Cloning and Expression of Human DDT

All SOD1 plasmids, including pCI-hSOD1^WT and pCI-hSOD1^G93^A were generated previously by our group, by inserting human SOD1 constructs into the pCI-NEO vector (Promega), between the EcoRI and the NotI sites. A DDT expressing plasmid pET-21b-hDDT, was generously received from the Bernhagen group (Munich, Germany). The sequence of human DDT was transferred from the pET-21b vector^{75 (link)} into the mammalian expression vector pcDNA3.1 (-) vector, between the BamHI and NotI restriction sites. Human DDT gene was amplified by PCR from pET-21b vector using following primers:
Forward primer: 5’-ATTACGCGGCCGCCACCATGCCGTTCCTGGAG-3’.
Reversed primer: 5’-GCTAGGATCCCTATAAAAAAGTCATGAC-3’.

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Alaskarov A., Barel S., Bakavayev S., Kahn J, & Israelson A. (2022). MIF homolog d-dopachrome tautomerase (D-DT/MIF-2) does not inhibit accumulation and toxicity of misfolded SOD1. Scientific Reports, 12, 9570.

Publication 2022

pCI-NEO vector

Ecori Human Mammalian Plasmid Primer Promega Vector

Corresponding Organization :

Other organizations : Ben-Gurion University of the Negev

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Variable analysis

independent variables

PCI-hSOD1^WT
PCI-hSOD1^G93A
PET-21b-hDDT

dependent variables

Expression of human SOD1 and DDT constructs

control variables

EcoRI and NotI restriction sites used for inserting human SOD1 constructs into pCI-NEO vector
BamHI and NotI restriction sites used for transferring human DDT sequence from pET-21b vector to pcDNA3.1 (-) vector

controls

Positive control: pET-21b-hDDT plasmid received from the Bernhagen group
Negative control: Not explicitly mentioned

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