One of the pairs of TALENs targeting the human HPRT1 gene was subcloned into the mammalian expression vector pCDNA3.1(-) (Invitrogen) using XhoI and AflII. These enzymes excise the entire TALEN from pTAL3 or pTAL4 and place the coding sequence under control of the CMV (cytomegalovirus) promoter. The resulting plasmids were introduced into HEK293T cells by transfection using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Cells were collected 72 h after transfection and genomic DNA isolated and digested with Hpy188I, which cuts in the spacer sequence of the TALEN target site. After digestion, a chromosomal fragment encompassing the target site was amplified by PCR. Upon completion, the reactions were incubated for 20 min at 72°C with 4 µl of Taq DNA polymerase. PCR products then were digested with Hpy188I and cloned in a TOPO TA vector (Invitrogen). Independent clones containing the full-length PCR product were sequenced to evaluate mutations at the cleavage site.
The TALENs targeting the Arabidopsis ADH1 gene were subcloned into the plant expression vector pFZ14 (27 (link)) using XbaI and SacI. These enzymes excise the entire TALEN from pTAL3 or pTAL4 and place the coding sequence under control of the CaMV (cauliflower mosaic virus) 35S promoter. Recombinant plasmids were transformed into Arabidopsis protoplasts as previously described (27 (link)). Forty-eight hours after transformation, DNA was prepared and digested with PflFI, which cuts in the spacer sequence of the TALEN target site. After digestion, a chromosomal fragment encompassing the target site was amplified by PCR and the reaction products were once again digested with PflFI and run on an agarose gel. The band corresponding in size to undigested product was excised and cloned and individual clones were sequenced to evaluate mutations at the cleavage site.
The TALENs targeting the Arabidopsis ADH1 gene were subcloned into the plant expression vector pFZ14 (27 (link)) using XbaI and SacI. These enzymes excise the entire TALEN from pTAL3 or pTAL4 and place the coding sequence under control of the CaMV (cauliflower mosaic virus) 35S promoter. Recombinant plasmids were transformed into Arabidopsis protoplasts as previously described (27 (link)). Forty-eight hours after transformation, DNA was prepared and digested with PflFI, which cuts in the spacer sequence of the TALEN target site. After digestion, a chromosomal fragment encompassing the target site was amplified by PCR and the reaction products were once again digested with PflFI and run on an agarose gel. The band corresponding in size to undigested product was excised and cloned and individual clones were sequenced to evaluate mutations at the cleavage site.
