High sensitivity of neonatal rat hepatocytes to retroviral-mediated gene transfer and their transplantation into the spleen of adult rat.

To optimize conditions for retroviral-mediated transfer of a recombinant gene to hepatocytes, the pMNSM-Tk-lacZ vector, which we had constructed to express the bacterial beta-galactosidase gene, was transduced to rat hepatocytes under various conditions, and the expression of beta-galactosidase activity was examined by cytochemical staining. Compared to the hepatocytes of adult rats, those of newborns were about 50-100 times more sensitive to transduction with the beta-galactosidase gene in vitro. The sensitivity was high in the newborn hepatocytes when the virus was infected on days 1 and 2 after initiation of culture. However, the sensitivity to infection did not correlate with the DNA synthetic activity. The gene transfer was feasible not only to hepatocytes in monolayer culture but also to those in spheroid culture. The spheroidal aggregates containing hepatocytes transduced with the beta-galactosidase gene could be transplanted into the spleen of syngenic adult rat, although the expression was very low.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

Generation of transgenic wheat plants producing high levels of the osmoprotectant proline

Plasmid DNA (pBI-P5CS), containing the selectable neomycin phosphotransferase-II `npt II' gene for kanamycin resistance and the reporter -glucuronidase `gus' gene as well as the Vigna aconitifolia ¹-pyrroline-5-carboxylate synthetase `P5CS' cDNA that encodes enzymes required for the biosynthesis of proline, was delivered into wheat plants using Agrobacterium-mediated gene transfer via indirect pollen system. Southern, northern and western blot analysis demonstrated that the foreign gene had been transferred, expressed and integrated into wheat chromosomal DNA. Salinity test indicated that proline acts as an osmoprotectant and its overproduction in transgenic wheat plants results in the increased tolerance to salt.     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Agrobacterium-mediated DNA transfer in sugar pine

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.     

Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction

Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.     

解毒酶基因的克隆及其在大肠杆菌和蓝藻中的表达(英文)

近几年来 ,在明确了杀虫药剂抗性机理的基础上 ,从杀虫药剂抗性的昆虫中分离出高抗性基因 (即解毒酶基因 ) ,将该基因克隆到表达载体 pRL 4 39上 ,得到表达载体 pRL B1,将其转化大肠杆菌HB10 1,获得了可以表达解毒酶基因的转基因工程菌株。同时构建了穿梭表达载体 pDC B1,并转化大肠杆菌HB10 1后 ,在抗生素氨卞霉素 (30 μg/mL)和卡那霉素 (30 μg/mL)平板上挑选阳性克隆 ,将阳性克隆的细胞、蓝藻和结合质粒以三亲结合转移的方式转入蓝藻。斑点杂交、Southern分析结果表明已经获得了Synechococcussp .PCC 794 2转基因工程藻。     

hGM-CSF基因穿梭表达载体的构建及其在鱼腥藻7120中的克隆

人粒-巨噬细胞集落刺激因子(hGM-CSF)作为一种造血生长因子,能够刺激T细胞和巨噬细胞增殖、成熟和分化,具有极其重要的免疫调解功能.本研究运用PCR方法,从质粒pAG-MT-8中克隆该基因,并在其5′端添加有利于在蓝藻细胞中高效表达的SD序列,然后插入到表达载体(pRL-439)强启动子PpsbA的下游,进一步与穿梭表达载体pDC-08相连构建成穿梭表达载体pDC-GM.利用三亲接合转移方法将该穿梭表达载体(pDC-GM)转入丝状鱼腥藻7120,通过相应抗生素筛选后得到能稳定遗传的转基因藻.以该转基因藻的基因组DNA为模板进行PCR检测,结果表明hGM-CSF基因已转入鱼腥藻7120.这是首次尝试把蓝藻作为制备重组hGM-CSF的新宿主,具有潜在的经济价值和社会效益.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.     

In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery

Transfer of the herpes simplex virus-thymidine kinase gene, followed by the administration of ganciclovir (HSV-tk/GCV), has been a major approach for cancer gene therapy. We investigated the antitumor effect of the HSV-tk/GCV strategy with the rat orthotopic hepatocellular carcinoma (HCC) model and the tumor-selective gene delivery by an adenovirus-mediated gene transfer through the hepatic artery. The complete antitumor effect was demonstrated, after the treatment with GCV in rat HCC established by the implantation of HSV-tk transferred rat HCC cells. The in vivo bystander effect was also observed. The marked infiltration of CD4+ and CD8+ T lymphocytes, macrophages and NK cells were found in the tumor area. After the injection of adenovirus carrying the LacZ gene into the hepatic artery, the selective expression of transgene in the tumor cell was achieved. These findings indicate that the HSV-tk/GCV strategy, using an adenoviral vector, could be a promising avenue for the treatment of hepatocellular carcinoma.     

p21基因的克隆及其对肺癌细胞生长的抑制

用反转录ＰＣＲ从正常人胚胎肺细胞中获得了ｐ２１基因ｃＤＮＡ,将其插入真核表达载体ｐＭＳＣＶｎｅｏ,构建成重组质粒,ｐＭＳ２１,并将其转染至肺癌细胞株Ａ５４９．通过集落形成观察到ｐ２１对肺癌细胞具有明显的抑制作用,经ＲＮＡ狭缝杂交、Ｗｅｓｔ－ｅｒｎｂｌｏｔ分析和免疫细胞化学实验证实这是ｐ２１表达的结果．荷瘤裸鼠实验也进一步证实了ｐ２１对肺癌细胞具有明显的抑制作用．为ｐ２１的深入研究提供了基础．