFP, pEGFP or the pmCherry vectors (Clontech - Takara, USA). (See the Appendix for more details).
Moutel S., Bery N., Bernard V., Keller L., Lemesre E., de Marco A., Ligat L., Rain J.C., Favre G., Olichon A, & Perez F. (2016). NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. eLife, 5, e16228.
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