Association of AIRE polymorphism and the susceptibility to multiple sclerosis in Iranian population

Background: Multiple Sclerosis [MS] is the most common cause of neurologic disability in young adults. Recently, the AIRE gene was identified as a genetic risk factor for several autoimmune diseases in genome wide association studies. The aim of this study was to further investigate the possible role of the AIRE gene in susceptibility to MS in Iranian population

Methods: A total of 112 MS patients and 94 ethnically matched controls were included in the study. The Single-Nucleotide Polymorphism [SNP] [rs1800520, C>G] with a global MAF=0.2282/1143 was selected and genotyped using HRM real-time PCR method

Results: Results showed that AIRE SNP rs1800520 was significantly less common in the MS patients than in healthy controls [17.8 vs. 28.7%, pc=0.032, OR=0.54,95% CI 0.279,1.042]. Also, the frequency of allele G was significantly higher among the control group than in the case group [37.77 vs. 25%, pc=0.014]. Interestingly, mRNA transcribed on the rs1800520 SNP showed decreased free energy than the wild type suggesting that its increased stability may be responsible for the different activities of the polymorphic AIRE molecule

Conclusions: This is the first study investigating the relationship between AIRE gene and the susceptibility to MS. These results indicated that the rs1800520 SNP is not a susceptibility gene variant for the development of MS in Iranian population

increase in protein and plasmid yields of E. coli with optimized concentration of ampicillin as selection marker

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid

Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized

Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG [0.5 mM] and incubation with 0, 100, 200 and 300 micro g.mL[-1] ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve

Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 micro g.mL[-1] ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07x10[9], 3.21x10[9], 2.32x10[10] , 8.11x10[8], respectively. The plasmid yields were 55 ng.micro L[-1], 69 ng.micro L[-1], 164 ng.micro L[-1] and 41 ng.micro L[-1], respectively

Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration [200 micro g.mL[-1]] was significantly [p < 0.01] higher than other doses

Real-time PCR: an appropriate approach to confirm ssDNA generation from PCR product in SELEX process

Background: Aptamers are single stranded DNA [ssDNA] or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment [SELEX] method is much dependent on the successful conversion of double stranded DNA [dsDNA] to ssDNA

Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the efficiency of another technique for confirming the ssDNA generation in comparison to the polyacrylamide electrophoresis [PAGE] analysis

Materials and Methods: Real-time PCR was employed in the present study for PCR amplification of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis

Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 [degree]C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds

Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation

gene expression level of IFN-gammaR1 and IFN-gammaR2 in a murine model treated with Toxoplasma gondii and its products

Aim: To evaluate the effect of active T. gondii tachyzoites and its products on the gene expression level of IFN-gammaR1 and IFN-gammaR2 in a murine model

Background: Many studies have shown that the parasite Toxoplasma gondii utilizes different mechanisms to inhibit the function of IFN-gamma, but the parasite effect on the function of IFN-gammaR1 and IFN-gammaR2 is still unclear

Patients and methods: Toxoplasma lysate product [TLP], excretory/secretory products [ESPs] obtained from cell free and cell culture media as well as active tachyzoites were injected separately to their respective group each consisted of 10 BALB/c mice. One control group of 10 mice received phosphate buffered saline [PBS]. All of the mice were euthanized three days after the last injection and then their peritoneal leukocytes were harvested separately. The total RNA was extracted from the samples, converted to cDNA, and the gene expression level of IFN-gammaR1 and IFN-gammaR2 was assessed in all of the treated groups relative to the control one

Results: There was no significant difference between each of the treated groups relative to the control group concerning the gene expression level of IFN-gammaR2 [P> 0.05]. Furthermore, the gene expression level of IFN-gammaR1 in two groups of TLP [P= 0.04] and ESP obtained from cell free medium [P= 0.008] showed a significant difference relative to the control group

Conclusion: Findings of this study revealed a new aspect of host-T. gondii interaction in that this parasite is able to downregulate IFN-gammaR1 to reduce the IFN-gamma effects on the infected cell

