Transfection of Lymantria dispar insect cell lines

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.     

含稳定整合pMCLacI／Neo质粒的NIH3T3细胞体外突变研究？…

目的 建立一个用于比较研究哺乳动物细胞内表达基因和沉默基因的突变机理的实验模型。方法 以脂质体转染法将线性化的ｐＭＣＬａｃＩ／Ｎｅｏ质粒导入ＮＩＨ３Ｔ３细胞,用Ｇ４１８筛选,造反一个药物抗性细胞克隆进行扩增,用基因组Ｓｏｕｔｈｅｒｎ杂交,ＲＴ－ＰＣＲ及ＲＴ－ＰＣＲ Ｓｏｕｔｈｅｒｎ杂交进行了分子鉴定。结果 （１）在此细胞克隆的基因组中整合有ｐＭＣＬａｃＩ／Ｎｅｏ质粒;（２）该质粒上的两个ｌａｃＩ靶     

Practicable recovery of plasmids carrying cDNAs encoding anti-apoptotic factors that had been introduced by particle bombardment into 5-week old rat hippocampal slices.

In the central nervous system, neuronal cells interact with glial cells and functionally differentiate, a process which can not be reproduced in cell culture. Identification of the novel factors involved in the growth and/or rescue of the differentiated neuronal cells has been impeded by a lack of methods for selecting the genes. In this study, hippocampal slices of a 5-week old rat were transiently introduced with plasmid DNA carrying anti-apoptotic rat bcl-2 or bcl-x cDNA by a particle-bombardment transfection procedure. The plasmid DNAs were expected not to be digested in living cells. Intact plasmid DNAs were recovered by PCR amplification from the slices with bcl-2 or bcl-x cDNAs but not from slices with empty vector or bax cDNA that promotes cell death. This study proposed that a technical combination of organotypic culture and particle-bombardment transfection is profitable for identifying novel genes that promote the survival of neuronal cells.     

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants

Summary.  Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-’79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-’85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-’79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-’85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation. Accepted April 13, 1999 Received January 24, 1999     

Methods of isolating and selecting recombinant vaccinia viruses expressing heterogenetic viral antigens

The paper describes a method using plasmid construction pSC11 for generation of recombinant vaccinia viruses supporting coexpression of heterologous genes and beta-galactosidase. The Ca2+-phosphate method of cell transfection by recombinant DNAs generated on the basis of pSC11, and selection of recombinant viruses from blue plaques of virus-infected cells in the presence of X-gala are reported at length.     

超声靶向微泡破碎介导EGFP基因转染肝癌细胞的影响因素研究

观察超声靶向微泡破碎(ultrasound targeted microbubble destruction,UTMD)在不同转染条件和细胞状态下对肝癌细胞HepG2转染率及细胞活性的影响.体外培养HepG2细胞,随机分为4组:质粒组、微泡+质粒组、超声+质粒组和超声+微泡+质粒组,各组再分为贴壁和悬浮状态2组.悬浮状态的超声+微泡+质粒组根据质粒和微泡浓度不同分为微泡浓度组和质粒浓度组,转染24h后在倒置荧光显微镜下观察绿色荧光蛋白在HepG2细胞中的表达,流式细胞仪测定细胞转染率,MTT法检测细胞活性.结果 在超声声强2w/cm2、占空比20％、照射时间60s的超声参数作用下,质粒5μg/ml、微泡:细胞20∶1时,悬浮状态细胞转染率为7.33％±0.98％,存活率为90.37％±1.80％贴壁状态细胞转染率为1.56％±0.81％,存活率为81.10±1.26％,两者比较有显著差异(P＜0.01).悬浮状态下,提高质粒和微泡浓度至质粒10 μg/ml、微泡:细胞40∶1时转染效率增至15.63％±1.81％,且细胞生存率＞80％.结论 相同转染条件下悬浮状态细胞转染率及存活率明显优于贴壁状态.优化质粒、微泡浓度可进一步提高超声微泡介导的基因转染效率和存活率.     

Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors

Mesenchymal stem cells (MSCs) have been proposed for use in combinatorial gene and cell therapy protocols for the treatment of disease and promotion of repair. The efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression with minimal cell death. The study was carried out on bone-marrow derived rat MSCs, and a range of vectors was tested on the same stem cell preparation. Adenovirus, adeno-associated virus (AAV; serotypes 1, 2, 4, 5, and 6), lentivirus, and nonviral vectors were compared. Lentivirus proved to be most effective with transduction efficiencies of up to 95%, concurrent with low levels of cell toxicity. Adenovirus also proved effective, but a significant increase in cell death was seen with increasing viral titer. Rat MSCs remained refractory to transduction by all AAV serotypes, in contrast to rabbit MSCs tested at the same time. Lipofection of plasmid DNA gave moderate transfection levels but was also accompanied by cell death. Electroporative gene transfer proved ineffective at the parameters tested and resulted in high cell death. High and moderate levels of cell transduction using lentivirus vectors did not affect the ability of the cells to differentiate down the adipogenic pathway.     

