Bikhof Torbati, Maryam, Khanahmad, Hossein, Jamshidi, Fatemeh, Sadeghizadeh, Majid, Karimipour, Morteza, Sadeghizadeh, Majid, Shokrgozar, Mohammad Ali, Amanzadeh, Amir, Zeinali, Sirus (2008). Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and β-globin gene: Introduction to gene therapy of major β-thalassemia. , 10(0), -.
Maryam Bikhof Torbati; Hossein Khanahmad; Fatemeh Jamshidi; Majid Sadeghizadeh; Morteza Karimipour; Majid Sadeghizadeh; Mohammad Ali Shokrgozar; Amir Amanzadeh; Sirus Zeinali. "Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and β-globin gene: Introduction to gene therapy of major β-thalassemia". , 10, 0, 2008, -.
Bikhof Torbati, Maryam, Khanahmad, Hossein, Jamshidi, Fatemeh, Sadeghizadeh, Majid, Karimipour, Morteza, Sadeghizadeh, Majid, Shokrgozar, Mohammad Ali, Amanzadeh, Amir, Zeinali, Sirus (2008). 'Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and β-globin gene: Introduction to gene therapy of major β-thalassemia', , 10(0), pp. -.
Bikhof Torbati, Maryam, Khanahmad, Hossein, Jamshidi, Fatemeh, Sadeghizadeh, Majid, Karimipour, Morteza, Sadeghizadeh, Majid, Shokrgozar, Mohammad Ali, Amanzadeh, Amir, Zeinali, Sirus Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and β-globin gene: Introduction to gene therapy of major β-thalassemia. , 2008; 10(0): -.

Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and β-globin gene: Introduction to gene therapy of major β-thalassemia

Pathobiology Reserach
Article 9, Volume 10, Issue 0, 2008    PDF (494.13 K)
Authors
Maryam Bikhof Torbati1; Hossein Khanahmad2; Fatemeh Jamshidi3; Majid Sadeghizadeh4; Morteza Karimipour3; Majid Sadeghizadeh4; Mohammad Ali Shokrgozar5; Amir Amanzadeh5; Sirus Zeinali* 3
1Department of Biology, Islamic Azad University-Science and Research Campus, Tehran, Iran
2Department of BCG, Pasteur Institute of Iran, Karaj, Iran
3Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran
4Department of Genetics, School of Basic Sciences, Tarbiat Modares University, Tehran, Iran
5Department of Cell Bank, Pasteur Institute of Iran, Tehran, Iran
Abstract
Objective: β-thalassemia is caused by absence or reduction of β-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and β-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and β-globin gene for transfer to the target cells for gene therapy of β-thalassemia.
Materials and Methods: HS2, HS3, HS4 segments (mini LCR) and β-globin gene with 5΄ and 3΄ UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids (Plp1, Plp2, Plp/VSVG) were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses.
Results: The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random integration of construct into the genome was evaluated.
Conclusion: The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene.
Keywords
miniLCR; β-thalassemia; β-globin gene
Statistics
Article View: 95
PDF Download: 48
Statistics
Number of Journals45
Number of Issues2,174
Number of Articles24,684
Article View25,076,094
PDF Download17,776,382