Development of a stable cell line, overexpressing human T-cell immunoglobulin mucin 1

Background: Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 [TIM-1] glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases

Objectives: In this study, we aimed to express TIM-1 protein on Human Embryonic kidney [HEK] 293T cell line in order to have an available source of the TIM-1 antigen

Materials and Methods: The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells [PBMC] and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA[TM]3.1/Hygro [+] and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR

Results: The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells

Conclusions: Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1

Monitoring and comparison of antibiotic resistant bacteria and their resistance genes in municipal and hospital wastewaters

Background: Human exposure to antibiotic resistant bacteria [ARB] is a public health concern which could occur in a number of ways. Wastewaters seem to play an important role in the dissemination of bacteria and antibiotic resistant genes [ARGs] in our environment. The aim of this study was to evaluate the occurrence of three groups of ARB and their resistance genes in hospital and municipal wastewaters [MWs] as possible sources

Methods: A total of 66 samples were collected from raw MWs and hospital wastewaters [HWs] and final effluents of related wastewater treatment plants [WWTPs]. Samples were analyzed for the detection of three groups of ARB including gentamicin [GM], chloramphenicol [CHL] and ceftazidime resistant bacteria and their ARGs [aac [3]-1, cmlA1 and ctx-m-32, respectively]

Results: The mean concentration of GM, CHL and ceftazidime resistant bacteria in raw wastewater samples was 1.24 × 10[7], 3.29 × 10[7] and 5.54 × 10[7] colony forming unit/100 ml, respectively. There is a variation in prevalence of different groups of ARB in MWs and HWs. All WWTPs decreased the concentration of ARB. However, high concentration of ARB was found in the final effluent of WWTPs. Similar to ARB, different groups of ARGs were found frequently in both MWs and HWs. All genes also detected with a relative high frequency in effluent samples of MWs WWTPs

Conclusions: Discharge of final effluent from conventional WWTPs is a potential route for dissemination of ARB and ARGs into the natural environment and poses a hazard to environmental and public health

[Effect of interleukin -27 on recovery from experimental autoimmune encephalomyelitis in C57BL/6 mice]

Multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis [EAE], are chronic inflammatory demyelinating diseases of CNS. This study aimed to examine the effects of IL-27coding plasmid on disease status and certain immunological parameters in EAE-affected C57BL/6 mice. IL-27 gene was subcloned in P240 plasmid. The recombinant P240-mIL27 and P240 plasmids were injected two times, each time 200 micrograms, to test and control EAE mice, respectively. The clinical signs of the treated mice were evaluated daily and scored according to a standard method. One week after the last injection, all mice were sacrificed. The ELISA and MTT tests were performed to evaluate the production of IL-4, IFN-? and IL-17 from and proliferative response of splenocytes against specific antigen challenge, respectively. Furthermore, to demonstrate the immune cells infiltration, histopathological exam was performed on the brainstem of mice. The P240-mIL27 plasmid could significantly improve clinical course of EAE in the test group. Also in this group, the level of IL-4 was greater than that in the control group, while the levels of IFN-? and IL-17 were lower than those of the control group. In MTT test, the splenocytes of the test group showed a significantly less proliferative response than the control group. Finally, less infiltration of immune cells was seen in the brainstem of EAE mice treated with P240-mIL27 plasmid. IL-27 by shifting the immune responses from inflammatory Th1/Th17 towards anti-inflammatory Th2-type responses could be a suitable candidate for the treatment of inflammatory diseases such as MS

Use of integrase-minus lentiviral vector for transient expression

Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein [GFP] gene expression by a fluorescence microscopy. Recombinant and wild lentiviruses titer was about 58×10[6] transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting

[Comparison of four different types of carriers for the growth of vero cells and propagating rabies virus for production of human rabies vaccine]

