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At least 541 records · Page 30Linked to original sources
PubMed1988

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice.

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

Animals↗
PubMed1997

Production of bioactive salmon calcitonin from the nonendocrine cell lines COS-7 and CHO.

To produce bioactive salmon calcitonin from the conventional nonendocrine cell lines, COS-7 and CHO, we devised a salmon calcitonin expression vector by combining the amino-terminus of human calcitonin precursor with a salmon calcitonin sequence, inserting the efficient furin-cleavable processing sequence Arg-X-Arg-X-Lys-Arg before salmon calcitonin, and deleting the carboxyl-terminal extension peptide. This chimeric calcitonin precursor terminates at glycine to easily receive an amidation reaction. COS-7 and CHO produced a high level of bioactive calcitonin by the resorption pit formation assay. Although amidating activity is highly expressed in CHO, but only a little in COS-7 cells, both cells produced a similar level of bioactive calcitonin. Thus, the engineered salmon calcitonin expression vector enables nonendocrine cells even with low amidation activity to produce bioactive calcitonin.

Animals↗
PubMed2004

Transgene expression in strawberries driven by a heterologous phloem-specific promoter.

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the beta-glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H+ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-microM thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.

Arabidopsis↗
PubMed1996

Construction of a stable expression vector carrying sop genes [ZJ1].

Mini-F, the fifth fragment of F plasmid from EcoRI digestion, is known to carry an efficient partitioning function. Two pBR322 plasmid derivatives, pDMC32 and pDMC311, have been constructed from this fragment. The plasmid pDMC32 carries all the relative genes for plasmid stability, ccd, repD, and sop genes (sopA, sopB, and sopC), along with oriS and oriV, while pDMC311 carries only sop genes (sopA, sopB, and sopC). The plasmid maintenance proportions for pDMC32 and pDMC311 in E. coli were 93% and 100%, respectively, after 100 generations continuous cultivation of cells harboring the derivatives, MI32 (pDMC32) and MI311 (pDMC311), in a phosphate-limited basal medium. As a control, the maintenance proportion of plasmid pBR322 dropped down to a low of 10% at generation 55 of continuous cultivation of E. coli MIR322 (pBR322) in the same medium. In order to make a stable expression vector that carries only sop genes, plasmid pDMC40 was constructed by adding a trp promoter from pDR720 to pBR322. The stable expression vector pDMC48 was then derived from pDMC40 by inserting sop genes into it from pDMC311. The maintenance proportion of plasmid pDMC48 in E. coli was still 100% after 100 generations of continuous cultivation of cells harboring the plasmid in phosphate-limited basal medium.

Ampicillin↗
PubMed2002

Adenovirus-based libraries: efficient generation of recombinant adenoviruses by positive selection with the adenovirus protease.

Adenoviruses (Ad) deleted in the protease (PS) gene are capable of only one round of replication in non-complementing cells. This feature was exploited to develop a positive selection method for constructing adenoviral recombinants using ectopic expression of the PS gene in the E1 region. Very low levels of PS were sufficient to ensure the rescue of a PS-deleted Ad genome (Ad(Delta)PS), thereby eliminating deleterious effects PS over-expression might exert on cell or virus growth. In addition to the standard co-transfection method, an alternative protocol was developed in which the Ad5-(Delta)PS viral DNA was delivered by infection before subsequent transfection of 293 cells with the transfer vector. Under optimal conditions, at least one recombinant Ad per 10(3) cells was generated with 100% of the plaques being recombinant. Since the infection/transfection protocol is readily scalable, this represents the first method that allows for the easy construction of adenovirus vector (AdV) libraries with high diversities. This approach addresses in a novel way the bottleneck encountered when converting plasmid libraries, constructed in E. coli using a variety of well-established strategies, into corresponding AdV libraries. It maintains high diversity while generating recombinant viruses with 100% efficiency.

Adenoviridae↗
PubMed1999

Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.

3T3 Cells↗
PubMed2002

[Construction, packaging and titration of recombinant adeno-associated virus vectors contaning antitumor genes].

