Cloning and Expression of β3-Integrin-GFP Fusion

Full-length mouse β3-integrin cDNA was provided by Dr. Patrick Ross (Washington University School of Medicine, St. Louis, MO) (Weerasinghe et al., 1998 (link); Legler et al., 2001 (link)). Fusion of the enhanced GFP (EGFP) coding sequence (CLONTECH Laboratories, Inc.) with β3-integrin cDNA was performed in two steps. First, a COOH-terminal fragment of β3-integrin, containing a unique EcoRV site (underlined), was amplified with a 5′-ATGGATCCAAGGGTCCTGATATCCTG-3′ forward and 5′-AATACCGGTGAAGTCCCCCGGTAGGTGATA-3′ reverse primer pair, in order to remove the stop codon. The amplified sequence was digested with BamHI (5′) and AgeI (3′) restriction enzymes and cloned into pcDNA3/EGFP, containing the EGFP cDNA sequence 3′ to an AgeI site. pcDNA3/EGFP was prepared by insertion of the HindIII/NotI EGFP containing fragment from pEGFP-N1 (CLONTECH Laboratories, Inc.) into pcDNA3 at these sites (Invitrogen). The remaining NH₂-terminal part of the β3-integrin cDNA sequence (5′ to the EcoRV site) was cut out of the original vector with BamHI and EcoRV, and inserted at the respective sites into pcDNA3/EGFP, resulting in full-length β3–GFP-integrin joined by a short spacer (SerProValAlaThr).