In this study, capability of Vero cells for growth on FibraCel disks were compared on 3 kinds of microcarriers including Cytodex-1, Cutodex-3, and Sigma Solohill in 500 ml Spinner flasks in both serum contained medium [DMEM+10% Fetal Calf Serum, FCS] and serum-free medium [VP-SFM]. The propagation of fixed PV [Pasteur Virus] strain of rabies in Vero cells, for production of rabies vaccine, grown under the above conditions were studied and compared. Stepwise perfusion mode in growth phase and batch mode were applied in the virus production step by the use of M199 +0.2% Bovine Serum Albumin [BSA] and VP-SFM as serum containing and serum-free media, respectively. The available surface area provided by the carriers, and primary cell density in the experiments were assumed the same [about 12,000 cm[2] and 12,500 cells/cm[2], respectively]. The highest cell density was achieved on FibraCel disks in DMEM equal to 7.1 +/- 0.7_10[6] cells /ml on day 9, while the lowest cell density was obtianed on Cytodex-3 in VP-SFM equal to 2.91 +/- 0.2_10[6] cells/ml. The highest virus titer [55.18 +/- 4.4_10[6] Fluorescent Focus Unit, FFU/ml] was gained in VP-SFM containing FibraCel disks, and the lowest titer [3.66 +/- 0.4 _10[6] FFU/ml] was resulted on Cytodex-3 in M199. In these experiments, FibraCel disks supported the growth of Vero cells better than the microcarriers, and the use of DMEM for propagation of Vero cells and VP-SFM for proliferation of rabies virus showed better results. The experimental vaccine prepared by collected virus from VP-SFM has an acceptable potency of 2.75 IU. Based on these results and to the relative ease for making FibraCel disks, we recommend the use of this carrier for propagation of Vero cells and production of rabies virus

Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. In this study, we expressed the extra membrane loop of hCD20 [exCD20] consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a[+] expression vector. The desired protein was expressed in fusion with thioredoxin and 6 His tag in E. coll Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin [exCD20] can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies

Design and construction of two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast

Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic responses in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction [PCR]. Finally they were cloned in yeast vector pPICZ alpha A containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression

[Production of camel polyclonal antibody against vascular endothelial growth factor receptor-2 and performance evaluation in ELISA test]

In molecular approach, serum of camel contains a unique type of antibodies devoid of light chains since the light chain is missing, the heavy-chain antibodies should bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain. Vascular endothelial growth factor receptor-2 [VEGFR-2] is one of the important proteins in angiogenesis which over-expressed in many types of tumors. Two male dromedaries [Camelus dromedarius] were immunized against VEGFR-2. ELISA was used to evaluate the immunization process. Camel immune sera were fractionated with protein A and G affinity chromatography. Heavy chain antibodies tested for its specific reactivity in cell-based ELISA in recognition of VEGFR-2 on the cell surface of three cell lines; 293/KDR, HUVECs and A431. In ELISA test, the camel sera in 1/12800 dilution had the acceptable results. Furthermore, cell-based ELISA demonstrated that the polyclonal heavy chain antibodies bind to VEGFR-2 in 293/KDR and HUVECs cell lines. Single chain polyclonal antibodies against VEGFR-2 can be used for detection of soluble form of this protein by ELISA, and also flowcytometry, western blotting and immunohistochemistry

Transient expression assay of [A]gamma-588 [A/G] mutations in the k562 cell line

In the previous study, we have shown that the presence of A allele at position -588 in [A]gamma -globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele [A allele at -588] could result in an increase in [A]gamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients. Three constructs containing ji locus control region, [A]gamma -globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of, [A]gamma -globin gene [A and G alleles at -588]. A construct with T to C base substitution at -175 of, [A]gamma -globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of, [A]gamma -globin gene was determined by quantitative real-time reverse transcription-PCR. There was not a significant increase in the expression of, [A]gamma -globin gene in the construct containing A allele comparing the one with G allele at -588. -588 [A>G] mutation does not play a major role in regulation of, [A]gamma -globin gene, suggesting that other factors may be involved

High expression of methylotrophic yeast-derived recombinant human erythropoietin in a pH-controlled batch system

