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. 2021 Jan 3;162(4):bqaa249. doi: 10.1210/endocr/bqaa249

Figure 2.

Figure 2.

Generation of the hPRL PitProKO-Luc BAC. Schematic illustration of the knockout of the prolactin proximal pituitary promoter using a 160kb hPRL BAC-Luc construct (hPRL WT). 5 kb pituitary promoter sequence was replaced with positive selection marker Galk, via a seamless BAC recombineering strategy to knockout the PRL pituitary promoter (A). Site-mutations were introduced in the 3 Pit-1 binding sites present in the proximal pituitary promoter (−256/+1) using hPRL 5 kb Luc plasmid (B). 256 bp DNA fragment containing mutated Pit-1 sites along with alternative acceptor site was amplified from hPRL 5kb Luc plasmid using BAC homology arms and inserted into the pituitary promoter knockout BAC replacing the Galk gene, thereby creating a construct expressing luciferase under the control of the extra-pituitary promoter (PitProKO) (C). GH3 cells containing hPRL WT or hPRL PitProKO BAC constructs were fixed in 1% formaldehyde and subjected to ChIP with either nonspecific immunoglobin G antibody or Pit-1 specific antibody. DNA was extracted and amplified by primers flanking the first and third pit-1 binding sites or a non-specific rat GAPDH sequence (D). Abbreviations: GalK, galactokinase; Luc, luciferase.