OBJECTIVE: To construct, package, and titrate vectors that express antitumor genes using adeno-associated virus (AAV) containing human alpha-fetoprotein (AFP) promoter. METHODS: Recombinant AAV vectors were constructed by blunted ligation of plasmids rAAV-AFP-GFP and pTR-UF5 and p16 cDNA and p21 cDNA. Packaging cell lines, 293 and C12 cells, were transfected with rAAV vectors, plasmid PspRC72, and adenovirus cosmid pAdc with the help of LipofectAMINE and then infected by wild adenovirus 48 hours later. The total viral particle titre was measured by PCR. Electron microscopy was used to observe the morphology of rAAV. C12 cells were infected with rAAV and wild adenovirus (MOI 10) and the titre of infectious virus was measured. by RCA method. Hepatocellular carcinoma cells were cultured and infected with rAAV containing green fluorescent protein (GFP), the infectious rate was observed 48 hours later. RESULTS: Recombinant vectors, rAAV-AFP-p16, rAAV-AFP-p21, rAAV-CMV-p16, and rAAV-CMV-p21, were constructed and verified by DNA sequencing and enzyme digestion. The total viral titre was 10(13) to 10(14)/ml. The titre of infectious rAAV containing GFP was 5 x 10(10)/ml. Electron microscopy showed that the rAVV was round and plaqued with a diameter between 20 and 30 nm. When the MOI was 100, the infectious rate of hepatocellular carcinoma cells was at least 70%. Four rAAV vectors carrying p16 or p21 genes have been constructed. rAAV packaging and titration methods were developed to produce highly-purified AAV stocks. These approaches help in research of gene therapy in liver cancer.

Cell Line↗
PubMed1997

Retrovirally transduced human dendritic cells express a normal phenotype and potent T-cell stimulatory capacity.

Dendritic cells are attractive candidates for vaccine-based immunotherapy because of their potential to function as natural adjuvants for poorly immunogenic proteins derived from tumors or microbes. In this study, we evaluated the feasibility and consequences of introducing foreign genetic material by retroviral vectors into dendritic cell progenitors. Proliferating human bone marrow and cord blood CD34+ cells were infected by retroviral vectors encoding the murine CD2 surface antigen. Mean transduction efficiency in dendritic cells was 11.5% from bone marrow and 21.2% from cord blood progenitors. Transduced or untransduced dendritic cell progeny expressed comparable levels of HLA-DR, CD83, CD1a, CD80, CD86, S100, and p55 antigens. Granulocytes, macrophages, and dendritic cells were equally represented among the transduced and mock-transduced cells, thus showing no apparent alteration in the differentiation of transduced CD34+ precursors. The T-cell stimulatory capacity of retrovirally modified and purified mCD2-positive allogeneic or nominal antigen-pulsed autologous dendritic cells was comparable with that of untransduced dendritic cells. Human CD34+ dendritic cell progenitors can therefore be efficiently transduced using retroviral vectors and can differentiate into potent immunostimulatory dendritic cells without compromising their T-cell stimulatory capacity or the expression of critical costimulatory molecules and phenotypic markers. These results support ongoing efforts to develop genetically modified dendritic cells for immunotherapy.

Antigen-Presenting Cells↗
PubMed1986

Gene replacement therapy for inborn errors of purine metabolism.

Effective retroviral vectors carrying the human HPRT and ADA genes have been described. Initial characterization of the retroviral gene transfer system using the HPRT vector allowed the delineation of several parameters important in viral titer, expression, and stability. Using the HPRT and ADA vectors, we have initiated experiments designed to insert these human genes into various tissues of the mouse and have demonstrated expression of both transduced genes in mouse bone marrow cells. Further work with these and other vector constructions is underway in the hope that this technique may allow safe and effective treatment of ADA and HPRT deficiencies, paving the way for treatments of other inborn errors of metabolism through somatic gene replacement therapy.

Adenosine Deaminase↗
PubMed1990

Construction and hormone regulation of a novel retroviral vector.

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.

Animals↗
PubMed1995

Molecular genetic manipulation of mosquito vectors.

Despite their central role in disease transmission, relatively little is known of the molecular biology of arthropod vectors. Modern molecular approaches will undoubtedly provide considerable information about gene regulation and expression in vectors and consequently a much better understanding of the biology and molecular biology of vectors. Such knowledge is essential for developing effective control strategies for vector-borne diseases. In this review, we focus upon techniques and approaches used at the Arthropod-Borne and Infectious Diseases Laboratory (AIDL) at Colorado State University to bioengineer mosquitoes with reduced vector competence. We have developed technologies and procedures that allow genetic manipulation of mosquitoes, including RNA and DNA virus gene-delivery vehicles and efficacious antiviral constructs, which will facilitate the development of pathogen-resistant, transformed mosquitoes. Many of the approaches, constructs, and technologies developed at AIDL will be applicable to molecular manipulation of other arthropod genomes.

