Peg it virus concentration reagent

Plasmid pTA-FLuc, containing a TATA-box basal promoter firefly luciferase reporter gene, was constructed as the normalized control, as described previously (Ariazi et al., 2007 (link)). A TATA-box promoter (TA) drove the expression of firefly luciferase downstream of p53-specific binding sites in multiple copies of a cis-acting enhancer element in a p53 reporter plasmid (Bellis et al., 2013 (link)). A p53 reporter plasmid together with lentiviral packaging vectors (pMDLGagPol, pRSV-Rev, pIVS-VSV-G) and jetPRIME (Polyplus-transfection, Illkirch, France; Duncan et al., 2014 (link)) were co-transfected into HEK-293T cells to produce lentivirus. Supernatants were collected after 48 h and centrifuged to remove cell debris. PEG-it virus concentration reagent (Systems Biosciences, Palo Alto, CA, USA) was used to concentrate the virus suspension and PBS used as a re-suspension medium.

pMRX- IRES-GFP retroviral vector, which contains a green fluorescent protein (GFP) reporter, was a kind gift from Dr. Leonid Pobezinsky (University of Massachusetts Amherst). The PRMT5 construct was cloned into the pMRX- IRES-GFP vector by GeneScript company (Piscataway, NJ). Empty pMRX-IRES-GFP vector was used as a control. Retroviral supernatants were produced by transfecting Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, Inc, San Diego, CA) using Transporter 5 transfection reagent (Polysciences, Warrington, PA). Retroviral supernatants were concentrated with PEG-it™ virus concentration reagent (System Biosciences, Palo Alto, CA) prior to transduction. CD4 T cells were retrovirally transduced 24 h after activation with 10x concentrated retrovirus supernatants by spin-infection (660 × g for 90 min at 37°C) in the presence of polybrene (4 μg/ml). Transduction media was replaced with Th1-like iTreg polarizing media 4 h after spin-infection. Transduced cells were analyzed by flow cytometry and qRT-PCR.

The Ccr2 open reading frame (ORF) was obtained from GenScript (plasmid ID OMu22965D) and Ccr5 cDNA was obtained from Dharmacon (clone ID 12774). Ccr2 and Ccr5 ORFs were cloned into the pMRX-IRES-GFP plasmid, containing a green fluorescent protein (GFP) reporter (67 (link)). Empty pMRX-IRES-GFP plasmids were used as controls. Retrovirus supernatants were produced by transfecting Platinum-E (Plate-E) retroviral packaging cells (68 (link)) using Transporter 5 transfection reagent (Polysciences). Retrovirus supernatants were concentrated 10x in lymphocyte culture media with PEG-it™ virus concentration reagent (System Biosciences) prior to cell transduction. 2D2Rag2KO CD4⁺ T cells were retrovirally transduced 24 h after activation with 10x-concentrated retrovirus supernatants by spin-infection (660 × g for 90 min at 37°C) in the presence of polybrene (4 μg/mL). Transduction media was replaced with pathogenic Th17-polarizing lymphocyte culture media 4 h after spin-infection. Analysis of transduced cells was performed by gating on the GFP⁺ cell population.