The full-length CDS of murine Nlrc5 was cloned through PCR using primers Nlrc5-Frw: 5′-GATTCTAGAGCCACCATGGACGCTGAGAGCATCC-3′ and Nlrc5-Rev: 5′-GCAATCGATTTAATTAATCAAAGAGTCTGCTGGTCAGTG-3′ with cDNA from B6 CD8 T-cell splenocytes. Nlrc5 cDNA was further subcloned into lentiviral expression vector lentiCas9-EGFP and further verified by DNA sequencing. Nlrc5 expression lentivirus (lentiNlrc5-EGFP) was generated as previously described65. As a control, an empty construct (lenti-EGFP) was generated encoding EGFP (enhanced green fluorescent protein) as a marker for transduction efficacy. Viral supernatants were used to transduce HEK-293T cell. HEK-293T cells were analyzed for MHC I expression by flow cytometry 48 h post transduction.

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