A Novel Single Step Double Positive Double Negative Selection Strategy For Β-Globin Gene Replacement




Introduction: Beta thalassemias are a heterogenous group of autosomal recessive disorders, characterized by reduced or absent production of the b-Globin chain by the affected allele. Transplantation of allogenic hematopoietic stem cells (HSC) is a curative approach but this therapeutic option is not available to the majority of patients. Transplantation of genetically corrected autologous HSC is an attractive approach for the cure of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy because it can precisely correct the mutant genes and restore their normal expression. In the present study a specific gene construction for b-Globin gene targeting was designed and constructed. This construction consists of: two homologous arms including, upstream and downstream regions of b-Globin gene, b-Globin gene as the target gene, hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase gene as negative selection marker.
Material and Methods: All segments were amplified by PCR and cloned in pTZ57T/A cloning vector and then sub cloned in pBGGT. The authenticity of cloning and sub cloning steps was checked by PCR, restriction analysis and finally by sequencing. The final plasmid was named pFBGGT. The mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing.
Results: The results of PCR, restriction analysis and sequencing during all cloning and sub cloning steps confirmed the authenticity of these steps. The results of sequencing of PCR products on selected cells, confirmed the occurrence of homologous recombination.
Conclusion: In this novel strategy, gene replacement was achieved in one step and by a single construct.