Pmys ires gfp retroviral vector

The lentiviral vector pCT-CD63-GFP (System Biosciences) and a packaging plasmid for generation of a CD-63-GFP MC3T3-E1 cell line or the pMYs-IRES-GFP Retroviral Vector (CELL BIOLABS) for generation of a MC3T3-E1 GFP cell line were transfected into HEK293T cells using PEI max (Polysciences). MC3T3-E1 cells were transduced with virus-containing medium from transfected HEK293T cells and polybrene (Merck) 48 h after transfection. Transduced MC3T3-E1 cells expressing GFP were selected using a cell sorter (SONY), and single cells were cultured to establish a cell line.

For retrovirus infection, Irf8 was cloned into pMYs-IRES-GFP retroviral vector (Cell Biolabs) as previously described^{8 (link)}. cDNA was amplified by PCR using the following pairs of oligo-nucleotide primers 5′-aactcgagaacaccatgtgtgaccg-3′ and 5′-tagtggcagattatcgccggcgat-3′. The ligation of DNA fragments was performed with T4 DNA ligase (M1801, Promega). The orientation of the insert was determined by PCR and restriction enzyme digestion.
ShRNA specific for Irf8 (5′-ccaggctttccgcatgtttttcaagagaaaacatgcggaaagcctgg-3′) was cloned into pMXs-U6-GFP retroviral vector (Cell Biolabs). BamHI and EcorI restrictions enzyme sites were introduced for subcloning. The ligation of DNA fragments was performed with T4 DNA ligase (M1801, Promega).
Retroviral particles were generated by transfecting the platinum-E cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 2 days, fresh virus supernatant was harvested and mixed with proliferative CD4⁺ naive T cells and 10 μg/ml protamine sulphate (APP Pharmaceuticals) in a 24-well plate and centrifuged for 90 min at 2,000 × g at 32 °C. The transduced naive CD4⁺ T cells were collected after 2 days and cell-sorted according to GFP expression. GFP⁺ cells were differentiated as described above. After 3 days, the cells were collected for PCR and ELISA assays.