Basic Gene Research Of Beta Thalassemia For Translating Human Beta-Globin Gene And Rna Interference Of Alpha-Globin Gene Expression By Lentivirus Vectors

BackgroundP-thalassemia is a serious hereditary hemolytic anemia disease to human health. Because of point mutations or deletions of the beta globin gene,P-globin synthesis reduce to decrease or stop completely. The pathogenesis of P-thalassemia has one type was called translated defect type:The coding sequence mutation caused by termination codon (chain termination) mRNA interrupt the beta globin gene,this will result in no synthesis of β-globin chain, i.e. β0-thalassemia, such as condon41-42(-CTTT) mutation and condon27/28(+C) mutation. This not only makes hemoglobin level decreas in patients, but also the original in the number of alpha globin relative surplus, the relative excess free α-globin chain does not form a stable four mer, they are deposited in red blood cells leading to a large number of invalid red blood cell formation and red blood cell membrane damage,then shorten the life of red blood cells, at last the nearly no expression of P-globin cause severe hemolytic Anemia. Any factor capable of reducing the α/β chain imbalance in a subject with affected P-globin gene may have an ameliorating effect on the clinical picture.Recently lentiviral vectors has been successfully used in the treatment of P-thalassemia, especially in Splice-switching eror type. Because the aberrant splicing can be corrected such as intron1(IVS-Ⅰ-110, IVS-Ⅰ-6, IVS-Ⅰ-5) and intron2(IVS-Ⅱ-654, IVS-Ⅱ-745) of by RNAi arroding lentivi-rus mediated, by which the error splice-switching squence can be recovered and normal globin synthesis can be reestablished. But the research of translated defect P-thalassemia was less than the other typies. This type can not be recovered the defect gene by RNAi. Only can improve the P-globin hemoglo-bin expression or decrease a-globin expression to adjust the α/β globin gene proportion. But the research of a-globin gene interference, while the proportion of control and long-term coordination has not been effectively verified. Mice model completely (th3/th3) died late in gestation, limiting their utilization as a model for Cooley’s anemia.This subject is based on K562cell research on translated defect β-thalassemia codon41-42which is the most common type in Guangxi province. This research designed into normal human β-globin supplement while reducing a-globin gene expression to adjust the α/β globin gene proportion, provide more effective evidence for the treatment of β thalassemia, which try a new strategy for gene therapy. Construction and various types of gene carrier preparation and design for further individual level translated defect type codon41-42mutation gene knocked mice model to do basic research.Objectives:1.To contract containing normal human P-globin gene lentiviral vector LV-β-globin(GFP), and to make virus particles packaging.2.To test the expression of a-globin gene expression in K562cells.3. Design and synthesis four a-globin gene RNAi (RFP). vectors will be transfection stably into K562cells.Real time PCR detect mRNA expression of a-globinand and interfere efficiency, screening the most effective one interference segments for follow-up experiments.4.The constructed human beta globin gene lentiviral and a-globin gene RNAi lentivirus transferred into K562cells stably. Real time PCR and Western-blotting detect mRNA and protein expression of a-globin and P-globin. In order to show the purpose of the experiment, whether reduced the expression of a-globin, and increase human β-globin, and to achieve treat β0thalassemia symptoms by adjusting the expression of α/β globin. Methods:1.Polymerase chain reaction method was used to obtain the target gene and SphI、NotI. two restriction sites were added, obtain the recombinant plasmid pUC57-β-globin after T vector cloned. The target gene fragment connect with pLVEF1a/GFP+Puro vector by means of enzyme digestion. The lentiviral expression vector LV-β-globin was obtained after screening followed by sequencing. The lentiviral vector was used to transferred into293T cells and package virus, and virus titers were determined.2.Human chronic myelogenous leukemia K562cell were cultured to the logarithmic growth period, at the same time human hepatocellular carcinoma cells, human breast cancer cells, human astrocyte cell were selected as control groups, a-globin