Cloning and Lentiviral Production of Mouse and Human Chemerin

A mouse chemerin cDNA made from groin fat RNA was used to amplify the chemerin coding sequence using the following primer set: forward primer 5′-ACCGAATTCAGGTGAAGCCATGAAGTGCT-3′, with an EcoRI restriction site, and reverse primer 5′-TGCGGCCGCCTGTCTAGGGCTTATTTG-3′, with a NotI restriction site. The chemerin fragment was then cloned into the pCDH-CMV-MCS-EF1-copGFP (CD511B-1) vector (LV-GFP, System Biosciences, U.S.A.) to produce LV-mChemerin-GFP. Similarly, the human chemerin coding sequence was amplified from JEG-3 cells cDNA by using the following primer set: forward primer, 5′-TGGAAGAAACCCGAGTGCAAA-3′ and reverse primer, 5′- AGAACTTGGGTCTCTATGGGG-3′. The PCR product was ligated into the pMD18-T Vector (Takara Bio Inc., Shiga, Japan), after which the chemerin coding sequence was liberated by digestion with EcoRI and NotI and cloned into LV-GFP to produce LV-hChemerin-GFP. The constructs were transfected into packaging HEK293T cells as previously described to produce lentiviral particles [17 (link),18 (link)], and the lentivirus-containing supernatant was harvested and tittered using the QuickTiter Lentivirus quantitation kit (Cell Biolabs Inc, San Diego, CA, U.S.A.).