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Results for “vector engineering”

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At least 523 records · Page 29Linked to original sources
PubMed1990

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Animals↗
PubMed1983

Easy identification of cDNA clones.

A set of six cloning vectors, pUR 278, 288, 289, 290, 291, 292 is presented. These vectors have the cloning sites, BamHI, SalI, PstI, XbaI and HindIII, in all frames at the 3' end of the lacZ gene. Insertion of cDNA in the proper cloning sites leads to a fusion protein of active beta-galactosidase and the peptide encoded by the cDNA. A simple immunoenzymatic assay can be used to identify clones in such a cDNA library.

Amino Acid Sequence↗
PubMed1999

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

Animals↗
PubMed1991

Vaccinia virus: a tool for research and vaccine development.

Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

Animals↗
PubMed1986

Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: removal of early region introns from polyoma virus mutants.

We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37 degrees C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.

Antigens, Viral, Tumor↗
PubMed1990

Use of the lac repressor in constructing sequential deletions and a new sequencing vector.

Large sequencing projects require an efficient strategy to generate a series of overlapping clones. This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion. We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence. To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector. Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences. Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31. Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions. This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions.

Base Sequence↗
PubMed1998

Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines.

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation.

Animals↗
PubMed1991

Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

A method for transforming Tetrahymena has been established earlier, but its application has been limited because of the lack of selectable markers other than the rRNA-encoding DNA (rDNA). Mutations in the yeast ribosomal protein L29 gene (CYH2) are known that confer cycloheximide resistance. We have cloned and sequenced the homologue of this gene from both a wild-type and a cycloheximide-resistant (ChxA) strain of Tetrahymena. Surprisingly, a comparison shows that the ChxA mutation is not present in the CYH2 homologue. We therefore created the yeast mutations in the Tetrahymena gene by site-directed mutagenesis and used them to transform Tetrahymena either with or without linking to an rDNA vector. All clones transformed by the rDNA vector also became resistant to cycloheximide when the rDNA contained the engineered mutant genes. Without the rDNA vector, the mutant genes transform approximately 1% of injected cells to become resistant to cycloheximide. DNA analysis indicates that transformation occurs by replacement of the host sequence and not by random integration of the injected sequence. The replacement occurs to some but not all copies of this gene in the polyploid macronuclear genome. Thus, transformation in Tetrahymena occurs by specific sequence replacement, and the injected mutant genes can serve as dominant selectable transformation markers in this organism.

Amino Acid Sequence↗
PubMed1992

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugates were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT0230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 x 10(-4) transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 M NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation.

Biotechnology↗
PubMed2002

Hexon gene switch strategy for the generation of chimeric recombinant adenovirus.

The usefulness of adenovirus as a vehicle for transgene delivery is limited greatly by the induction of neutralizing anti-adenoviral immunity following the initial administration, thereby resulting in shorter-term and reduced levels of transgene expression. In this paper, we outline a strategy for the generation of recombinant Ad5-based adenovectors that have undergone a complete hexon exchange in an effort to circumvent pre-existing anti-vector humoral immunity. Eighteen different chimeric adenoviral vectors (from subgroups A, B, C, D, and E) have been constructed using a combination of direct cloning and bacterial homologous recombination methods. However, only chimeric Ad5-based constructs in which the hexons from Ad1, Ad2, Ad6, and Ad12 are incorporated in place of the Ad5 hexon were successfully rescued into viruses. Despite several attempts, the remaining fourteen chimeric adenovectors were not rescuable. In vivo rodent studies using transgenes for human immunodeficiency virus type 1 (HIV-1) gag and secreted human alkaline phosphatase (SEAP) suggest that the Ad5/Ad6-gag chimera (wherein Ad5 hexon was replaced with that of Ad6) is able to evade neutralizing antibodies generated against Ad5 vector efficiently. However, it appears that cross-reactive cytotoxic T lymphocytes (CTL) may also play a role in controlling in vivo infectivity of Ad5/Ad6-gag chimera. The Ad5/Ad12 chimera was found to be extremely ineffective in the i.m. delivery and expression of HIV-1 gag in mice compared to the Ad5/Ad6 construct. Implications of these results will be discussed.

Adenoviruses, Human↗
PubMed1990

In vivo expression and survival of gene-modified T lymphocytes in rhesus monkeys.

Lymphocytes can be readily transduced with retroviral vectors and the gene-modified lymphocytes will stably express the inserted genes in vitro for long periods. As a prelude to studies in humans, we evaluated the survival of gene-modified T lymphocytes and the expression of the introduced genes in nonhuman primate T lymphocytes both in vitro and in vivo to determine if lymphocytes could be a potential cellular gene therapy vehicle. Rhesus peripheral blood T-lymphocytes and/or lymph node lymphocytes were transduced with a retroviral vector that contained a bacterial neomycin resistance (NeoR) gene or both NeoR and the human adenosine deaminase (hADA) genes. The cells were then selected for NeoR expression by growth in the neomycin analogue G418 and the autologous gene-modified T cells were reintroduced into the donor animals. T lymphocytes were periodically regrown from the blood and selected in G418. Gene-modified cells persisted in 1 animal for 727 days as detected by analysis for vector DNA by polymerase chain reaction (PCR). Evidence for expression of the human ADA or NeoR genes has also been detected up to 727 days after cell infusion. These findings suggest that gene-modified T lymphocytes can survive and circulate for long periods in vivo and can continue to express the introduced genes.

Adenosine Deaminase↗
PubMed1987

Towards gene therapy for hemophilia B.

Hemophilia B is an X-chromosome-linked bleeding disorder resulting from lack of clotting factor IX activity and affects about 1 in 30,000 males. Current therapy involves injection of crude factor IX prepared from pooled human plasma. Treatment is complicated by viral contaminants in factor IX preparations, such as non A-non B hepatitis and the AIDS virus, and by the practical difficulties of chronic injections. An alternative therapy might include the insertion of a factor IX expression vector into the somatic cells of affected individuals to allow continued production of factor IX. Toward this end, we have constructed a retrovirus vector for transfer and expression of factor IX. Despite the fact that factor IX is normally synthesized in hepatocytes and requires extensive post-translational modification for activity, we have shown that fully active factor IX can be made by human skin-derived fibroblasts. These results open the way to testing the use of skin grafts for gene therapy of hemophilia B.

Biological Assay↗
PubMed1987

Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase.

The gene for firefly luciferase (luc) can be used as a generalized genetic probe. A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here. Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction. Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay. This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.

Animals↗
PubMed2003

Stability of plasmid pA387 derivatives in Amycolatopsis mediterranei producing rifamycin.

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.

Actinomycetaceae↗
PubMed1995

Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

Animals↗
PubMed1996

Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells.

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.

Animals↗
PubMed1995

Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli.

A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.

Base Sequence↗