Psbbi bla hace2

The Sleeping Beauty transposase system was used for the generation of BHK-21_hACE2, CHO-K1_hACE2, HEK293-FT_hACE2, and A549_hACE2 cells as previously described (32 (link), 33 (link)). Briefly, a semi-confluent 60-mm plate was seeded with each cell line and cotransfected with 0.5 μg of pCMV(CAT)T7-SB100 (transposase vector; Addgene, no. 34879) and 5 μg of pSBbi-Bla hACE2 (transposon vector), using TransIT-LT1 Transfection Reagent (Mirus Bio), following the manufacturer’s instructions. After 24 hours, cells were transferred to a T-75 flask and selected with blasticidin (250 to 500 μg/ml) for 2 weeks. The surface expression of hACE2 was confirmed by flow cytometry using anti-human ACE2 Alexa Fluor 647–conjugated Ab (R&D Systems, no. FAB9332R). The expression was further confirmed by immunoblot using hACE2 Ab (Cell Signaling Technology, no. 4355). The ORF of hACE2 (provided by S. Best from NIAID/NIH) was cloned into pSBbi-Bla vector (Addgene, no. 60526) as described (32 (link)).

The Sleeping Beauty transposase system was used for the generation of BHK-21_hACE2, CHO-K1_hACE2, HEK293-FT_hACE2 and A549_hACE2 cells as previously described ^{31 (link),32 (link)}. In brief, a semi-confluent 60 mm plate was seeded with each cell line and co-transfected with 0.5 µg of pCMV(CAT)T7-SB100 (Transposase vector, Addgene # 34879) and 5 µg of pSBbi-Bla hACE2 (Transposon vector), using TransIT-LT1 Transfection Reagent (Mirus Bio), following manufacturer instructions. After 24 h, cells were transferred to a T-75 flask and selected with 250–500 μg/ml of blasticidin for two weeks. The surface expression of hACE2 was confirmed by flow cytometry using anti-human ACE2 AlexaFluor 647-conjugated Ab (R&D Systems, # FAB9332R). The expression was further confirmed by immunoblot using hACE2 Ab (Cell Signaling Technology, # 4355). The open reading frame of hACE2 (kindly provided by Sonja Best from NIAID/NIH) was cloned into pSBbi-Bla vector (Addgene # 60526) as described ^{31 (link)}.