DNA oligos (Integrated DNA Technologies, Inc.) containing sgRNA sequences (see Supplementary Table 2 ) were annealed and ligated into lentiCRISPRv2 (Addgene, #52961, gift from Feng Zhang). Lentivirus was packaged by co-transfection of constructs with the 2nd generation packaging plasmids pMD2.G and PsPax using jetPEI (Polyplus-transfection) into six-well plates with HEK293T cells according to protocol. Sixty hours post-transfection supernatants were collected, centrifuged at 1500 rpm for 5 min, and filtered through a 0.45 μm filter. Calu-3 or Vero-E6 cells were transduced with lentiviruses in the presence of 7 ug/ml polybrene (TR-1003, Sigma) and then selected with puromycin for 7 days. Knockout was confirmed by western blot, Real-Time PCR, or sequencing. For GATA6 complementation experiment, GATA6 knockout cells were complemented by transfection of 2.5 µg pBabe 3XFLAG-wt GATA6-3XAU1 puro vector (Addgene #72607) using Lipofectamine 3000 (L3000015 ThermoFisher) according to manufacturer’s instructions. Seventy-two hours post-transfection, the expression of GATA6 was analyzed by western blot.
Pbabe 3xflag wt gata6 3xau1 puro vector
Manufactured by Addgene
PBabe 3XFLAG-wt GATA6-3XAU1 puro vector is a plasmid construct that contains a 3XFLAG-tagged wildtype GATA6 gene and a puromycin resistance cassette. The vector is intended for ectopic expression studies.
Automatically generated - may contain errors
2 protocols using pbabe 3xflag wt gata6 3xau1 puro vector
1
Lentiviral CRISPR-Cas9 Knockout and Complementation
2
CRISPR-Mediated Gene Knockout and Complementation
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DNA oligos (Integrated DNA Technologies, Inc.) containing sgRNA sequences (see Supplementary Table 2) were annealed and ligated into lentiCRISPRv2 (Addgene, #52961, gift from Feng Zhang). Lentivirus was packaged by cotransfection of constructs with the 2nd generation packaging plasmids pMD2.G and PsPax using jetPEI (Polyplus-transfection) into 6-well plates with HEK293T cells according to protocol. Sixty hours post transfection supernatants were collected, centrifuged at 1500 rpm for 5 minutes and filtered through a 0.45 μm filter. Calu-3 or Vero-E6 cells were transduced with lentiviruses in the presence of 7 ug/ml polybrene (TR-1003, Sigma) and then selected with puromycin for 7 days.
Knockout was confirmed by western blot, Real-Time PCR or sequencing. For GATA6 complementation experiment, GATA6 knockout cells were complemented by transfection of 2.5 µg pBabe 3XFLAG-wt GATA6-3XAU1 puro vector (Addgene #72607) using Lipofectamine 3000 (L3000015 ThermoFisher) according to manufacturer's instructions. Seventy-two hours post transfection, the expression of GATA6 was analyzed by western blot.
Knockout was confirmed by western blot, Real-Time PCR or sequencing. For GATA6 complementation experiment, GATA6 knockout cells were complemented by transfection of 2.5 µg pBabe 3XFLAG-wt GATA6-3XAU1 puro vector (Addgene #72607) using Lipofectamine 3000 (L3000015 ThermoFisher) according to manufacturer's instructions. Seventy-two hours post transfection, the expression of GATA6 was analyzed by western blot.
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