Genetic Manipulation of MOLT-3 Cells

MOLT-3 cells were obtained from the ATCC. To obtain MOLT-3 cells lacking LCK, preassembled ribonucleotide complexes consisting of purified Cas9 (Synthego) and chemically modified synthetic sgRNA (target sequence: 5′-GCTCCGCGTCCTTGCGGCTC-3′) (Synthego) were delivered into MOLT-3 cells via nucleofection using the Cell Line Nucleofector kit V with the Nucleofector II device (Lonza). Clones obtained by limiting dilution were screened by Western blotting with anti-LCK antibody 3A5 (Santa Cruz Biotechnology; sc-433). Protein loading was assessed with anti-actin antibody (Santa Cruz Biotechnology; sc-47778).
MOLT-3 cells lacking PAK2 were obtained by transiently transfecting a plasmid expressing an sgRNA targeting PAK2 (target sequence, 5′-GATTTCGTATGATCCGGTCG-3′) by nucleofection, along with a plasmid expressing Cas9. Clones obtained by limiting dilution were screened by Western blotting with anti-PAK2 (Cell Signaling Technology; 2608) and anti-actin antibodies. To obtain MOLT-3 cells lacking all group I PAKs, Cas9/sgRNA complexes targeting PAK1 (target sequence, 5′-AGGCACCGTGTACACAGCAA-3′) were delivered into PAK2 KO MOLT-3 cells by nucleofection. Clones obtained by limiting dilution were screened by Western blotting with anti-PAK1 (Cell Signaling Technology; 2602) and anti-actin antibodies.
MOLT-3 double-KO cells lacking SERINC3 and SERINC5 (MOLT-3 S3/5 double-KO cells) have been described (26 (link)). To obtain MOLT-3 cells lacking SERINC3, SERINC5, and CD4 (M3 triple-KO cells), Cas9/sgRNA complexes targeting CD4 (target sequence, 5′-GAGGTGCAATTGCTAGTGTT-3′) were delivered into MOLT-3 S3/5 double-KO cells by nucleofection. Clones obtained by limiting dilution were screened by flow cytometry after staining with anti-CD4 antibody (BioLegend; 300502) and PE-conjugated secondary antibody (Jackson ImmunoResearch; 115-116-146).
M3 triple-KO/CD4 and M3 triple-KO/CD4_ΔCT cells were obtained by retroviral transduction of M3 triple-KO cells with pCXbsrCD4 and pCXbsrCD4_ΔCT, respectively, followed by selection with blasticidin (5 μg/mL). The ectopic expression of CD4 or CD4_ΔCT was confirmed by flow cytometry. To examine the effects of Nef on CD4 surface levels, M3 triple-KO/CD4 and M3 triple-KO/CD4_ΔCT cells were transduced with empty MSCVpuro or MSCVpuroNef_LAI, followed by selection with puromycin (1 μg/mL). To facilitate the entry of R5-tropic viruses, M3 triple-KO/CD4_ΔCT cells were transduced with pCX4pur-synCCR5, followed by selection with puromycin (1 μg/mL).
M3 triple-KO cells expressing reduced amounts of the μ2 subunit of AP-2 (AP2M1) were obtained by delivering Cas9/sgRNA complexes targeting AP2M1 (target sequence, 5′-CGATGTCATCTCGGTAGACT-3′) into M3 triple-KO cells by nucleofection. Clones obtained by limiting dilution were screened by Western blotting with anti-AP50 (BD Biosciences; 611351) and anti-actin antibodies. To restore AP2M1 expression, clones expressing reduced amounts of AP2M1 were transduced with pCX4purAP2M1, followed by selection with puromycin (1 μg/mL).