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 retrovira...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.展开更多
THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system...THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of展开更多
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 w...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.展开更多
目的 :探讨人凝血因子 (F )内含子 1片段正向插入逆转录病毒载体对骨骼肌细胞表达 F 的影响。 方法 :将含F 内含子 1片段的 F 微小基因 m 1和 m 2正向插入 L SN逆转录病毒载体的 Bam H 位点 ,取代 F c DNA,获得 m 1SN和 L m 2 SN载...目的 :探讨人凝血因子 (F )内含子 1片段正向插入逆转录病毒载体对骨骼肌细胞表达 F 的影响。 方法 :将含F 内含子 1片段的 F 微小基因 m 1和 m 2正向插入 L SN逆转录病毒载体的 Bam H 位点 ,取代 F c DNA,获得 m 1SN和 L m 2 SN载体 ,在小鼠成肌细胞进行瞬时和稳定表达试验。结果 :L m 1SN的 m 2 SN载体的病毒滴度、在成肌细胞中瞬时表达和稳定表达 F 的水平 ,均比不含内含子的 L SN增高 1～ 2倍 ,小部分病毒 RNA中的内含子在包装细胞中未被剪接去除而转入了靶细胞。结论 :人 F 内含子 1片段正向插入逆转录病毒载体对提高病毒滴度和 F 的表达水平有一定作用。展开更多
基金This work was supported by a grant from the State High Technology Development Program 863-102-17-40
文摘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.
文摘THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of
基金Project Supported by the state High Technology Development Program
文摘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.
文摘目的 :探讨人凝血因子 (F )内含子 1片段正向插入逆转录病毒载体对骨骼肌细胞表达 F 的影响。 方法 :将含F 内含子 1片段的 F 微小基因 m 1和 m 2正向插入 L SN逆转录病毒载体的 Bam H 位点 ,取代 F c DNA,获得 m 1SN和 L m 2 SN载体 ,在小鼠成肌细胞进行瞬时和稳定表达试验。结果 :L m 1SN的 m 2 SN载体的病毒滴度、在成肌细胞中瞬时表达和稳定表达 F 的水平 ,均比不含内含子的 L SN增高 1～ 2倍 ,小部分病毒 RNA中的内含子在包装细胞中未被剪接去除而转入了靶细胞。结论 :人 F 内含子 1片段正向插入逆转录病毒载体对提高病毒滴度和 F 的表达水平有一定作用。
