CD69 Retroviral Overexpression in EL4 Cells

The full open reading frame (ORF) of CD69 was amplified by PCR using specific primers as listed in Table 1 (Sangong Biotech, Shanghai, China). Subsequently, the CD69 ORF was cloned into the retroviral vector pMX containing internal ribosomal entry site-green fluorescent protein (IRES-GFP) to generate the plasmid of pMX-CD69. The pMX-CD69 and control pMX were transfected into Plat-E cells to generate retrovirus with pMX-CD69 or pMX, respectively. Retroviral transfection was performed as described previously^{27 (link),28 (link)}. Briefly, EL4 cells were transfected with retrovirus at MOI (multiplicity of infection) of 50 in the presence of 5 μg/ml polybrene (Millipore) for 48 h, then the cells were collected for further experiments.