Construction and identification of eukaryotic vector incorporated with wild-type PTEN

Aim: To construct and identify the eukaryotic vector incorporated with wild-type PTEN (pcDNA 3.0 GA PTEN). Methods: A pair of primers matching the each end of PTEN cDNA and supplemented with appropriate endonuclease sites were designed according to the sequence published in NCBI GenBank. The PTEN mRNA was extracted from peripheral blood lymphocytes, and used as the template to synthesize the first strand of PTEN cDNA. The PTEN gene with complete expression sequence was amplified, which was cloned into pcDNA 3.0 vector after being double digested by endonucleases. The recombinants were identified with double GA endonuclease digestion, specific PCR and sequencing. Results: The results of double-endonuclease digestion and specific PCR showed that a 1.2 kb fragment had been cloned into pcDNA 3.0 vector, which was further identified by sequencing and NCBI BLAST analysis. Conclusion: pcDNA 3.0-PTEN has been successfully constructed which may provide the base for further study of PTEN in tumorigenesis mechanism.