Pcdna3.1 topo

The coding regions corresponding to full-length mouse and human Lhx1 were amplified by PCR from pineal cDNA and subcloned into pcDNA3.1-TOPO (Invitrogen, CA, USA) to yield expression plasmids mouse Lhx1/pcDNA3.1 and human Lhx1/pcDNA3.1, respectively. Site-directed mutagenesis (Stratagene, CA, USA) was performed to construct mouse Lhx1/pcDNA3.1 to generate a point mutation changing asparagine (amino acid 230) to serine. A DNA fragment corresponding to ∼1 kb of mouse Vip promoter was amplified by PCR from mouse genomic DNA and cloned into pGL3 basic vector (Promega) to yield the Vip reporter vector.

Total RNA was isolated from swine spleen using Trizol® (Invitrogen) and used for cDNA synthesis using Superscript II reverse transcriptase (Invitrogen). Sequences encoding full length swine CD40 (swCD40) or the extracellular domain (swCD40ED) was PCR amplified with Accuprime Pfx DNA Polymerase (Invitrogen) using primers based on GenBank sequence AF248545.1. The PCR product encoding the swCD40ED was ligated into PCR-TOPO vector (Invitrogen), and the ligation mix was used to transform E. coli TOP 10 cells (Invitrogen). Following colony screening and DNA sequencing of positive clones, the construct encoding the authentic swCD40ED was modified by overlap extension PCR to incorporate a secretory signal sequence at the 5’ terminus and the FLAG-tag at the 3’-terminus. The resultant gene encoding the swCD40ED and the PCR product encoding full length swCD40 were sub-cloned into the eukaryotic expression vector pcDNA3.1-TOPO (Invitrogen) and verified by sequencing. A construct encoding full length bovine CD40 (boCD40) was similarly generated. Recombinant swCD40ED was expressed as a FLAG-tagged protein by transfecting HEK-293 Free-Style cells (Invitrogen) and affinity purified using anti-FLAG M2-agarose affinity chromatography (Sigma) as previously described [30 (link), 31 (link)].