Cell and Tissue Journal

Abstract Aim: The aim of this research was cloning and expression of human Midkine coding gene (mdk) in Escherichia coli that achieved in a laboratory-scale experiment.
Material and Methods: Methods were included cell culture, RNA extraction, cDNA synthesis, cloning techniques, induction of expression by IPTG (isopropyl thiogalactosidase), expression evaluation using polyacrylamide gel and confirmation by Western blot techniques.
Results: Midkine gene was cloned in pET-21a (+) and then transformed into Origami strain of E. coli. This growth factor was expressed in cytoplasmic level by a colony containing pETmdk recombinant after 16 hours incubation at 18°C and 250 rpm mixing. Expression of histidine tagged 13 kD protein confirmed by Western blotting technique.
Conclusion: Because Origami strain is trxB and gor genes mutant strain its cytoplasm is an oxidizing environment. Due to this, it enhances disulfide bond forming, therefore it seems that after expression of midkine, cysteine residues make an intra-molecular disulfide bridge and remains in soluble form. These conditions provide suitable environment for the proper folding of the protein and consequently solubilization of the protein.

Abstract Aim: The aims of this study were subcloning of Dj-1(PARK7) gene to lentiviral transfer vector, recombinant lentiviruses production and target cells infection with these viruses.
Material and methods: DJ1 gene obtained from pcDNA-DJ1 vector using restriction enzymes EcoR1 and Xho1. Lentiviral transfer vector was coincidental digested using EcoR1 and Sall enzymes. DJ-1 gene was interred into the lentiviral transfer and upstream of Jred gene using T4-DNA ligase. As DJ1-IRES-Jred sequence was placed in downstream and control of CMV promoter. To production of recombinant lentivruses, the made vector is coincidental transferred into the HEK-293T (Human Embryonic Kidney) cells with two packaging and envelope lentiviral vectors. Produced virus was used for target cells infection.
Results: For integrity of subcloning, enzymatic tests and PCR were used.
Fluorescent microscopy also used to show expression of Jred reporter gene that showed the success of our gene transferring .Then RT-PCR was done for showing overexpression of  DJ1 gene in transduced cells compare with normal cells.
Conclusion: This study show the lentiviral vectors success in gene transferring to eukaryotic cells and clear that this vectors can be used in treatment of nervous system diseases.

Abstract Aim: The purpose of present research was extraction and cloning of tropinonereductase-II gene (tr-II) at antisense direction in pBI121 binary vector to provide transgenic plants with low rate of tropinonereductase-II enzyme and high production of scopolamine and hyoscyamine for future projects.
Material and methods:  Total RNA was extracted from Iranian native Hyoscyamusnigerroots, and the interest gene after cDNA synthesis and cloning at antisense direction in pBI121 binary vector, was transfered to Agrobacterium tumefacience. Accurate cloning was studied through 3 methods; enzymatic digestion, PCR and DNA sequencing. The bioinformatic characters of the gene were then surveyed.
Results: Three used methods confirmed true cloning in high efficiency. Nucleotide sequence of the gene revealed the 783 bp in length, encoding a polypeptide of 260 amino acid residues, with high similarity to that one registered in NCBI. The predicted molecular mass and isoelectric point of deduced polypeptide were 28437.3 Da and 5.46, respectively. Protein structures were not completely similar to those previously reported at PDB data base. Also, phylogenic study demonstrated that this gene belongs to the group I of TRs.
Conclusion: Due to successful cloning and high similarity of nucleotide and polypeptide sequences of gene with those recorded in world data bases; it is expected to get success in access to main purpose.