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Expression of human clotting factor IX with EBV shuttle vector

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Chinese Science Bulletin 1995年第21期40卷 1829-1833页

作者：王宏伟郑冰薛京伦邱信芳 nstitute of: Genetics Fudan University Shanghai 200433 China

Culture of patient somatic cells, transfected with therapeutic genes into genomic DNA with retroviral vectors, followed by colony selection, amplification, and reimplantation of transformed cells into patient, has been Widely used for clinical trial of gene therapy in the past years. But the disadvantages of this protocol are obvious. (i) To a great extent the expression level of the transfected cells depends on the different integration sites,which cause the various expression rates from different colonies; besides, the life span

关键词： EBV shuttle plasmid clotting factor IX expression.

RAPID DETECTION OF EXOGENOUS HUMAN clotting FACTOR IX cDNA IN TRANSFERRED CELLS BY POLYMERASE CHAIN REACTION

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Chinese Science Bulletin 1990年第23期35卷 1995-1999页

作者：周洁民戴一凡邱信芳薛京伦 Institute of Genetics Fudan University Shanghai 200433 PRC

Polymerase chain reaction (PCR) is a recently devoloped molecular biological technique, using specific oligonucleotide primers and DNA polymerase to amplify the specific fragment of DNA in vitro. This technique has been successfully introduced in gene diagnosis, mutation detection, polymorphism analysis and nucleotide sequencing. In the study of gene transfer and gene therapy, it is necessary to analyse DNA in the

关键词： polymerase chain reaction human clotting factor Ⅸ cDNA exogenous cDNA detection.

Polymerase Chain Reaction (PCR) in Detection of Transgenic Mice Harbouring Human clotting Factor IX cDNA

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Chinese Science Bulletin 1994年第13期39卷 1133-1138页

作者：胡以平邱信芳薛京伦

DNA-DNA molecular hybridization is commonly used in the detection of transferred foreign gene in transgenic mice, which is an effective tool in the detection of transgenic mice due to its high sensitivity and reliabil... 详细信息

关键词： PCR human clotting factor IX cDNA transgenic mice.

High expression of human clotting factor IX cDNA in the bone marrow stroma cells of hemophilia B patient

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Chinese Science Bulletin 1995年第16期40卷 1374-1377页

作者：卢大儒邱信芳郑冰薛京伦 Institute of Genetics. Fudan University Shanghai 200433 China

Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion... 详细信息

关键词： human clotting factor Ⅸ retroviral vector bone marrow stroma cells gene transfer.

TRANSFER AND EXPRESSION OF HUMAN clotting FACTOR IX GENE IN CHINESE HAMSTER OVARY CELLS

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Chinese Science Bulletin 1990年第1期35卷 60-63页

作者：戴一凡刘坚邱信芳薛京伦 Institute of Genetics Fudan University Shanghai 200433 PRC Institute of Genetics Fudan University Shanghai 200433 PRC Clotting factor Ⅸis an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder--hemophilia B or Christmas disease. Factor Ⅸis synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently several groups have cloned factor ⅨcDNA and reported that in the presence of vitamin K active factor Ⅸcould be secreted in human and mouse fibroblasts

clotting factor Ⅸ is an essential member in the intrinsic dotting pathway and its defect may cause an inherited X-chromosome linked bleeding disorder——hemophilia B, or Christmas disease. Factor Ⅸ is synthesized in liver hepatocytes where it undergoes complex vitamin K-dependent post-translational modifications which are essential for the activation of factor Ⅸ. Recently, several groups have cloned factor Ⅸ cDNA and reported that, in the presence of vitamin K, active factor Ⅸ could be secreted in human and mouse fibroblasts,

关键词： clotting factor Ⅸ gene transfer.

Regulation of human clotting factor IX cDNA expression in transgenic mice

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Science China Chemistry 1995年第7期38卷 825-833页

作者：胡以平邱信芳薛京伦刘祖洞 Institute of Genetics Fudan UniversityShanghai 200433China

To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured *** of the recombinant constructionswere found to he expressed well in *** were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic ***,none of them was expressed in any *** results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

关键词： human clotting factor IX cDNA transgenic mice regulatory expression.

Cloning and expression of canine clotting factor Ⅸ cDNA in vitro mediated by retroviral vector

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Science China(Life Sciences) 1999年第4期42卷 370-375页

作者：高啸波邱信芳卢大儒薛京伦

Oligonucleotide of cFIX cDNA (canine FIX, cFIX) was used to transcript mRNA of dog liver cell to cD-NA by RT-PCR, and further construct it on the plasmid vector pGEM-T. The correct sequence of cFIX cDNA was obtained which covered the entire cFIX coding region. Furthermore, GINaCcIX (driven by hCMV promoter) and GlNaMBcLX (driven by MCK enhancer and β-actin promoter) were constructed using the retroviral vector backbone of GINa. Canine skin fibroblast (CSF) was used as target cell, transduced with the above constructors respectively. The results showed that these modified CSF cells could express cFIX and that the expression levels were 173 ng/106 cell/24 h (GINaCcIX) and 211 ng/106 cell/24 h (GINaMBcIX) respectively. Those data offered a promising result for further animal study.

关键词： canine clotting factor IX canine skin fibroblast retroviral vector gene transfer gene expression.

HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN clotting FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

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Science China Chemistry 1992年第2期35卷 183-193页

作者：戴一凡邱信芳薛京伦刘祖洞 Institute of Genetics Fudan University Shanghai 200433 PRC

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.

关键词： gene therapy retroviral vector clotting factor Ⅸ.

High expression of human clotting factor IX cDNA in myoblasts C2C12 cells and C3H mice

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Science China(Life Sciences) 1997年第4期40卷 371-378页

作者：王宏伟包赟卢大儒邱信芳 JerryL.Hsueh 薛京伦 Institute of Genetics Fudan University Shanghai 200433 China

Mouse myoblast C2C12 cell was used as target cell for gene transfer study of human clotting factor IX (hFIX) cDNA. In addition to the previously constructed retroviral vectors XLIX, LNCIX and GINaCIX, GlNaMCIX with hFIX driven by muscle creatine kinase (MCK) enhancer and human cytomegalovirus (CMV) was constructed, based on the retroviral vector GINa. These four retroviral vectors were used to transduce mouse my-oblasts C2C12. With ELISA assays, it has been found that the expression levels of human clotting factor IX detected in those transduced C2C12 cells are GlNaMCIX>GlNaCIX> LNCIX>XLIX. Mixed colonal cells transduced with GlNaMCIX expressed hFIX protein at the level of 640 ng/106 cell every 24 h. The modified C2C12 cells transduced with GlNaMCIX were implanted into skeletal muscle of the hindlegs of C3H mice; a stable expression of hFIX was detected and lasted for 35 d, with a maximum level of 206 ng/mL plasma. The regulation of hFIX cDNA expression in myoblasts was discussed and it was strongly suggested that a myoblast-mediated gene delivery system had the poten-tial to be optimized as a safe and effective therapeutic modality for hemophilia B.

关键词： myoblasts human clotting factor Ⅸ gene transfer gene therapy.