重组人pEGFP—heNOS质粒转染人内皮祖细胞及其条件优化

目的用脂质体介导重组人pEGFP—heNOS质粒转染人内皮祖细胞（endothelial progenitor cell,EPC）,并对其转染条件优化以获得较高转染效率。方法脂质体介导重组人pEGFP—heNOS质粒转染人骨髓来源的内皮祖细胞,改变质粒及脂质体剂量,在荧光显微镜下观察荧光并计算转染效率;转染48h后逆转录聚合酶链反应（RT～PCR）和Westernblot方法检测heNOS基因在内皮祖细胞中的表达。结果重组人pEGFP—heNOS质粒体外成功转染入人内皮祖细胞中,质粒：脂质体为1：1时转染效率最高。结论成功地将重组人pEGFP—heNOS质粒体外转染入内皮祖细胞中,通过优化转染条件提高了转染效率,为下一步基因治疗提供了试验基础。     

基因枪介导不同真核质粒DNA转染体外细胞系的实验研究

目的:建立基因枪子的弹制备及将不同质粒DNA转染体外培养MCF-7细胞系的方法。方法:以亚精氨-氯化钙沉淀法制备子弹,采用基因枪方法分别将真核表达质粒pEG-FP,Pmcherry转染对照组和实验组MCF-7细胞,转染24h后,利用荧光显微镜观察细胞中红、绿荧光蛋白的表达情况。结果:制备了基因枪子弹,基因枪介导的pEGFP、Pmcherry能够分别和共同转染入体外培养的MCF-7细胞中,转染后24h可检测到红、绿色荧光,而对照组则没有荧光蛋白的表达。结论:基因枪能够有效介导外源基因转移并能够实现两种基因的共同转移,基因枪转染的MCF-7细胞能够有效表达报告基因。     

Development of RD-tat cell lines: use in HIV recombination studies

The transactivator (tat) gene of human immunodeficiency virus (HIV) plays an essential role in the replication cycle of HIV. Previous studies have evaluated the extent and mechanistic aspects of tat-mediated transactivation using lymphoid and adherent non-lymphoid cells. We have exploited the transactivation property of the tat gene to achieve high levels of hybrid HIV resulting from recombination between HIV DNAs. For this purpose, we have generated stably transformed human rhabdomyosarcoma (RD) cell lines expressing tat gene product of HIV-1. Functional analysis of the cell lines for the presence of tat protein by transfecting HIV-long terminal repeat (LTR) linked to chloramphenicol acetyl transferase (CAT) revealed low, moderate, and high tat producer cell lines. RD-tat cell lines also showed enhanced virus production upon transfection of HIV-1 proviral DNA. Further, tat producer cell lines showed a high amount of hybrid virus in comparison to the control RD cells upon transfection of truncated viral DNAs. Thus, RD-tat cell lines would be valuable target cells for generating homogeneous viruses upon transfection of viral DNA.     

Molecular cloning of infectious proviral genomes of bovine leukemia virus

Covalently closed circular DNA molecules of bovine leukemia virus were cloned in the lambda phage vector lambda gtWES-lambda B and subsequently in the plasmid vector pUC12. Proviral DNAs of 8.3 kb which have one copy of a long terminal repeat and uniform restriction endonuclease sites were preferentially obtained. Four randomly selected clones were examined for their biological activities by DNA transfection experiments. The ovine embryonic cells transfected with these clones formed syncytia which represent the expression of BLV genes. The infectious virions could be recovered from the transfectants.     

A gene transfection for rat mesenchymal stromal cells in biodegradable gelatin scaffolds containing cationized polysaccharides

The objective of this study was to design three-dimensional scaffolds of bone marrow-derived stem cells capable of their gene transfection and evaluate the transfection extent. Three-dimensional scaffolds of gelatin and β-tricalcium phosphate (β-TCP) were prepared. Spermine-introduced pullulan (spermine-pullulan) was prepared as the non-viral carrier of gene transfection. The spermine-pullulan was mixed with a luciferase plasmid DNA to prepare their polyion complex. The scaffolds were treated with succinylated gelatin (suc-gel) at different concentrations, treated in different methods of freeze-drying and dehydrothermal treatment, and placed in the aqueous of complexes to prepare various scaffolds containing the complex. When the stem cells were seeded into the scaffolds to evaluate the gene transfection of cells, the level of plasmid DNA transfection depended on the method of complex containing scaffolds preparation. The complexes were released with time from the scaffolds although the release profile depended on the type of scaffolds. The order of gene transfection for the stem cells in the scaffolds was in good accordance with that of plasmid DNA released. It is possible that cells were transfected with the complexes released from the scaffolds.     

