Amplification of TRIM27.1 and TRIM27-L from duck lung cDNA infected with highly pathogenic IAV (VN1203) (AmpliTaq® Gold PCR Master Mix, Invitrogen) was done using gene specific primers with 3′- and 5′-modifications for directional cloning and inclusion of an epitope tag (C-terminal-V5, amino acid sequence GKPIPNPLLGLDST) to confirm protein expression in transiently transfected cells. A 1574 bp PCR fragment of TRIM27.1 with C-terminal V5 epitope and a 1493 bp PCR fragment of TRIM27-L with C-terminal V5 epitope were cloned into pCR2.1-TOPO first, then cloned using restriction sites NotI/NheI into pcDNA3.1/Hygro+ expression vector (Invitrogen). For analysis of immunomodulation of the MAVS pathway we used two additional constructs, one which contains the glutathione-S-transferase (GST) gene fused to the two tandem CARD domains from duck RIG-I (d2CARD) and one containing the GST-I protein only as control (Miranzo-Navarro & Magor 2014 (link)).
Pcdna3.1 hygro expression vector
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The PcDNA3.1/Hygro+ expression vector is a plasmid used for the expression of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter for high-level expression and a hygromycin resistance gene for selection of transfected cells.
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4 protocols using pcdna3.1 hygro expression vector
1
Amplification and Cloning of Duck TRIM27 Isoforms
2
Generation of FPN1 Expressing Cell Lines
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Full‐length FPN1 complementary DNA was generated by RT‐PCR amplification of human fibroblast RNA. DNA gene sequencing by Sanger confirmed that the donor human fibroblast SCL40A1 gene was WT. The FPN1 coding region was sequenced and subcloned into the pcDNA3.1/Hygro expression vector (Invitrogen, Groningen, The Netherlands), in order to obtain FPN1 untagged or tagged at the C‐terminal with V5 epitope. The pA77D FPN1 was obtained by mutagenesis using the QuickChange Site‐Directed Mutagenesis kit (Stratagene, Milan, Italy). Madin‐Darby canine kidney cells (MDCK) cells stably expressing WT, pA77D FPN1, and FPN1‐V5 were obtained by transfecting MDCK cells with the Transfection Reagent Selector Kit Effectane (Qiagen, Milan, Italy), according to the manufacturer's instructions.
3
PCR-Based Cloning of Human BATF Gene
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The human BATF gene was PCR-amplified from cDNA of RPMI8226 cells using the following primers: 5'-GGC GCTAGCGCCACCATGCCTCACAGCTCCGAC-3' and 5'-GCCCTCGAGTCAGGGCTGGAAGCGC-3'. The amplified products were digested using the NheI and XhoI restriction enzymes, and were ligated into the pCDNA3.1- hygro expression vector (Invitrogen, Carlsbad, CA, USA). The resulting expression construct was sequenced to verify the cDNA sequence vectors were used in the reporter assays.
4
Stable LINC00313 Overexpression in HuCCT1 Cells
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Human LINC00313 (NR_026863.1) was synthesized and cloned into the pcDNA3.1/Hygro (+) expression vector (Invitrogen), by Eurofins (Eurofins Genomics, Germany). The sequence of the plasmid was verified by double-strand DNA sequencing. For the generation of stable LINC00313 over-expressing HuCCT1 clones, cells were transfected with pcDNA3.1-LINC00313 for 48 h. Then, transfected cells were grown, for 2 weeks, in fresh selection medium, consisting of 10% FBS/RPMI, in the presence of 650 µg/ml hygromycin B Gold (Invivogen, ant-hg-1). By using limiting dilution assay, individual clones from the stable LINC00313 over-expressing pool were seeded in 96-well plates and grown in selection medium. The same protocol was followed, in order to establish stable HuCCT1 clones over-expressing an empty vector (pcDNA3.1), which served as control for gain-of-function experiments. Human HA-tagged ACTL6A plasmid (VB211129-1064fwh) was constructed by Vectorbuilder. M50 Super 8x TOPFlash (Addgene plasmid #12456) and M51 Super 8x FOPFlash (TOPFlash mutant) (Addgene plasmid #12457) were a gift from Randall Moon. HuCCT1 or HEK293T cells were transfected with plasmid DNA, using Lipofectamine 2000 reagent (ThermoFisher Scientific, 11668019), according to the instructions by the manufacturer.
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