gene (Hbα2) RNA was detected in these cells. The target gene and reference gene GAPDH primer were designed, Hba2mRNA was detected by real-time PCR in K562cell.3.According to the gene sequence, Four best RNA interference sequences was designed by the siRNA design principle, design experience and design software evaluation,which named Hba2-RNAi (1), Hba2-RNAi (2), Hba2-RNAi (3) and Hba2-RNAi (4). Synthesis single stranded DNA oligo of interference sequences, annealing pairing double chain, then connected DNA to lentiviral vector PGCSIL-017and digested and connected by their same enzyme sites Agel and FcoRI.the connecting products were transferred to competent cell. Positive recombinants were sent to sequencing after identification of PCR.4.The human β-globin gene lentiviral LV-β-globin with high MOI value were added into K562cells.3days after infection, the infection efficiency higher than80%. The fluorescence expressions of transferred β-globin in K562cell was observed as well. K562cells were inoculated to the suitable density with LV-β-globin after lentiviral transferred stably, Than a-globin gene RNAi lentiviral with high MOI value were added into K562cells. The infection efficiency of fluorescence and expressions were observed three days later, a-globin gene(Hba2) and β-globin gene (HBB) mRNA were detected by RT-PCR method after two lentivirus transferred stability into K562cells.The a-globin and β-globin protein expression detected as well. Results1.A length of2128bp with SphI and NotI target gene sequence was obtained by PCR. The pUC57vector was connected to the lentiviral vector lentiviral vector LV-P-globin builded successful, sequence was correct. Refered to successful build containing normal P-globin gene lentiviral vector.2.The results showed Hba2expression abundance in K562was higher than in MCF-7, HEPG2and U87cells.3. Result shows the positive clone PCR fragments which connect-ed into vshRNA size was342bp,and not connected into the vshRNA fragment was307bp.The PCR results were confirmed as correct cloning by blast. Four lentiviral vectors were packed and transferred into293T cells. The first lentiviral vector LV-Hba2-RNAi (1) packaged titer was3×108TU/ml, the second lentiviral vector packaged titer LV-Hba2-RNAi (2) was2×108TU/ml, the third lentiviral vector LV-Hba2-RNAi (3) packaged titer was2×108TU/ml, the last lentiviral vector LV-Hba2-RNAi (4) packaged titer was3×108TU/ml. Four lentiviral vector particles were transferred to K562cell after adjusted of K562cell state. Expressions were observed by fluorescence microscope after3days transfection. Transfection rate was more than50%and fluorescence rate reached above80%. Transfection cells were collected to extract RNA and reversed transcription to cDNA. The RNA expression level of target gene a-globin were detected by Real time PCRReal time PCR results showed the group KD2(LV-Hbα2-RNAi/2) Fbα2gene knockdown efficiency reached76.5%(P<0.05) compared to NC negative control group in K562cells. The lentiviral vector Hbα2-RNAi/2is considered to be effective interference targeting to a-globin gene Hba2. This Lentiviral vector was packaged to be more and the concentrated titer was6×108TU/ml finally4. the human β-globin gene lentiviral LV-β-globin was transferred into K562cells after72h infection, the efficiency is greater than94.8%, the cells emitted green fluorescence, revealed that a large number of fluorescent expression. The control group, no fluorescence expression of virus to K562cells after stable transfection. a-globin gene RNAi lentivirus was transferred and expressed beta globin gene in K562cell stably, the infection efficiency is91.3%after72h infection. The cells emit red fluorescence, revealed that a large number of fluorescent expression, found at the same time using green fluorescent microscope, the same cells expressing green fluorescence, which showed two lentivirus transferred and expression stably in cells. No red fluorescence expression in control group after transfection, only the expression of green fluorescence. P-globin gene was amplified to specific band of227bp by RT-PCR,the expression of RT-PCR a-globin gene was amplificated to specific band of85bp as well.2-△△Ct value of β-globin gene mRNA was4.080±0.078and2-△△Ct value of a-globin gene mRNA was0.274±0.023.Application of monoclonal antibody to human P-globin by Western-blotting. Found in the stable transferred K562cells expressed human