Publication protocol
The full-length cDNAs for αV Integrin (GeneService Ltd cat. # 40146451), β5 Integrin (OpenBiosystems cat. # MHS1011-169792), Fos (OpenBiosystems cat. # MHS1011-60754), and Jun (OpenBiosystems cat. # MHS1010-97228128) were purchased for PCR amplification of the appropriate domains. A 2,971bp fragment of integrin αV encoding only the extracellular domain was PCR amplified (iProof High-Fidelity DNA polymerase; BIORAD cat. # 172-5302) with primers (see Table 1) designed with restriction endonuclease cloning sites Nco I and Eco RI. The following thermal cycler parameters were used for all PCR amplification steps: (1) 98°C – 2 min, (2) 98°C – 30 sec, (3)58°C – 1 min, (4) 72°C – 4 min, and (5) 72°C – 10 min, with 35 cycles run between steps 2 – 4. Following amplification, the cDNA fragment was excised from an agarose gel (BIO-RAD cat. # 161-3102), extracted (Qiagen cat. # 28706), restriction enzyme digested with Nco I and Eco RI, and ligated into the pQE-TriSystem Vector (Qiagen cat. # 33903) using the same cloning sites. A 3:1 insert to vector molar ratio was used for ligation. The ligation reaction was transformed into JM109 competent cells (Stratagene cat. # 200235) and streaked on an agarose plate with ampicillin (50 µg/ml) for overnight incubation at 37°C. Colonies were selected and grown in 5 ml of LB media containing ampicillin. Clones were purified (Qiagen cat. # 27106) and identified by restriction digest and verified by DNA sequencing. A similar cloning strategy was employed for the remaining cloning procedures. A 153 bp fragment of the Fos gene involved in forming a leucine zipper with Jun was PCR amplified from the full length cDNA with primers (Table 1) designed with restriction sites Eco RI and Xho I, and a five glycine linker. This linker region between the αV integrin extracellular domain and Fos was added to incorporate flexibility between the two expressed domains. The amplified Fos fragment was restriction digested, and cloned in frame utilizing the same cloning sites in the pQE-TriSystem vector. The completed construct was verified by DNA sequencing.
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