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Pcdh cmv msc ef1 copgfp

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The PCDH‐CMV‐MSC‐EF1‐copGFP is a lentiviral vector that expresses copGFP under the control of the Elongation Factor 1 (EF1) promoter. The vector contains a Protocadherin (PCDH) gene promoter and Cytomegalovirus (CMV) promoter for gene expression in mesenchymal stem cells (MSC).

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Lab products found in correlation

Polybrene, by GenePharma (1 mentions) Cold fusion kit, by System Biosciences (1 mentions) Phusion enzyme, by Thermo Fisher Scientific (1 mentions)

2 protocols using pcdh cmv msc ef1 copgfp

1

Lentiviral Transduction of CD4+ T Cells

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The full‐length coding sequence of human Bach2 or IRF4 was cloned into pCDH‐CMV‐MSC‐EF1‐copGFP (System Biosciences, Mountain View, CA, USA) vector, generating the vectors, LV‐Bach2 and LV‐IRF4. The empty pCDH‐CMV‐MSC‐EF1‐copGFP vector (LV‐NC) served as control. pCDH‐CMV‐MSC‐EF1‐copGFP‐mediated short hairpin RNA (shRNA) Bach2 (LV‐sh Bach2) and nontargeting plasmids (LV‐sh NC) were generated by GenePharma. CD4+ T cells were then infected with the lentiviral vectors in the presence of polybrene (GenePharma, Suzhou, China).
Sheng Y., Zhang J., Li K., Wang H., Wang W., Wen L., Gao J., Tang X., Tang H., Huang H., Cai M., Yuan T., Liu L., Zheng X., Zhu Z, & Cui Y. (2020). Bach2 overexpression represses Th9 cell differentiation by suppressing IRF4 expression in systemic lupus erythematosus. FEBS Open Bio, 11(2), 395-403.
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2

Cloning Linc-RoR and miR-145 Mutants

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PCR reactions for cloning purpose used Phusion enzyme from ThermoFisher Scientific (Pittsburgh, PA, USA). Different fragments of Linc-RoR were cloned into pCDH-CMV-MSC-EF1-copGFP (System Biosciences) with a strategy described previously (13 (link)). For example, to clone Linc-RoR E4, we first amplified the entire exon 4 by PCR using primers RoR-E4-R1–5.1 and RoR-E4-Not1-3.2 (Supplementary Table S1) and then cloned into the designated vector at EcoR I and Not I sites using Cold Fusion kit (System Biosciences). To make miR-145 binding site mutant clones, we carried out a two-step amplification procedure as described previously (19 (link)), using primers RoR-miR145-BS1-m-5.1 and RoR-miR145-BS1-m-3.1; RoR-miR145-BS2-m-5.1 and RoR-miR145-BS2-m-3.1 (Supplementary Table S1). All PCR products were verified by DNA sequencing. RoR-E4 clone mutated at two putative binding sites was made through the gBlock method from IDT.
Huang J., Zhang A., Ho T.T., Zhang Z., Zhou N., Ding X., Zhang X., Xu M, & Mo Y.Y. (2015). Linc-RoR promotes c-Myc expression through hnRNP I and AUF1. Nucleic Acids Research, 44(7), 3059-3069.
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