β-globin was significantly higher than that blank control group. But the synthesis of a-globin was significantly lower than the blank group. β-globin grey value was1.063±0.082, expression increased by Western-blotting, and the a-globin grey value was0.327±0.042, expression decreased, the different had significant statistically. Conclusions: This study first make builded β-globin gene lentivirus and a-globin gene siRNA lentivirus transfection into K562cells stably and successfully. The P-globin gene mRNA is higher than the control group was4.08times and protein level was3.6times higher, a globin gene mRNA inhibition rate reached72.3%, protein inhibition rate was68.3%after two lentivirus transfection, significantly higher than the previous research both at home and abroad. Implementation to adjust the α/β, the expression of globin gene therapy concept, in order to improve the β0thalassemia symptoms, laid the foundation treatment for the β0knockout mice model of gene therapy to implement ready to cell based research. Objectives:To construct recombinant vector pcDNA3functioning as a eukaryotic expression vector and carrying the CD41-42mutation responsible for human P-thalassemia. The locus control region (LCR), which consists of HS2, HS3, and the CD41-42mutation-containing fragmentwere used as the target fragments, respectively. This work provides the foundation for the building of cell model and a transgenic mouse model for the gene therapy of human P-thalassemia.Methods:DNA extraction was performed in eight P-thalassemia patients who were confirmed to carry the homozygous CD41-42mutation. The specific primers were designed for PCR amplification of P-globin gene LCR and CD41-4mutation-containing fragment. The amplified products were directionally inserted into the eukaryotic expression vector pcDNA3.1. The positive recombinant vector was screened by restriction enzyme analysis and PCR identification.Results:Specific LCR and CD41-42mutation-containing fragment were amplified, with the length of approximately5.5kb and2.1kb respectively. Restriction enzyme analysis and sequencing results indicated that the recombinant vector contained the P-globin gene LCR and CD41-42mutation-containing fragment, which were cloned successfully at the desired position.Conclusions:The recombinant vector carrying human P-globin gene LCR and CD41-42mutation-containing fragment was successfully constructed. Objectives:To construction of recombination vector pEGFP-C2-CD41-42of β thalassemic CD41-42mutation and its stable transferredK562cell, to provide the basis for cell and mice model of β-thalassemia CD41-42mutation type and its further gene therapy.Methods:The DNA of βCD41-42homozygote mutation patient was extracted and the DNA frament was amplified by PCR,then was subcloned into pEGFP-C2vector. The recombined plasmid pEGFP-C2-βCD41-42was sequenced and transferredinto K562cell by lipofectamineTM2000. Fluorescence expression of βCD41-42in K562cell was observed.The expression of βCD41-42in K562cell was identified by RT-PCR method. Results: Recombined vector pEGFP-C2-βCD41-42transferredinto K562expressed many of fluorescence expression after24hs and screening culture by G418. RT-PCR227bp special PCR products was showed,but the blank pEGFP-C2plasmid didn’t express in K562.Conclusion K562CD41-42cell has is successfully which provids provide the basis for cell and mice model of P-thalassemia CD41-42mutation type and further gene therapy studies on the function of βCD41-42invitro. Objectives:To contract containing βthalassemic CD41-42gene lentiviral vector LV-βCD41-42, and to make virus particles packaging.Methods:Polymerase chain reaction method was used to obtain the target gene and SphI、NotI two restriction sites were added, after T vector cloning,the gene was transformed into competent cells. The gene fragment and LV vector cloning,the gene was transformed into competent cells by means of enzyme digestion. The lentiviral expression vector LV-βCD41-42was obtained after screening followed by sequencing. The lentiviral vector was used to transfect293Tcells and package virus, and virus titers were determined.Results:A length of2100bp with SphI and NoA target gene sequences was obtained by PCR. The pUC57vector was connected to the lentiviral vector LV.Conclusions:Constructed lentiviral expression vector LV-βCD41-42was corresponded to the expected.The lentiviral particles were successfully packaged.