ساخت سازه ي ژني حاوي ژن گاما گلو بين و مناطق تنظيمي خوشه ژني بتا جهت بررسي موتاسيونهاي موثر در فنوتيپ HPFH

Making a construct containing of y-globin gene and control regions of beta gene cluster aiming for studying of effective mutations in HPFH phenotype

In p-thalassemia patients are mutations or deletions in beta globin gene which due to absence or reduction of p-globin chain synthesis. Adult hemoglobin (HbA) contains 2 Alfa Globin and 2 Beta Globin chains (a2p2) while Fetal hemoglobin (HbF) contains 2 Alfa Globin and 2 Gamma Globin chains (a2y2). HbF switches to HbA in early weeks after the birth. A kind of p-thalassemia is HPFH phynotype. In these patients the level of HbF will be high in after the birth continuously. The gamma globin gene is in beta globin gene cluster and its expression will be control by locus control region (LCR) which located in upstream of beta gene cluster. The LCR composed of five DNase I hyper sensitive sites (HS1-5) in Erythroid cells. We amplified HS2, HS3, HS4 segments (mini LCR) and y-globin gene from the genomic DNA of a normal individual by PCR. Each of segments cloned in pTZ57R/T vector and then subcloned in the pBGGT vector, a derivative of PUC19 plasmid. This construct is a positive control and containing 5f HS4,HS3,HS2 3ʹ segments (mini LCR, 4 kb) and A Gamma gene (2 kb). That could be used in studying of some mutations which due to HPFH phenotype and affect expression of y-globin gene and also for induction of HbF production in gene therapy of p- thalassemia.