a novel single step double positive double negative selection strategy for β-globin gene replacement
نویسندگان
چکیده
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.
منابع مشابه
Introduction of the negative selection marker into replacement vectors by a single ligation step.
Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an enrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selectio...
متن کاملA Novel Compact Ultra-Wideband Antenna with Single and Double Band Rejection
Band-notch characteristic has been of great interest recently to overcome the electromagnetic interference of Ultra-wideband systems (UWB) with other existing ones. In this paper, we present a novel microstrip-fed antenna with band rejection property appropriate for UWB applications. Band-notch characteristic has been achieved by adding a rectangular resonant element to the ground section. A pr...
متن کاملCo-inheritance of --MED double gene deletion and αααAnti3.7 triplication on α-globin gene in Mazandaran at 2016
Alpha Thalassemia is one of the most prevalent disorders worldwide with a [T1] high carrier rate in Mazandaran province (north of Iran). Carriers of --MED double gene deletion are at risk of having a child with hemoglobin haemoglobin[T2] H (HbH) disease, if they marry a silent carrier. Co-inheritance of αααAnti3.7 triplication that cannot be detected using hem...
متن کاملA set-covering formulation for a drayage problem with single and double container loads
This paper addresses a drayage problem, which is motivated by the case study of a real carrier. Its trucks carry one or two containers from a port to importers and from exporters to the port. Since up to four customers can be served in each route, we propose a set-covering formulation for this problem where all possible routes are enumerated. This model can be efficiently solved to optimality b...
متن کاملDesign of Dual-Band Double Negative Metamaterials
A dual-band artificial magnetic material and then a dual-band double-negative metamaterial structure based on symmetric spiral resonators are presented. An approximate analytical model is used for the initial design of the proposed structures. The electromagnetic parameters of the proposed metamaterial structure retrieved using an advanced parameter retrieval method based on the causality princ...
متن کاملمنابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
cell journalجلد ۸، شماره ۲، صفحات ۹۸-۱۰۵
میزبانی شده توسط پلتفرم ابری doprax.com
copyright © 2015-2023