Sub cloning of mouse mousculus glucocerebrosidase enzyme gene in lentiviral vector and transfer to HEK cell line

Gene therapy is an important technique in clinical research which offers new visions for the treatment of genetic deficiencies. Gaucher disease caused by genetic deficiency of glucocerebrosidase (Gba) enzyme has attracted special consideration in gene therapy. The aim of this project is cloning and transfering of glucocerebrosidase enzyme gene to HEK cell line by enhanced lentiviral vector. The cDNA of glucocerebrosidase enzyme gene was synthesized, amplified with specific primers by PCR methods, cloned in non-expressing vector and sequenced. The recombinant gene was subcloned in enhanced lentiviral vector by GFP reporter gene. After culturing the HEK cell line, the recombinant lentiviral vector was transferred to them and the transfer of Gba gene was examined by GFP reporter gene. The amplification and cloning of glucocerebrosidase enzyme gene was confirmed by restrictive enzymes. The sequence of Gba gene was compared correctly by its reported sequence. Subcloning of Gba gene in lentiviral vector was confirmed by different restrictive enzymes. The transfer of Gba recombinant gene was confirmed by reporter gene with flurecent proteins. This project is a part of gene therapy protocol performed by the transferring of mouse glucocerebrosidase enzyme gene to HEK cells by lentiviral vector.