To accomplish the worldwide demand for recombinant human erythropoietin [rHuEpo] as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris [P. pastoris] rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZ alpha A vector under control of AOX[1] promoter, downstream of the secretion-alpha-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin [1.0 mg/ml], followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDSPAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems

Potential mutations of thyroid peroxidase gene in children with congenital hypothyroidism in Isfahan province

Congenital hypothyroidism [CH], the most common congenital endocrine disorder, in childhood and one of the causes of mental retardation, may be caused by defects in the enzymatic cascade of thyroid hormone synthesis, called thyroid dyshormonogenesis, of which thyroid peroxidase gene [TPO] mutations are one of the most common causes. The aim of this study was to assess frequency of TPO gene defects in patients with thyroid dyshormonogenesis in Isfahan province. This was a cross sectional study conducted on 40 patients with permanent congenital hypothyroidism, due to thyroid dyshormonogenesis. Genomic DMA was extracted from the peripheral blood of these patients, using the salting out method. The 17 exonic region of the TPO gene was amplified and mutation screening was performed by single-strand conformational analysis [SSCP] and sequencing. Results demonstrated one missense mutation in the [G2669A] location of exon 15 in one patient and seven different single nucleotide polymorphisms [SNPs] in exons 1, 7, 8, 11 and 15 of the TPO gene. Frequency of TPO gene mutation in this study was lower in comparison to other similar studies. It remains possible that in these patients, the disorder was caused by a TPO gene defect in regulatory or intronic regions. In addition, methods besides SSCP analysis and detection of other gene defects in thyroid dyshormonogenesis need to be further investigated in this field

[Transfection of human hematopoietic stem cells by a gene targeting construct containing the beta-globin gene]

Replacment of CD133+ cell's beta globin gene by using a gene targeting construct containing the beta globin gene and essential elements for homologous recombination. pFBGGT was amplified, then digested using the NheI and XhoI restriction enzymes, and finally, a 13.3 kb band [naked DNA] was extracted from the agarose gel. Biological activity of positive and negative selection markers were checked by transfection of COS-7 cells with linear plasmid. Hematopoietic stem cells [HSCs] were separated and transfected with linear plasmids using lipofection followed by positive and negative selection. Polymerase chain reaction [PCR] were done on DNA from the selected cells and the products were sequenced. The results of biological activity assays showed that selection markers were active. PCRs for hygromycin, neomycin and joining segments were positive but PCRs for TK1 and TK2 genes were negative. Sequencing PCR product joining segment confirmed the formation of homologous recombination. In this novel strategy gene replacement was achieved and biological activities of its components were observed

[HIV type 1-based particles effect in transduction of HT1080 cell line: a technique in beta-thalassemia gene therapy]

One of the most effective methods in the treatment of beta-thalassemia is gene therapy by viral vectors. The aim of this study was to design a recombinant lentivirus containing mini LCR and beta-globin gene for transferring normal beta-globin gene into hematopoetic stem cells. In this basic-applied study, each segment was cloned into a lenti transfer vector and confirmed by restriction digestion and sequencing. Transfer vector and three packaging plasmids were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock was determined in a HT1080 cell line. Transduction of target cells was increased by polybrene until 2 fold. Transduced HT1080 colonies remained after 2-week antibiotic selection. The remained transduced HT1080 colonies were expanded and DNA was extracted. PRC evaluated random integration of construct into the genome in this gene transfer technique. PCR evaluated random integration of construct into the genome in this gene transfer technique. Optimum MOI for HT1080 cell line was determined. Lenti viruses can be used for effective and permanent gene transferring in mammalian cells such as hematopoetic stem cells in order to accomplish gene therapy of genetic diseases like beta thalassemia and cancers

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

Beta-thalassemia is caused by absence of reduction of beta-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 beta-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and beta-globin gene for transfer to the target cells for gene therapy of beta-thalassemia. HS2, HS3, HS4 segments [miniLCR] and beta-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 contransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. 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 intergration construct into the genome was evaluated. 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