A cDNA for the BiP deleted with the KDEL sequence was obtained by inserting a stop codon into a rat BiP cDNA (a gift from Dr. H.R.B. Pelham, MRC Laboratory of Molecular Biology, UK) just before the region encoding the carboxyl terminal KDEL sequence with a PCR reaction. The PCR product was subcloned into a pcDNA3.1 myc-His vector (Invitrogen). A PCR-generated fragment corresponding to the wild-type human SOD1 cDNA from human cDNA library was cloned into the plasmid pGFP vector (Clontech). The DNA sequences were verified using the Applied Biosystems ABI Prism 310 genetic analyzer. Transfection was performed with Fugene 6 (Roche, Basel, Switzerland).
Pgfp vector
Manufactured by Takara Bio
The PGFP vector is a plasmid-based expression system designed for the production of recombinant proteins in mammalian cell lines. The vector contains a cytomegalovirus (CMV) promoter for high-level transgene expression and a green fluorescent protein (GFP) reporter gene to facilitate monitoring of transfection efficiency and recombinant protein expression.
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Lab products found in correlation
2 protocols using pgfp vector
1
Expressing BiP Mutant and Wild-type SOD1
2
Generating dsRNA for Vg and GFP
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We prepared dsRNA against Vg and GFP as described previously (Guidugli et al. 2005 (link)). Briefly, we derived the dsRNA constructs for Vg from cDNA clone AP4a5, and for GFP from the pGFP vector (Clontech, Palo Alto, CA). Primers were fused with T7 promoter sequence (underlined): for Vg:
Fwd: 5′-TAATACGACTCACTATAGGGCGAAC GACTCGACCAACGACTT-3′
Rev: 5′-TAATACGACTCACTATAGGGCGAAA CGAAAGGAACGGTCAATTCC-3′;
and for pGFP:
Fwd: 5′-TAATACGACTCACTATAGGGCGATT CCATGGCCAACACTTGTCC-3′
Rev: 5′-TAATACGACTCACTATAGGGCGATC AAGAAGGACCATGTGGTC-3′.
PCR reactions were performed according to standard procedures. Products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA), and RNA was prepared using the Promega RiboMax T7 system (Promega, Madison, WI), and purified using TRIzol LS reagent (Invitrogen, San Diego, CA) before renaturation and resuspension.
Fwd: 5′-TAATACGACTCACTATAGGGCGAAC GACTCGACCAACGACTT-3′
Rev: 5′-TAATACGACTCACTATAGGGCGAAA CGAAAGGAACGGTCAATTCC-3′;
and for pGFP:
Fwd: 5′-TAATACGACTCACTATAGGGCGATT CCATGGCCAACACTTGTCC-3′
Rev: 5′-TAATACGACTCACTATAGGGCGATC AAGAAGGACCATGTGGTC-3′.
PCR reactions were performed according to standard procedures. Products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA), and RNA was prepared using the Promega RiboMax T7 system (Promega, Madison, WI), and purified using TRIzol LS reagent (Invitrogen, San Diego, CA) before renaturation and resuspension.
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