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

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 sug     

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes was studied. 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.     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector. Project supported by the State High Technology Development Program and Shanghai Science and Technology Commission.     

A study of gene transfer and expression of human clotting factor IX in hemophilia B mice mediated by mini-adenoviral vector

Vector Gti'IX 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 Gti'IX, and then was transfected with helper-adenovirus AdLC8, thus mini-Ad virions AdGTi'IX were obtained. At the same time, previous normal adenoviral vector pAdSPi'IX containing viral genome and hFIX mini-gene was constructed, and then previous adenovirus (pre-Ad) AdSPi'IX was obtained as control. The ratio of helper-adenovirus among purified virons AdGTi'IX was less than 0.8%. 3T3 cells were transfected with AdGTi'IX and AdSPi'IX at a MOI of 50 per cell and ELISA result showed that transient expression level in vitro was 1.4±0.2 μg /106@24 h and 1.6±0.3 μg/106@24 h respectively. Each hemophilia B (FIX knock-out) mouse received celiac injection of 1×1010pfu AdGTi'IX or AdSPi'IX. 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 μL to 60 μL. The results of anti-Ad IgG assays indicated that immune response triggered by AdGTi'IX was obviously weaker than that triggered by AdSPi'IX. These results indicated that, compared with previous adenovirus (pre-Ad), the mini-Ad vector system prolonged the expression time of hFIX and reduced immune response, thus offering a promising result for further pre-clinical study.     

Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β-casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and Lac2 gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ-carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β-casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β-casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.     

Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.     

A study of gene transfer and expression of human clotting factor IX in Hemophilia B mice mediated by mini-adenoviral vector

Hemophilia B is an X-linked disorder caused by the deficiency of clotting factor IX (FIX) activity. Compared with conventional treatment, gene therapy offers an attractive approach by which the goal of prophylactic hemostasis may be achieved without extreme costs, infectious and thrombotic risks. It is the key for?successful somatic gene therapy to effectively and safely ex-press target gene in cell, animal or human as well mediated by appropriate vectors. Adenoviral vectors are currently a…     

bcl-2高表达细胞株的构建

利用基因转染技术,将人的bcl-2基因cDNA克隆于逆转录病毒载体pLXSN,通过PA317细胞包装成病毒后感染HeLa细胞。经PCR及蛋白质印迹证明转染成功,得到bcl-2稳定高表达株Hbc17。用顺铂诱导凋亡,Hbc17比对照细胞株H1具有明显的抗凋亡能力。为进一步研究顺铂诱导肿瘤细胞凋亡的机理奠定基础。     

LMO3逆转录病毒表达载体的构建及其对SK-N-AS细胞增殖的影响

目的构建包含LM03（LIM-only3,LM03）全长基因的逆转录病毒表达载体,感染人神经母细胞瘤SK-N-AS,检测LM03对SK-N-AS细胞增殖的影响。方法将质粒pEGFP-Cl-一LM03经EcoRI和BamHI双酶切后亚克隆至逆转录病毒载体pLXSN,构建重组逆转录病毒表达载体pLXSN-LMO3,导人包装细胞pA317,获得逆转录病毒颗粒,感染SK-N-AS细胞,用RT-PCR及Western印迹鉴定,检测LM03感染后细胞的增殖及细胞周期分布情况。结果获得了能正确表达LM03基因的重组逆转录病毒表达载体pLX-SN-LMO3;LM03基因被逆转录病毒成功导入SK-N-AS细胞后,与对照组细胞相比,LM03感染组G1／G1期细胞减少,S期细胞增加,感染48h后,LM03感染组细胞的增殖能力显著高于空载体对照组及SK-N．AS组（P〈0．05）。结论成功构建了LM03基因的逆转录病毒表达载体,LMO3可以通过促进SK-N-AS细胞由G0／G1期进入S期,从而促进细胞的增殖。     

逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal ,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。     

重组逆转录病毒载体GTK的构建及HyTK基因转移的研究

用逆转录病毒载体将单纯疱疹病毒胸苷激酶基因（ＨＳＶｔｋ）导入恶性肿瘤细胞,随后可应用药物９－（１,３－二羟基－丙氧基－甲基）鸟嘌呤（ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）选择性地杀死肿瘤细胞．将ＨｙＴＫ基因替换逆转录病毒载体ＧｌＮａ中的ｎｅｏ基因,构建成重组逆转录病毒载体ＧＴＫ,转染混合包装细胞（双噬性ＰＡ３１７细胞和单噬性ＧＰ＋Ｅ－８６细胞）,通过“乒乓效应”获得高滴度重组病毒．用该重组病毒转染小鼠恶性黑色素瘤细胞系Ｂ１６细胞,用ｈｙｇｒｏｍｙｃｉｎＢ筛选出阳性细胞克隆（ＨｙＴＫ＋）,经ＰＣＲ方法检测证明ＨｙＴＫ基因已成功地导入肿瘤细胞中,且不含可复制的辅助病毒．分别用不同浓度的ＧＣＶ作用于ＨｙＴＫ－及ＨｙＴＫ＋的Ｂ１６细胞,光镜下观察２４ｈ和４８ｈ后细胞形态及进行活细胞计数．结果表明,ＧＣＶ浓度大于０．１μｍｏｌ／Ｌ时即对Ｂ１６／ＨｙＴＫ＋细胞有显著的杀伤作用     

Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer

The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor IX (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is evaluated. The muscle creatine kinase enhancer (MCK) and β-actin promoter (βA) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high titer (2.5 × 10¹¹ vector genomes/mL) of rAAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-mediated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.     

逆转录病毒载体介导表达伪狂犬病病毒PK基因MDBK细胞系的建立

细胞凋亡 (apoptosis)是机体在生理条件下受刺激后 ,经过多种途径的信号传导导致细胞产生一系列形态和生物化学方面的改变而引起的细胞自我消亡的过程 ,是程序性细胞死亡 (programmedcelldeath ,PCD)的主要机制 ,它对于机体的发育 ,内外环境的平衡等都具有重要的作用[1,2 ] .研究表明 ,许多病毒具有诱导或抑制细胞凋亡的功能 ,但其发生的机理仍然不清 .随着分子生物学研究的深入 ,人们发现病毒的某些基因与细胞凋亡相关 ,其编码的基因产物或因病毒感染诱导细胞表达的蛋白抑制或诱导细胞发生凋亡 .最近研究表明 ,伪狂犬病病毒 (psedorabies…     

携带HSV-1TK基因的逆转录病毒载体构建及在HeLa细胞中的表达

目的:构建携带单纯疱疹病毒脱氧胸腺嘧啶激酶基因(herpes simplex virus thymidine kinase,HSV-TK)的逆转录病毒,用于宫颈癌的治疗研究。方法:用限制性内切酶从质粒pcDNA3.1/HA-myc-His(-)Z-TK切下HSV-1TK cDNA序列,亚克隆入逆转录病毒载体pLXSN得到重组质粒pLXSN-TK,鉴定正确的阳性重组质粒经PA317细胞包装,G418筛选,在NIH3T3细胞进行病毒滴度测定。然后用病毒感染人宫颈癌细胞HeLa。PCR、RT-PCR和Western blotting方法检测HSV-1TK基因在HeLa中的整合和表达情况。结果:重组质粒pLXSN-TK经PA317细胞包装后收获病毒上清,感染HeLa细胞,检测发现HSV-1TK基因整合到细胞基因组DNA中,并且能有效的转录和翻译。结论:成功构建了逆转录病毒pLXSN-TK,该病毒能有效感染HeLa细胞,并使携带的治疗基因HSV-1TK在细胞中表达,为今后HSV-1TK基因治疗宫颈癌的研究奠定基础。     

Netrin-1慢病毒表达载体的构建及鉴定

目的:构建携带有Netrin-1基因的逆转录病毒载体,为研究Netrin-1在神经发育中的作用奠定基础。方法:PCR扩增Netrin-1基因片段后,将其克隆入慢病毒表达载体pLXSN;通过PCR、酶切、测序鉴定重组质粒。重组质粒转染PA317包装细胞后获得包装的病毒颗粒。病毒颗粒感染人脑胶质瘤细胞SW038-C2,经Western blot证明重组病毒在真核细胞内表达Netrin-1的情况。结果:经PCR扩增、酶切和测序验证,重组质粒构建正确,命名为pLX-NT。Western blot证明在感染细胞泳道有一特异性条带。结论:成功构建了能表达Netrin-1的慢病毒载体。     

Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer

An autoregulatory bidirectional expression cassette encoding all components necessary for regulated gene expression in a one-step gene transfer was evaluated for use in adenoviral vectors. Adenoviral vectors transducing this cassette provide about 1000-fold regulation. Regulation could be further improved by integrating the cassette as a retroviral vector into the adenoviral backbone. Moreover, with these adeno/retroviral hybrid vectors, the frequency of chromosomal integration is enhanced and about 1% of infected cells show stable chromosomal integration of the autoregulated cassette. In these stably transduced cells high regulation capacity is maintained. To elucidate the molecular mechanism underlying this unexpected observation we investigated the regulation capacity of these cassettes in a viral and non-viral vector background after stable integration into the host's DNA. While naked cassettes show regulated expression that is strongly influenced by the chromosomal surrounding sequences the regulatory capacity of LTR flanked cassettes is highly comparable amongst different cell clones. This strict regulation with little influence from the flanking sequences is obtained when LTR-flanked cassettes are transduced as DNA, by retroviral or by adenoviral infection.     

Long-term expression of gene introduction into normal human T-lymphocytes by retroviral-mediated gene transfer

Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.