A transforming plasmid from HSV-2 transformed cells contains rat DNA homologous to the HSV-1 and HSV-2 genomes

Morphologically transformed rat (3Y1) cell lines were established following transfection with HSV-2 mtrII DNA sequences (0.585 to 0.601 map units). The mtrII sequences were cloned in plasmids containing the neor gene. Cells resistant to the antibiotic G-418 were passed into soft agarose, and clonal lines were established from individual colonies. The DNAs from two cell lines examined by Southern blot hybridization were shown to contain the original transfected viral DNA sequences in a fashion consistent with a multiple and complex pattern of integration. From one cell line, an approximately 20-kbp plasmid was isolated after transformation of bacteria with Hirt supernatant DNA. This plasmid was capable of rapidly transforming rat cells at a greater than 1000-fold higher frequency than the mtrII DNA. This plasmid consists mainly of unique sequence rat DNA with two copies of the HSV-2 mtrII region DNA (HSV-2 genomic map unit location of ca. 0.595) present at sites distant from each other. The rat DNA in the rescued plasmid is homologous to the putative focus-forming sequences present in the HSV-2 mtrIII (0.53 to 0.58 map units) and the colinear HSV-1 DNA. The genomic copy of these rat sequences in four HSV-2 mtrII transformed cell lines appears to have undergone rearrangement. These data provide evidence that the HSV-2 mtrII sequences are involved in transformation, and that the HSV-2 mtrII region may affect transformation by rearranging the cellular sequences that are homologous to mtrIII.     

丙型肝炎病毒核心蛋白转染HepG2细胞对其p53表达的影响

目的 研究丙型肝炎病毒核心蛋白（HCV—core protein，HCV—C）对HepG2细胞周期、细胞凋亡和n53蛋白表达的影响。方法 表达HCV—C的真核表达质粒pcDNA3.1-core，通过Lipofectamine基因转染法转染HepG2细胞，经G418筛选获得稳定转染HepG2细胞，经Westem Blot证实HCV核心蛋白阳性表达。利用四甲基偶氮唑蓝比色（MTT）法，平板克隆实验和流式细胞术，蛋白印迹和免疫细胞化学法检测HCV核心蛋白对细胞生长增殖率、细胞周期、细胞凋亡率和p53蛋白表达的影响。结果 HCV—C转染组细胞增殖率显著高于空白质粒转染组和未转染组；HCV—C转染组细胞S期所占百分率高于未转染组；HCV-C转染组细胞凋亡率显著低于未转染组；HCV—C转染组细胞突变型p53蛋白的表达高于空白质粒转染组和未转染组。结论 HCV核心蛋白可能通过促进突变型p53蛋白的表达，促进HepG2从G0／1期进入S期，促进细胞生长增殖，抑制细胞凋亡。     

α-1,2岩藻糖基转移酶基因293C＞T和658C＞T突变真核细胞稳定表达的研究

目的 建立α-1,2岩藻糖基转移酶基因(α-1,2-fucosyltransferase gene,FUT1)293C＞T和658C＞T突变真核表达细胞,阐明FUT1突变引起H抗原减弱的机制.方法 抽提类孟买型先证者基因组DNA扩增FUT1全长编码序列,扩增片段与真核表达载体pcDNA3.1连接构建重组表达质粒.采用脂质转染技术将重组质粒转染COS7细胞并进行稳定表达筛选.采用实时荧光定量PCR检测mRNA表达量,应用HPLC检测酶活性,采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和Western印迹技术鉴定蛋白.结果 构建了pcDNA3.1-FUT1野生型、pcDNA3.1- FUT1 293C＞T、pcDNA3.1-FUT1 658C＞T真核表达重组质粒,转染后通过G418筛选获得了稳定表达FUT1的COS7细胞.以野生型重组体转染细胞中FUT1 mRNA量作为对照,293C＞T和658C＞T重组体转染细胞FUT1 mRNA量分别为野生型的97.10％和104.74％.经SDS-PAGE电泳和Western印迹检测显示细胞表达相对分子质量约46000大小的目的蛋白片段,pcDNA3.1-FUT1野生型重组体转染细胞表达蛋白可催化相应的酶促反应,而pcDNA3.1-FUT1 293C＞T、pcDNA3.1- FUT1 658C＞T转染细胞蛋白完全失去酶活性.结论 体外实验提示293C＞T和658C＞T突变并不影响FUT1 mRNA转录和蛋白生成,但表达蛋白的酶活性明显下降,从而导致H抗原生成减弱.     

