ABSTRACT
The aim of the present study is to construct the recombinant eukaryotic expression and short hairpin RNA (shRNA) expression vectors of microRNA-21 and phosphatase and tensin homolog deleted on chromosome ten (PTEN). According to the gene sequence of microRNA-21 and PTEN, we designed and synthesized two pairs of single-stranded siRNA oligonucleotides and PCR primers. After annealing, the double-stranded DNA oligonucleotides were cloned into vector Psilencer4. 1-CMV. In addition, the gene sequences encoding pre-miR-21 and PTEN were amplified from colorectal cancer cell HCT-116 by RT-PCR. Then the PCR products were digested with restrictive endonuclease enzyme and cloned into vector pEGFP-N1. The constructed recombinant vectors were identified by restrictive digestion and DNA sequence analysis. The positive clone was confirmed by double enzyme digestion, and the enzyme fragments were consistent with the vector and purpose gene sequence. DNA sequencing confirmed that the purpose oligonucleotide fragments were correctly inserted in to the eukaryotic expression plasmids. It could be concluded that the microRNA-21 and PTEN eukaryotic expression and shRNA expression vectors have been successfully constructed, providing a foundation for further study on the effect of miR-21 on human colorectal cancer.
