Prsv rev

To produce lentivirus, pCSII-EF-mVenus-hGem, pCgpV (Cell Biolabs), pRsv-Rev (Cell Biolabs), and pCMV-VSVG (Cell Biolabs) were transfected to HEK293Ta cells. The supernatant was concentrated using an ultracentrifuge and added to mESCs. mVenus-hGemini expressing clone was selected using fluorescence. To establish the stable cell line of p27-2A-mRFP, pSMPUWneo-TRET-p27-TetOff was digested by XhoI, and the linearized plasmid was transfected into miPSCs [23 (link)] with Lipofectamine 2000 (Life Technologies). p27-2A-mRFP expressing clone was selected by resistance to Geneticin (100 μg/ml; Roche) at first, then selection was done using fluorescent active cell sorting (FACS). The stable cell line was designated as miPSCs-p27. Transient transfection of pSMPUW-TRET-p27-TetOff to miPSCs and mESCs was done with Xfect (Clontech), Lipofectamine 2000 (Life Technologies), or Lipofectamine 3000 (Life Technologies) transfection reagents.

For the production of recombinant lentiviruses, third-generation packaging vectors based on the pRSV-Rev and pCgpV plasmids (Cell Biolabs, San Diego, CA, USA), the obtained lentiviral expression vectors, and pCMV-VSV-G (Cell Biolabs) for pseudotyping were co-transfected into Lenti-X HEK293T cells using CalPhos mammalian transfection kit (Takara/Clontech). For recombinant retroviruses, a transfer plasmid (pBABEpuro-based or pLZRS-IRES-ΔNGFR-based) and pCMV-VSV-G were co-transfected into GP2-293 packaging cells as above. Twenty-four hours after transfection the medium was refreshed; for lentiviruses it was supplemented with 1 mM sodium butyrate (Sigma-Aldrich, Saint Louis, MO, USA). Virus-containing supernatants were collected after 48 h, concentrated with PEGit (System Biosciences), and used for transduction in the presence of 0.01 mg mL⁻¹ polybrene (Sigma-Aldrich). MJS cells with TAP1 or TAP2 knock-outs were stably reconstituted with the wt or fluorescent TAP1 or TAP2 constructs using lentivirus vectors and cell-sorting for GFP- and MHC I-positive cells. The cells were subsequently transduced with a retrovirus coding for BoHV-1 UL49.5 and sorted for nerve growth receptor (NGFR)-positive cells or with a retrovirus coding for HSV-1 ICP47, HCMV US6, VZV UL49.5, or CPXV012, and selected with puromycin (2 µg mL⁻¹) (Sigma-Aldrich).

These steps were performed with the help of the GIGA Viral vectors platform (University of Liège, Belgium). In brief, Lenti-X 293T cells (Clontech) were co-transfected with pcgpV (Cell Biolabs), pRSV-Rev (Cell Biolabs), and VSV-G (Cell Biolabs) encoding vectors together with pLenti U6gRNA NME1-Cas9-GFP-Puro or pLenti U6gRNA NME2-Cas9-GFP-Puro or pLenti CRISPR-NT CONTROL. Lentiviral supernatants were collected 48–96 h post-transfection, filtrated, and concentrated 100x by ultracentrifugation. Lentivirus stocks were titrated with qPCR Lentivirus Titration (Titer) Kit (abm) and used to transduce cells. After 72 h, cells were selected with 2 μg/ml puromycin (Cayla/Invivogen). Then, cells expressing GFP were isolated and cloned by FACS on a FACSaria III 4L sorter (BD Biosciences). Each clone was tested by Western blotting. Clones that were negative for NME1 or NME2 expression were selected for further experiments.