| Hemophilia B is a X chromosome-linked recessive bleeding disorder which results from a deficiency in the coagulation factor IX. In recent ten years ,in spite of some pogress has been made on hemophilia B with gene therapy,the expression of FIX fails to sustain long in circulation and the expression level hasn't reached to its therapeutic levels.Short time of expression is because of the unstable expression of FIX which has something to do with vector in gene therapy. Present gene therapy uses virus vector which has not a high insertive rate after transfecting. Some vector which lies in nucleolus in the form of accessory can't inherit stably with the split of the cell. So FIX gene can't express stably in the long time. Human source gene vector ,which can target gene to short arms of D,G group chromosomes,was constructed using BM-specific fragments as BM was from short arms of D,G group chuomosomes in our lab. So it can solve the problem of short expressive time. But , when we target FIX cDNA to human source gene vector and transfect into HT1080,we confront another problem of gene therapy for hemophilia B that the expressive quantity of FIX is not high. Therefore, the problem of enhancement of the expression of FIX is to be solved in the corner based on the stbale expression of human source gene vector.Intron I of hFIX gene is often reported as a cis-acting element which can improve expression. Intron I has been successfully used as virus vector to improve expression of FIX in the research of gene therapy for hemophilia B. In our research ,intron I is targeted to ordinary vector and human source gene vector. We study the enhancement of expression of FIX of intron I,especially in the human source gene vector for the sake of stable and effective expression of FIX.We amplified the related fragments of intron I from normal genome DNA by means of PCR. We acquired the minigeneFIXa and the minigeneFIXb after a series of subcloning. The Intron Ia inserted in the former with a length of 1.424kb was truncated 4.8kb of the middle of intron I. The Intron Ib inserted in the latter with a length of 299bp was truncated 5.93kb of the middle of intron I.The expressing vectors contained minigeneFIXa,minegeneFIXb and FIX cDNA was transfected into HT1080 by Lipofectamine?2000 after they were verified by sequencial analysis. The expression of transient product of FIX was tested. Then ,we inserted minigeneFIXa, minegeneFIXb and their expressing element into human source gene vector (we constructed the human source gene vector containing FIX cDNA). We selected one cis-plasmid and one reversal-plasmid (we named cis-plasmid when the neo gene has the same transcribe direction as minigeneFIX and FIX cDNA; we named reversal -plasmid when the neo gene has the different transcribe direction from minigeneFIX and FIX cDNA). After sequencing and linearing ,the vectors were transfected into HT1080 by electroporation. Positive cell clones were obtained after selecting by G418 with the concentration of 400ug/ml for 3 weeks. We tested the expression of FIX of product of poly-clone and extracted mRNA and gDNA from poly-clone. Test the product of mRNA by RT-PCR and identify the foreign of gDNA.The results: both minigeneFIXa and minigeneFIXb had the same gross expression of FIX and active FIX under the condition of transient or stable expression. In transient expression,the expression of minigene is 3-5 folds as high as that of FIX cDNA. In stable expression,the gross expression of the minigeneFIX of cis-plasmid is 3-5 folds as high as that of FIX cDNA,and the expression of active FIX is 3-4 folds; there are 15-20 folds and over 20 folds respectively in the reversal-plasmid. Test expression of mRNA of poly-clone by RT-PCR and the results are accordance with the above. We succussfullly amplified the targeted gene by using gDNA from positive cell clones as templates.These results show : 1. Intron I can obviously enhance the expression of FIX whether it was inserted into pGEM-T vector or human source gene vector. 2. Intron la had the... |
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