Psectag frt v5 his topo

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.

All materials were purchased from Sigma-Aldrich Merck (Gillingham, UK) unless otherwise stated. Vectors pCR8/GW/TOPO and pSecTag/FRT/V5-His-TOPO, and the Gateway (GW) vector conversion system were obtained from Invitrogen (Carlsbad, CA, USA). LR Clonase II and the Flp-In-CHO cell line were also from Invitrogen (Carlsbad, CA, USA). FITC-labelled lectins SNA-I and MAA were from EY Laboratories (San Mateo, CA, USA) and biotinylated SNA-I, RCA-I, and MAA lectins were from Vector Laboratories (Peterborough, UK). Oligonucleotide primer synthesis and DNA sequencing was carried out by Eurofins. Proof-reading Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) was used for PCR amplification of cDNA during construction and standard Taq polymerases (Promega, Madison, WI, USA) for screening. PNGase F was from New England Biolabs (Ipswich, MA, USA).