Transfection of Human Hematopoietic Stem Cells by a Gene Targeting Construct Containing the β-Globin Gene

• Main Authors: Sirous Zeinali, Bahman Zainali, Mohamadali Shokrgozar, Hossein Khanahmad, Seyedeh Soghra Moosavi, Esmat Kamali, Masood Soleimani, Mojhgan Shaikhpoor
• Format: Article
• Language: English
• Published: Royan Institute (ACECR), Tehran 2010-01-01
• Series: Cell Journal
• Subjects: Beta Thalassemia; Gene Targeting; Homologous Recombination; Gene Therapy
• Online Access: http://celljournal.org/library/upload/article/SheikhPoor.pdf
• ISSN: 2228-5806; 2228-5814

Objective: Replacment of CD133+ cell’s β globin gene by using a gene targeting constructcontaining the β globin gene and essential elements for homologous recombination.Materials and Methods: pFBGGT was amplified, then digested using the NheI and XhoIrestriction enzymes, and finally, a 13.3 kb band (naked DNA) was extracted from theagarose gel. Biological activity of positive and negative selection markers were checkedby transfection of COS-7 cells with linear plasmid. Hematopoietic stem cells (HSCs) wereseparated and transfected with linear plasmids using lipofection followed by positive andnegative selection. Polymerase chain reaction (PCR) were done on DNA from the selectedcells and the products were sequenced.Results: The results of biological activity assays showed that selection markers wereactive. PCRs for hygromycin, neomycin and joining segments were positive but PCRs forTK1 and TK2 genes were negative. Sequencing PCR product joining segment confirmedthe formation of homologous recombination.Conclusion: In this novel strategy gene replacement was achieved and biological activitiesof its components were observed.