AatII (10 U/µL)

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	To create a crippled CMV promoter, the majority of the enhancer region was deleted from EGFP-N1 (Clontech, Takara) using the restriction enzyme AatII (Thermo Fisher) to generate the plasmid delCMV-EGFP-N1 described in Watanabe and Mitchison (77).

Protocol tips
To create a crippled CMV promoter, the majority of the enhancer region was deleted from EGFP-N1 (Clontech, Takara) using the restriction enzyme AatII (Thermo Fisher) to generate the plasmid delCMV-EGFP-N1 described in Watanabe and Mitchison (77).

Publication protocol

"The plasmid mApple-Lifeact-7, a gift from Michael Davidson (Florida State University, Tallahassee, Florida, USA) (Addgene plasmid 54747), was subcloned into the XhoI and NotI restriction sites of the pHR-SIN lentiviral vector. For virus production, 6 μg pHR-SIN-mApple-Lifeact-7 was transfected with 4 μg pMDG VSV-G (envelope plasmid) and 4 μg pCMV delta 8.9 (packaging plasmid) into HEK293T cells (at 70% confluency) using TransIT transfection reagent (Mirrus) and cultured overnight. Supernatant was collected after 48 hours, centrifuged, and filtered through a 0.45 μm filter before concentration with LentiX concentrating solution (Clontech, Takara). 107 CTLs were transduced with concentrated viral particles in the presence of 6 μg protamine sulfate (P4020-1G, MilliporeSigma) in 1 mL of T cell media and left for 4 hours at 37°C. CTLs were diluted to 106 cells/mL before stimulation at a 1:1 ratio with irradiated PBMCs in the presence of 1 μg/mL PHA.

To generate the EGFP-ARPC3 construct, mRNA was extracted from OT-I CTLs using an RNEasy Mini Kit (QIAGEN). OT-I cDNA was generated from this using the AccuScript High-Fidelity First Strand cDNA Synthesis Kit (Agilent). To create a crippled CMV promoter, the majority of the enhancer region was deleted from EGFP-N1 (Clontech, Takara) using the restriction enzyme AatII (Thermo Fisher) to generate the plasmid delCMV-EGFP-N1 described in Watanabe and Mitchison (77). ARPC3 primers were used to amplify the coding region based on NCBI Reference Sequence NM_019824.4: forward, 5′-ATGCCGGCATACCACTCTTCTCTC-3′; and reverse, 5′-CTGCCCAGGCCCCGAAAGAC-3′ with XhoI and BamHI restriction sites added. The product was ligated into delCMV-EGFP-N1, and 5 μg of DNA was nucleofected with 5 μg mApple-Lifeact-7 using the P3 Primary Cell Kit (V4XP-3024, Lonza) following the manufacturer’s recommendation for the 4D nucleofector (Lonza)."

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