Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro,thus showing relatively high systemic delivery efficiency of various transgene products. The authors investig...Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro,thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK,Me2) were inserted in forward or reverse orientation at NheI site of 3’ long terminal repeat (LTR),resulting in two hybrid vectors,which were designated as LMe2IXm_2SN(F) and LMe2IXm_2SN(R),respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm_2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm_2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo,and MCK enhancer should be positioned in the same orientation as that of LTR promoter.展开更多
This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site ...This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.展开更多
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...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 reliability. The method, however, is very laborious and time-consuming, and requires a large number of DNA samples展开更多
Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein ...Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.展开更多
Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hF...Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.展开更多
Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glacto...Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glactosidase expression lasted at least 3 months, and hFIX expression lasted 1 month. These results will be helpful to organically combine the advantages of both skin fibroblasts and muscle, and provide a new way for somatic gene therapy of hemophilia B and other genetic diseases.展开更多
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 be...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展开更多
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 I...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 cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These 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.展开更多
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...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 is expensive and associated with a high risk展开更多
The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined wi...The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients' skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantily of factor IX proteins to cause the phenotypic modification for hemophilia B patients.展开更多
Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense...Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense mutation.Methods Three nonsense mutants were obtained by using PCR mutagenesis to ana-展开更多
文摘Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro,thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK,Me2) were inserted in forward or reverse orientation at NheI site of 3’ long terminal repeat (LTR),resulting in two hybrid vectors,which were designated as LMe2IXm_2SN(F) and LMe2IXm_2SN(R),respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm_2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm_2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo,and MCK enhancer should be positioned in the same orientation as that of LTR promoter.
文摘This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.
基金State High-Technology Development ProgramState Commission of Education, PRC
文摘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 reliability. The method, however, is very laborious and time-consuming, and requires a large number of DNA samples
基金supported by the State High Technology Development Program(863).
文摘Vector GtiIX containing human clotting factor IX cDNA with intron 1 (hFIX mini-gene or Fi’IX) driven by CMV promoter was constructed based on the mini-adenoviral vector GT2073 (mini-Ad vector) with all viral protein coding sequences deleted. Mini-Ad packaging cell 293Cre4 was first transduced with GTiIX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTiIX were obtained. At the same time, previous normal adenoviral vector pAdSPiIX containing viral genome and hFIX mini-gene was constructed, and then previous ade-novirus (pre-Ad) AdSPiIX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTiIX was less than 0.8%. 3T3 cells were transfected with AdGTiIX and AdSPiIX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 mg /106·24 h and 1.6±0.3 mg/106·24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 11010pfu AdGTiIX or AdSPiIX. The highest expression level of hFIX in mouse plasma was 590 ng/mL and 690 ng/mL respectively, and the expression time lasted for 16 weeks and 9 weeks respectively. The bleeding time reduced from over 30 min to 7.5 min, and 5-min blood lost reduced from 430 mL to 60 mL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTiIX was obviously weaker than that triggered by AdSPiIX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector sys-tem prolonged the expression time of hFIX and reduced immune response, thus offering a prom-ising result for further pre-clinical study.
基金the State High Technology Program "863", the National Natural Science Foundation of China (Grant No. 39880019) and Shanghai Scientific Phosphor Program.
文摘Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.
文摘Primary rabbit skin fibroblasts (RSF) containing lacZ reporter gene or human clotting factor IX (hFIX) cDNA could long termly exist and be expressed after being implanted in rabbit muscles. Among them, the beta glactosidase expression lasted at least 3 months, and hFIX expression lasted 1 month. These results will be helpful to organically combine the advantages of both skin fibroblasts and muscle, and provide a new way for somatic gene therapy of hemophilia B and other genetic diseases.
基金Project supported by High Technique Research of the State.
文摘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
文摘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 cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These 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.
基金Project supported by the "863" State High Technology Development Program.
文摘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 is expensive and associated with a high risk
文摘The construction of the high liter and highly expressed safety retroviral vector carrying human clotting factor IX cDNA is reported. Retroviral vectors LNCTX, LIXSN and LCTXSN, driven by hCMV, LTR and hCMV combined with LTR promoter respectively, were constructed, based on the retroviral vector LNL6, and transferred into packaging cell line PA317 with electroporalion. Human dolling factor IX was delected in the cultured cells transduced with LNCIX and LIXSN but not in the cells transduced with LCIXSN. The viral titer of PA317/LNC1X was 800000 CFU per mL. With ELISA detection, it was found that the cells transduced with this vector can express human clotting factor IX at the level of 3.3μg per 106 cells in 24 h in human fibrosarcoma cells HT-1080 and 2.5μg per 106 cells in 24 h in hemophilia B patients' skin fibroblast HSF cells, and more than 80% of them were biologically active. The viral liter and expression of human FIX were increased, and the construction of retroviral vector backbone was improved and the safety was guaranteed as compared to those vectors used previously. These vectors may produce a sufficient quantily of factor IX proteins to cause the phenotypic modification for hemophilia B patients.
文摘Objective To construct human coagulation factorⅨmini-gene(Mini-h F9)and some nonsense mutants,detect the levels of the Mini-h F9 mRNA,and analyze the molecular mechanism of microRNA125 regulating F9gene with nonsense mutation.Methods Three nonsense mutants were obtained by using PCR mutagenesis to ana-
