High efficiency gene transfer into mammalian kidney cells using baculovirus vectors

Gene delivery into kidney cells is essential to the development of gene therapy for nephropathy. This paper describes the use of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as a vector for gene delivery into several mammalian kidney cells. High-level expression of a reporter gene encoding for the green fluorescent protein (GFP) under a heterogonous promoter was observed in kidney cells from mouse, hamster, monkey, pig, and human. The level of transgene expression exhibited viral dose dependence and was enhanced by the addition of butyrate. Baculovirus transduction could also provided long-term target gene expression in kidney cell lines without cytotoxic effects. High efficiency transduction was also observed in primary mouse kidney cells. These results indicate that baculovirus can be used as a vector for kidney-directed gene transfer.     

High efficiency gene transfer into mammalian kidney cells using baculovirus vectors

Summary. Gene delivery into kidney cells is essential to the development of gene therapy for nephropathy. This paper describes the use of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as a vector for gene delivery into several mammalian kidney cells. High-level expression of a reporter gene encoding for the green fluorescent protein (GFP) under a heterogonous promoter was observed in kidney cells from mouse, hamster, monkey, pig, and human. The level of transgene expression exhibited viral dose dependence and was enhanced by the addition of butyrate. Baculovirus transduction could also provided long-term target gene expression in kidney cell lines without cytotoxic effects. High efficiency transduction was also observed in primary mouse kidney cells. These results indicate that baculovirus can be used as a vector for kidney-directed gene transfer.     

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.     

Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells

Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients.     

乳酸乳球菌同时发送外源性蛋白质/DNA到哺乳动物细胞

目的:建立乳酸乳球菌同时发送外源蛋白/DNA到哺乳动物细胞的系统.方法:分别将红色荧光蛋白基因、绿色荧光蛋白真核表达框整合于乳酸菌穿梭载体pMG36e中相关部位.重组质粒电转乳酸乳球菌后,经甘氨酸消弱乳酸乳球菌细胞膜后与哺乳动物细胞293T细胞共培养.结果:红色和绿色荧光蛋白均能在293T细胞表达.结论:借助乳酸乳球菌成功实现同时发送蛋白质/DNA到哺乳动物细胞.     

2种诱导iPSC向神经干细胞分化方法的比较

目的:整体比较2种促进诱导性多能干细胞(induced pluripotent stem cells,i PSC)向神经干细胞(neural stem cells,NSC)分化的方法,确定一种稳定、高效的获得NSC的方法,并对NSC进行系统鉴定。方法:方法A:SB431542和drosomophorin的浓度均为5μmmol/L,诱导初始密度100%;方法 B:SB431542的浓度为5 mmol/L,drosomophorin的浓度为1 mmol/L,诱导初始密度为40%。比较及鉴定方法:镜下观察诱导获得NSC的状态;realtime PCR比较神经干细胞相关基因Pax6、nestin、Sox1、Sox2等表达量;流式细胞术分析诱导第16天Pax6阳性率;免疫荧光定性分析神经干细胞相关蛋白的表达及其自发分化的能力。结果:方法 A获得的NSC悬起后成球趋势明显,圆形,透明;方法 B诱导获得NSC形状不规则,色灰暗。Real-time PCR结果证明方法 A诱导获得的细胞神经干细胞相关基因的表达量高于方法 B。流式细胞术分析证明第16天,PAX6的阳性率,方法 A高于方法 B。经鉴定,方法 A获得的神经干细胞高表达Pax6、nestin、Sox2等基因自发分化30 d,形成明显的神经纤维束,表达TUJ-1、MAP2及GFAP等神经元和胶质细胞的特异性标志物。结论:方法 A整体优于方法 B,我们推荐方法 A作为诱导i PSC向神经干细胞分化的方法。     

Comparison of mammalian cell expression vectors with and without an EBV-replicon

Summary We have characterized the properties of an Epstein-Barr virus vector (EBV-CMV) and compared its expression potential with a respective integrating vector (CMV). These vectors were used to express chloramphenicol acetyltransferase (CAT) gene in human HeLa, 293, monkey CV-1, dog MDCK, and hamster R 1610 cells. The EBV-CMV-cat DNA replicates extrachromosomally in HeLa, 293 and CV-1 cells, where also high expression of CAT gene was observed. The EBV-CMV vector integrated in MDCK and R 1610 cells and the CMV vector integrated in all cells tested. Integration yielded mostly clones with low CAT expression. In all cell lines, except HeLa cells, the existence of the extrachromosomal but not the integrated vector DNA is strictly dependent on the Hygromycin B selection pressure. The extrachromosomal state of the EBV vector is a prerequisite for good expression particularly in human and monkey cells.     

