Transient transfection and stable cell line generation using lentiviruses

QG Qin Gong
KR Kim Robinson
CX Chenrui Xu
PH Phuong Thao Huynh
KC Kelvin Han Chung Chong
ET Eddie Yong Jun Tan
JZ Jiawen Zhang
ZB Zhao Zhi Boo
DT Daniel Eng Thiam Teo
KL Kenneth Lay
YZ Yaming Zhang
JL John Soon Yew Lim
WG Wah Ing Goh
GW Graham Wright
FZ Franklin L. Zhong
BR Bruno Reversade
BW Bin Wu
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293T-ASC-GFP cells were described previously8. All other 293T-GFP responder cell lines were constructed by lentiviral transduction (pCDH vector backbone, System Biosciences). All transient expression plasmids were cloned into the pCS2+ vector using standard restriction cloning. Polyclonal Cas9/CRISPR knockout cell lines were generated with lentiCRISPR-v2 (Addgene #52961) and selected with puromycin. Knockout efficiency was tested with western blot 7–10 days after puromycin selection. Site-directed mutagenesis was carried out with QuickChangeXL II (Agilent #200522).

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