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Results for “vector engineering”

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At least 919 records · Page 51Linked to original sources
PubMed1992

Genetically engineered bacteria to identify and produce anti-viral agents.

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Tetr mutants are discussed.

Antiviral Agents↗
PubMed2004

Toward novel vaccines and therapies based on negative-strand RNA viruses.

The study of negative-strand RNA viruses has suggested new strategies to produce more attenuated viruses. Reverse genetics has allowed the implementation of the strategies, and new or improved monovalent vaccines are being developed. In addition, recombinant viruses expressing foreign proteins or epitopes have been produced with the aim of developing multivalent vaccines capable of stimulating humoral and cellular immune responses against more than one pathogen. Finally, recombinant viruses that selectively enter cells expressing tumor markers or the HIV envelope protein have been engineered and shown to lyse target cells. Preclinical and clinical trials of improved and multivalent vaccines and therapeutic (oncolytic) viruses are ongoing.

Animals↗
PubMed1993

Genetically improved potatoes: protection from damage by Colorado potato beetles.

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

Amino Acid Sequence↗
PubMed1989

Stable co-transformation of maize protoplasts with gusA and neo genes.

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

DNA↗
PubMed1990

Transgenic tobacco plants and their progeny derived by microprojectile bombardment of tobacco leaves.

Transgenic tobacco plants and progeny carrying coding sequences for neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) were recovered following microprojectile bombardment of tobacco leaves. Transgenic plants were regenerated from bombarded leaf pieces of tobacco cvs. 'Xanthi' and 'Ky 17' which were cultured in the presence of 100 or 200 micrograms/ml kanamycin for six to eight weeks. Among 160 putative transgenic plants from at least 16 independent transformation events 76% expressed NPTII, and 50% expressed GUS. Southern analysis of plants expressing either one or both of the enzymes indicated DNA in high molecular weight DNA in 8 of 9 independent transformants analyzed. Two independent transformants and their progeny were analyzed in detail. Analysis of progeny for quantitative enzyme levels of NPTII and GUS, and Southern analysis of parents and progeny clearly demonstrated that the genes were transmitted to progeny. One transformant demonstrated Mendelian ratios for seed germination on kanamycin-containing medium while the other transformant had non-Mendelian ratios. DNA analysis of progeny indicate complex integration of the plasmid DNA, and suggest that rearrangements of this DNA has occurred. These results are consistent with other methods of direct DNA uptake into cells, and verify that the microprojectile bombardment method is capable of DNA delivery into intact plant cells which can give rise to transgenic plants and progeny.

Blotting, Southern↗
PubMed1990

Deletion analysis of a zein gene promoter in transgenic tobacco plants.

A zein gene (Z4) promoter containing 886 bp upstream from the transcription start site has been shown previously to be active specifically in the endosperm of transgenic tobacco seeds. To investigate the region required for this tissue-specific activity, deletions of the Z4 promoter were constructed and placed upstream of the beta-glucuronidase (GUS) reporter gene. When these deletions were tested in transgenic tobacco plants, seed-specific GUS activity, which reached a peak between 15 and 19 DAP, was observed for promoters extending from -886 to -174. Interestingly, the 174 bp promoter lacked the complete 15 bp consensus sequence found in the same position in all zein genes so far sequenced. With the next shorter promoter in the deletion series (79 bp), which just included the CAAT and TATA elements, negligible GUS activity was observed in seeds. The results demonstrated that 174 bp upstream of the transcription start site are sufficient for tissue-specific and temporally regulated activity of the Z4 promoter in tobacco. At most, two-fold enhanced activity was observed with additional 5' sequences up to -886.

Base Sequence↗
PubMed1995

Osteopontin (OPN) may facilitate metastasis by protecting cells from macrophage NO-mediated cytotoxicity: evidence from cell lines down-regulated for OPN expression by a targeted ribozyme.

Osteopontin (OPN) is a GRGDS-containing phosphoglycoprotein that is capable of facilitating cell adhesion and modulating gene expression via integrin receptors. Three hammerhead ribozymes designed to target three different regions of OPN mRNA were shown to cleave the message catalytically in vitro. Plasmid vectors that had been engineered to express the ribozymes in mammalian cells were used to generate stably transfected T24 H-ras-transformed NIH3T3 cells that normally express OPN at high levels. Northern and Western blot analyses showed that OPN mRNA and protein expression were reduced in a subset of these anti-OPN ribozyme-expressing cell lines. Cells whose ability to produce OPN had been impaired exhibited greater sensitivity to the cytotoxic action of activated RAW264.7 macrophage-like cells; they were also less effective at suppressing macrophage NO production. In agreement with previous reports, they were also less tumorigenic and metastatic in an experimental metastasis assay. These results are consistent with the hypothesis that OPN serves as a defense against NO-mediated host cell cytotoxicity and thereby augments the metastatic phenotype.

Animals↗