不同方法提取基因组DNA的比较

目的通过对不同方法的比较建立一种遗传工程鼠基因组DNA鉴定提取最简单快速的方法。方法以大鼠和小鼠的鼠尾及脚趾为实验材料,采用酚氯仿、煮沸法、涡旋离心法提取基因组DNA;通过琼脂糖凝胶的方法检验DNA质量,通过对时间的统计分析比较3种方法提取DNA的速率。利用PCR技术检测DNA的扩增效果。结果煮沸法、涡旋离心法和酚氯仿提取的DNA比较,电泳条带均较弱。涡旋离心法所耗费的时间最短,煮沸法次之,酚氯仿法耗时最长。除了煮沸法外,涡旋离心法和酚氯仿方法提取的DNA在用于PCR检测时都能得到清晰的目的条带。结论在使用PCR进行基因型初步鉴定时,涡旋离心法是三种方法中最简单快速的首选方法。     

Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant-albeit only 1.7-fold-increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120(h)(E)) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120(h)(E). M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.     

Nonviral DNA vectors for immunization and therapy: design and methods for their obtention

The use of plasmid DNA for vaccination and therapy is a relatively novel technology, with advantages and limitations as with other gene transfer techniques. The technology is based on DNA vectors designed for administering genes coding for relevant proteins into a given organism, fulfilling requirements of the regulatory agencies that once properly formulated and delivered the desired vaccine/therapeutic effect can be achieved. Starting from conventional plasmid DNA vectors currently tested in clinical trials, improvement resulted in bacterial element-less vectors, increasing the complexity of the developmental process. The present review focuses on systems described for generating these nonviral DNA vectors for immunization and therapy from bacterial hosts (conventional and conditionally replicating plasmids, nonreplicating minicircles, and linear dumbbell-shaped expression cassettes) in vivo or in vitro. Additionally, nontherapeutic genetic sequences with a negative or positive effect according to the specific application are described, bringing a better comprehension of the technologys state of the art.Abbreviations COR Conditional origin of replication - ISS Immunostimulatory sequences - MIDGE Minimalistic immunogenic defined gene expression - PCR Polymerase chain reaction     

三种测序DNA模板制备方法的比较○

背景：DNA模板质量对DNA序列测定起着至关重要的作用。 目的：为基因组DNA或甲基化DNA测序寻找一种经济,简便的方法。 方法：分别采用96管集合板及96孔板提取质粒,并且针对质粒设计一对包含目的片段的引物,扩增后纯化PCR产物,通过以上3种方法制备DNA测序模板进行测序。 结果与结论：实验所采用的3种方法对于基因组DNA测序效果无差异(P > 0.05)。对于甲基化DNA测序效果,96管集合板法优于其他2种方法(P < 0.05)。说明3种方法均适用于基因组DNA的测序,而96管集合板法更适用于甲基化DNA的测序。      

Infection of bovine cells of embryonic origin by amphotropic retroviral vectors

Two amphotropic-based mouse retroviral vectors carrying the neomycin-resistance gene were used to infect four bovine cell lines. Two cell lines, bovine kidney and spleen cells, were refractory to the infection while two independent bovine cells of apparent embryonic origin were infected by the amphotropic retroviral vectors at a measurable liter. Southern blot analysis reveals the presence of neomycin-resistance gene in the G418- resistant bovine cells. The results demonstrate the successful transfer of a gene to bovine cells of embryonic origin using a murine retroviral vector system.     

Purification and characterization of bovine herpesvirus-1 isolates and virus DNA utilizing bovine embryonic lung cells

Summary Techniques are described for the culture of bovine embryonic lung (BEL) cells by the primary explant technique and mild enzymatic disaggregation of lung tissue. Preparations of bovine herpesvirus-1 (BHV-1) isolates with titers of 10⁹ plaque-forming units/ml are purified by sucrose and cesium chloride gradient centrifugation after replication in primary cultures of BEL cells. BHV-1 isolated by velocity sedimentation bands in 35% sucrose and sediments at a density of 1.23 g/cc in cesium chloride density gradients. The DNAs of all virus isolates have a velocity sedimentation coefficient of approximately 54S. Virus DNAs from all isolates sediment to equilibrium in cesium chloride at 1.730 g/cc. Differences between virus isolates can be demonstrated by digestion of virus DNA with the restriction endonucleaseKpnI and by polyacrylamide gel electrophoresis of virion polypeptides. These techniques are useful for production of purified high titer virus free of host cell contaminants.     

Comparison of methods for concentration and purification of bovine viral diarrhea virus

The NADL strain of bovine viral diarrhea virus (BVDV) was concentrated by hollow fiber ultrafiltration or polyethylene glycol and purified by centrifugation through sucrose or potassium tartrate gradients. The protein content of polyethylene glycol concentrates was much lower than that of ultrafiltration concentrates. Conversely, recoveries of infectivity were greater using polyethylene glycol (100%) as compared to ultrafiltration (50%). Sucrose or potassium tartrate density gradients appeared comparable in purification of BVD virus. Peak infectivity fractions in both gradients corresponded quite well, having densities of 1.12-1.14 g/cm3, and showed a 150-fold reduction of protein when compared to crude viral supernate. Further examination by negative stain electron microscopy revealed integral pleomorphic, roughly spherical particles in both purified virus preparations. Small knob-like projections could be seen on viral particles.