Design and Constructing of a Vector Containing EGFP Reporter Gene for the Purpose of Gene Targeting by Means of Homologous Recombination in Beta Globin locus.

Beta-thalasseamia is one the most prevalent genetic disorder in Iran. At the present, more than 15000 affected individuals exist in Iran, while no permanent cure is obtainable yet. Recently, gene therapy by homologous recombination (HR) seems to be the only safe way to treatment the beta-thalasseamia disorder. HR replace the damaged gene with wild type, without causing any changes elsewhere in the genome. In the present study, in order to evaluation of gene targeting by HR in Beta globin locus, a specific construct was designed. This construct contained enhanced green fluorescent protein (EGFP) gene which was controlled by the beta-globin promoter. Hence it will just be expressed in erythroid cells and consequently expressed cells will emit green florescence light. In order to avoid non-homologous recombination cells selecting, neomycin (Neo) resistant and thymidin kinase (TK) genes were included as positive and negative selectable markers, respectively. To facilitate HR process the upstream (US) and the downstream (DS) of beta-globin gene were included within construct. The fragments were amplified using polymerase chain reaction (PCR) method and then cloned in the following order TK1-US-Promoter-EGFP-Neo-DS-TK2. The final construct was confirmed by digestion analysis using certain restriction enzymes and sequencing. On account of the fact that the frequency of HR is low, thus Hematopoetic stem cells (HSC) should be expanded befor and after gene targeting by HR. In this study HSCs were also separated from cord blood and then to find out the high expansion level of the HSCs, different culture mediums with different growth factors were evaluated. Results revealed that the serum free media was the best medium for the expansion of HSCs. Finally HSCs will be transfected by designed construct and its expression and integration will be evaluated.