Animals↗
PubMed2002

Artificial extension of the adenovirus fiber shaft inhibits infectivity in coxsackievirus and adenovirus receptor-positive cell lines.

Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 beta-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype 5 (Ad5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the Ad5 shaft. We show that "longer-shafted" Ad vectors (up to 32 beta-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.

Adenoviruses, Human↗
PubMed2002

Replication competent helper functions for recombinant AAV vector generation.

Adeno-associated virus (AAV) is a promising gene transfer vector tested in both animal studies and human clinical trials. However, current production methods are generally inefficient and require improvements to meet the increasing clinical need for economical, high titer and high quality rAAV vectors. The inefficiency of the current systems largely arises from the AAV helper function, which contains only the AAV coding region but lacks inverted terminal repeats. The terminal repeats were originally removed to prevent replication competent AAV contamination. Here we designed a novel and highly efficient rAAV helper function containing AAV terminal repeats. The new helper function not only mimics the wild-type AAV growth as it replicates along with the vector plasmid, but also restores the cis regulating function of the AAV terminal repeats. Addition of heterologous introns to the helper genome and use of a mutant AAV terminal repeat defective in packaging effectively controls the contamination of replication competent AAV particles. This new strategy also performs better in AAV producing cell lines than those based on non-replicating AAV rep and cap genome.

Cell Line↗
PubMed1992

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.

Biological Transport, Active↗
PubMed1990

Vectors and genes for improvement of animal strains.

Strain improvement of agriculturally important animals will require efficient techniques for gene delivery, the ability to regulate the expression of the newly introduced genes and, most important, the identification of genes whose appropriate expression could cause improvement of the animal. We have developed a series of avian retroviral vectors that can be used to introduce new genetic information into the germ line of chickens, for which transgenics cannot be created by direct microinjection of DNA into fertilized eggs. We have identified a 220-bp segment of the chicken skeletal muscle alpha-actin gene that can cause other genes to be expressed specifically in striated muscle. This chicken promoter shows correct tissue specificity in transgenic mice and presumably could be used in other mammalian species. The skeletal muscle alpha-actin promoter has been inserted into the avian retroviral vectors and the promoter is functional in cultured cells infected by these retroviral vectors. The tissue specificity of the expression of the skeletal muscle alpha-actin promoter carried by the retroviral vectors will soon be tested in vivo. We are studying two types of genes that might be useful in strain improvement; genes that could produce dominant resistance to infection by pathogenic viruses, and genes that could play critical roles in muscle development. Expression of the envelope glycoprotein of retroviruses can specifically block the cellular receptor that viruses use to infect a susceptible cell. Expression of the avian leukosis virus subgroup A envelope in transgenic chickens prevents infection by pathogenic viruses of the same subgroup. We are attempting to block reticuloendotheliosis virus infection by expressing the reticuloendotheliosis envelope glycoprotein. We have shown that we can block infection in cultured cells, and we are now creating retroviral vectors for experiments in vivo. We have also begun to study the cellular homologue of the ski oncogene, which has been shown to stimulate the differentiation of quail myoblasts in vitro. Biologically active cDNAs have been isolated; we have now begun to analyse the effects of expressing the c-ski proteins in the whole animal.

Animals↗
PubMed1987

Retroviral-mediated gene transfer into mammalian cells.

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.

Adenosine Deaminase↗
PubMed2002

Generation of hepatitis C virus-like particles by use of a recombinant vesicular stomatitis virus vector.

Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.

Animals↗
PubMed1983

Gene cloning in the actinomycetes.

The production of antibiotics by soil-borne micro-organisms, the actinomycetes, has considerable economic importance. The manipulation of antibiotic producers has become a prime target for the application of recombinant DNA technology. Certain technical requirements have had to be met for gene cloning to be successful in the actinomycetes. These requirements, including the development of cloning vectors and transformation procedures, have been satisfied, in part, for some members of the Streptomyces genus. Some problems including sequence rearrangement and stability of plasmid maintenance are now being recognized. A number of genes have been cloned in Streptomyces and some preliminary results characterizing the gene for a Streptomyces-derived beta-galactosidase-like activity were described.

Actinomyces↗