Jetpei macrophage in vitro dna transfection reagent

pIRES-Ugi-eGFP and pIRES-hUNG2-eGFP reporter plasmids were constructed by replacing the neoR cassette for eGFP using SmaI and XbaI cloning sites of pIRESneo3 (Clonetech, Mountainview, CA). The empty pIRES-eGFP plasmid was used as a transfection control (shown). Primary macrophages derived from blood monocytes were cultured in the presence of M-CSF for 7 days prior to transfection using jetPEI-Macrophage in vitro DNA transfection reagent (Polyplus). Cells expressing GFP were sorted by FACS 48 hr post-transfection. GFP⁺ cells (indicating the presence of hUNG2 or its inhibitor protein) were allowed to adhere for 24 hr and then spin-infected with HIV^SF162(CCR5) virus. Cells were harvested 3 days post-infection, and DNA was extracted using QIamp DNA kit (Qiagen, Valencia, CA) for PCR and Ex-PCR analyses. Due to the low efficiency of transfection using MDM target cells, it was not possible to obtain immunoblots for Ugi or hUNG2 expression levels in these samples. However, the specific and opposite effects of the Ugi and hUNG2 transfection, the absence of an effect upon transfection with pIRES-eGFP plasmid, and the alternative approach of deleting vpr are strongly supportive of our interpretations.

The primers for MGF360-11L siRNAs are listed in Table 2. siRNA transfection was conducted using jetPEI^®-macrophage in vitro DNA transfection reagent (Polyplus) according to the instructions. SiMGF360-11L was transfected into PAMs for 24 h, and the cells were infected with ASFV (MOI = 1) for another 12 h or 24 h. IFN-β, ISG15 and ISG56 mRNA levels were measured by RT–PCR. The cytokines IFN-β, IL-1β and IL-6 in the cell culture supernatants were measured by commercial ELISA kits (Thermo Fisher). RT-PCR and Western blotting was used to measure the expression level of the P72 protein.Table 2

siRNA sequences used in this study.

Primers	Sequence (5′ → 3′)
siMGF360-11L-forward	CAAAUACUGGUACGCGAUAdTdT
siMGF360-11L-reverse	UAUCGCGUACCAGUAUUUGdTdT
siNC-forward	UUCUCCGAACGUGUCACGUTT
siNC-reverse	ACGUGACACGUUCGGAGAATT