Large-scale production means for the manufacturing of lentiviral vectors

Lentiviral vectors become more and more famous for the use as gene vector for gene therapy purposes for the treatment of acquired or inherited diseases. In this review, the present state of the art of the production of lentiviral vectors is presented with particular emphasis on the large scale production of these vectors for preclinical and clinical purposes. In contrast to oncoretroviral vectors which are produced using stable producer cell lines, clinical grade lentiviral vectors are essentially produced by transient transfection of 293 or 293T cells grown in Cell Factories. The main reason is that these production processes have been developed when good and safe LV producer cell lines were not available. With respect to the purification of lentiviral and in agreement with actual developments in the biotech industry, rather sophisticated downstream processing protocols have been established in order to remove any potentially dangerous process derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review presents large scale production means for LV vectors, the different downstream processing steps as used for the purification of LV vectors as well as LV specific safety issues. Published large scale production and purification processes of lentiviral vectors and their process performances are compared.     

脂质体介导法转染mIMCD-3细胞的可行性及最佳转染条件

背景：近年来阳离子脂质体成为基因转移较为常用的载体,但应用脂质体对mIMCD-3细胞株进行转染的相关报道较少。 目的：探讨脂质体法转染mIMCD-3细胞株的可行性,并通过优化脂质体转染效率的影响因素,获得mIMCD-3的最佳转染条件。 方法：以绿色荧光蛋白作为报告基因,采用脂质体Lipofectamine^TM 2000包裹pIRES2-EGFP质粒转染mIMCD-3细胞,分别按照细胞接种密度、DNA用量、DNA与脂质体的比例、不同的质粒抽提方法、血清有无设定分组,观察以上因素对mIMCD-3细胞转染效率的影响。 结果与结论：采用脂质体Lipofectamine^TM 2000转染mIMCD-3细胞,在2×10⁸ L^-1细胞接种浓度、1 μg DNA用量、1∶3的DNA与脂质体比例时,转染效率最高。采用美国OMEGA公司生产的E.Z.N.A. Endo-Free Plasmid Mini Kit抽提的质粒能获得更高的转染效率。转染前漂洗细胞后换入无血清培养基比有血清组转染效率高(P < 0.01),而转染后6 h加入血清不影响转染效率。结果显示,脂质体法转染mIMCD-3细胞株是可行的,通过优化脂质体转染效率的影响因素,获得了mIMCD-3的最佳转染条件,可作为有关研究或应用的参考。     

Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania

Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania. RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei. For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively. This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids. However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L. donovani. Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect. Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct. These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae. The implications to parasite biology, gene amplification, and genetic analysis are discussed.     

Optimized protocol for high-titer lentivirus production and transduction of primary fibroblasts

The lentivirus–short hairpin RNA (shRNA) system is a widely used tool for RNA interference. Multiple factors may affect the RNA interference efficiency during lentivirus production and transduction procedures. Thus, an optimized protocol is required to achieve high-titer lentivirus and efficient gene delivery. In the present study, lentivirus was produced by transfecting lentiviral transfer and packaging plasmids into HEK 293T cells. The factors affecting lentiviral titer were assessed, including lentiviral plasmid ratio, lentiviral transfer plasmid type, serum type for cell culture, transfection reagent–plasmid mixture incubation time, and the inoculation density of 293T cells for transfection. The high-titer lentivirus was achieved when plasmids were transfected at a molar ratio of 1:1:1:2, and the transfection reagent–plasmid mixture was replaced 6–8 h after transfection. The pLVX-shRNA2 lentiviral transfer plasmid was associated with the highest lentiviral titer, while both pLVX-shRNA2 and psi-LVRU6GP plasmids were associated with efficient RNA interference in target cells. The serum type for 293T cell culture affected the lentiviral titer significantly, while the inoculation density of 293T cells showed no influence on transfection efficiency or lentiviral titer. Moreover, the human primary fibroblasts infected with lentivirus, using the centrifugation method, achieved higher transduction efficiency than those infected with the non-centrifugation method. In conclusion, this study helped optimize lentiviral production and transduction procedures for more efficient gene delivery.