Isolation and cloning of predicted microrna in pten gene into pegfp-c1 vector

Phosphatase and tensin homolog (PTEN) has great important role in tumor cell migration and proliferation by its phosphatase activity (1). Down regulation of the PTEN gene is frequently occurred in different cancers (2-4). Recent evidences indicated that intragenic miRNAs may regulate the expression of their host genes. miRNAs are small non-coding RNAs which play their role through complementarities to target mRNAs (5-7). Alteration in the expression level of these regulatory molecules has been observed in different diseases including cancers (8). In order to confirm the presence of proposed miRNA-like structures, experimental studies are inevitable (9). The aim of the present study was to identify and clone the predicted miRNAs in PTEN gene. Human genomic DNA was extracted from peripheral blood cell through standard protocol. To clone the predicted miRNA, the PTEN region containing 389 bp putative miRNA was PCR amplified. Then, PCR product was cloned into the linear pTG19-T vector and subjected to enzymatic digestion and finally ligated into the pEGFP-C1 expression vector. Finally, the recombinant pEGFP-C1 vector has been sequenced for verification of intended insert. Different softwares such as gene runner and primer blast were used to perform primer designing. Among the highly conserved structures predicted previously, three met the desired criteria for bearing a putative miRNA. One out of these, selected for primer designing because the rest were GC rich and were excluded from further analysis. Following TA cloning, appropriate colonies were selected by colony PCR. Then the fragment was isolated from TA vector by enzymatic digestion and ligated into pEGFP-C1 vector. Alignment of the sequencing result of recombinant vectors confirmed the accuracy of amplified region. As introducing even one wrong base pair could affect the accuracy of miRNA processing in cell, the accuracy of this sequencing result is a prerequisite step before cell transfection. We are designing further studies to investigate the effect and function of this predicted miRNA in vitro

کلیدواژه ها:

Gene and Cancer ، Cell and Cancer ، Multidisciplinary Cancer Research ، Cancer Genetics ، Solid Tumors