Paptag2

Dll1-AP and Jag1-AP were constructed by cloning the sequences encoding amino acids 1–540 of mouse DELTA-LIKE PROTEIN 1 (NP_031891.2) and amino acids 1–1067 of mouse JAGGED1 (NP_038850.1), respectively, in frame with human placental AP from pAPtag-2 (GenHunter Corp., Nashville, TN). Total cDNA from mouse tissues was used as a template for PCR to generate modified cDNA ends with HindIII site in 5′ and BglII or BamHI site in 3′. After subcloning into pGEM-Teasy (Promega Corp.) and enzymatic digestion with appropriate restriction enzymes, purified fragments were directly cloned into pAPtag-2 using HindIII and BglII cloning sites to obtain the constructs referred to as pAPtag2-Dll1 and pAPtag2-Jag1. According to a previously described cloning technique [65 (link)], the pAPtag-2 was modified with prehybridized oligonucleotides encoding a signal peptide promoting secretion of recombinant Ctrl-AP. Eight prehybridized oligonucleotides encoding the same Igκ-chain leader sequence as found in commercial pSecTag/FRT/V5-His-TOPO (Invitrogen, Thermo Fisher Scientific) were cloned between HindIII and BglII cloning sites downstream from the cytomegalovirus promoter in pAPtag-2. The plasmidic constructs were sequenced and were used to transiently transfect COS-7 cells.