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At least 955 records · Page 53Linked to original sources
PubMed2004

Chemical nursing: phytosulfokine improves genetic transformation efficiency by promoting the proliferation of surviving cells on selective media.

The relative growth rate of plant cells in vitro is considerably affected by initial cell density. This troublesome effect has interfered with the establishment of efficient plant cell culture systems, especially when only a small number of cells are expected to survive, such as in the genetic transformation of cells under antibiotic selection. To improve the recovery of antibiotic-resistant cells, we examined the use of the peptide plant hormone phytosulfokine (PSK), which has been shown to promote cellular growth and development in vitro. The addition of PSK to selective media increased the recovery of transformed callus from Agrobacterium-infected carrot hypocotyl explants from 7% to 39%, which is more than a fivefold improvement over the control. Most calluses developed into normal plantlets with cotyledons and primary roots and, eventually, formed foliage leaves. Thus, chemical nursing using PSK shows promise as a tool for basic research in plant biology and biotechnological applications.

Anti-Bacterial Agents↗
PubMed2004

Transformation of Saussurea medusa for hairy roots and jaceosidin production.

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.

Agriculture↗
PubMed2005

Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.

Agrobacterium tumefaciens↗
PubMed1997

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants.

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.

Agrobacterium tumefaciens↗
PubMed1998

DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice.

In order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after final vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after final booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells.

Animals↗
PubMed2005

Mice with the enhanced green fluorescent protein gene knocked in to chromosome 11 exhibit normal transmission ratios.

Meiotic recombination between homologous chromosomes can be suppressed within a chosen segment by a regional inversion. In mice, this feature can be engineered and conveniently used in genetic screens to maintain chemically induced mutations within the homologous chromosome. The efficiency of an inversion-based mutagenesis screen can be substantially enhanced provided that the inversion chromosome and its wild-type (WT) homologue are both visibly tagged by two different coat color markers. Dual tagging eliminates labor associated with molecular genotyping. Previously, we reported the generation of the In(11)10Brd strain of mice carrying K14-agouti tagging a 30-cM inversion between the Trp53 and Egfr loci on mouse chromosome 11. Since K14-agouti causes yellowing of ears and tails, the In(11)10Brd mice are easily distinguishable from their WT littermates. In this paper, we describe the construction of a second strain of mice that carry the enhanced green fluorescent protein (EGFP) transgene at the Egfr locus. The EGFP carriers are visually recognizable by emitting green fluorescent light upon UV illumination. We found that the EGFP function was transmitted from one generation to another with expected Mendelian frequencies, and no detrimental effects of EGFP expression were detected in hemizygous or homozygous animals. The EGFP mice together with the previously generated In(11)10Brd inversion carriers constitute a complete set of reagents required for initiation of a regional ENU mutagenesis screen to address functionally more than one-third of mouse chromosome 11.

Animals↗
PubMed2006

Enhancement and selective production of oligomycin through inactivation of avermectin's starter unit in Streptomyces avermitilis.

Oligomycin and its analogues, produced by Streptomyces avermitilis and other actinomycetes, are of interest for their potent and selective biological activities. PCR-mediated gene replacement, targeting bkdF, one of avermectin's starter unit encoding genes in S. avermitilis, was performed to yield an oligomycin producer, BIB0423. The engineered strain produced oligomycin A at 2.3 mg/ml compared to the wild type strain at 0.1 mg/ml. This resulting mutant was genetically stable and should be useful for the industrial production of oligomycin.

Gene Expression Regulation, Bacterial↗
PubMed2006

Polymeric gene transfection on insulin-secreting cells: sulfonylurea receptor-mediation and transfection medium effect.

PURPOSE: In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/ transplantation. Insulin-secreting insulinoma cell lines or pancreatic beta-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection. METHODS: Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca2+ and K+. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection. RESULTS: For an insulin-secreting cell line (RINm5F), polyplexes in Ca2+--containing KRH medium (Ca2+(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K+ or depleting Ca2+ from Ca2+(+)KRH, poly(L-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection. CONCLUSION: The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.

ATP-Binding Cassette Transporters↗
PubMed2006

High lysine and high tryptophan transgenic maize resulting from the reduction of both 19- and 22-kD alpha-zeins.

The major maize seed storage proteins, zeins, are deficient in lysine and tryptophan content, which contribute to the poor nutritional quality of corn. Whether through the identification of mutations or genetic engineering, kernels with reduced levels of zein proteins have been shown to have increased levels of lysine and tryptophan. It has been hypothesized that these increases are due to the reduction of lysine-poor zeins and a pleiotropic increase in the lysine-rich non-zein proteins. By transforming maize with constructs expressing chimeric double-stranded RNA, kernels derived from stable transgenic plants displayed significant declines in the accumulation of both 19- and 22-kD alpha-zeins, which resulted in higher lysine and tryptophan content than previously reported for kernels with reduced zein levels. The observation that lysine and tryptophan content is correlated with the protein levels measured in transgenic maize kernels is consistent with the hypothesis that a pleiotropic increase in non-zein proteins is contributing to an improved amino acid balance. In addition, a large increase in accumulation of free amino acids, consisting predominantly of asparagine, aspartate and glutamate, was observed in the zein reduction kernels.

Amino Acids↗
PubMed1985

Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture.

Mouse L fibroblasts and other mammalian cells are killed by the translation inhibitor hygromycin B. We have modified the gene conferring resistance against hygromycin B in E. coli in such a way that it can be transcribed in mammalian cells from the promoter of the HSVtk gene. The resulting plasmid, pHMR272, was transfected into mouse L fibroblasts and HeLa cells by the calcium phosphate method and upon selection produced clones resistant against hygromycin B. The transfection rate was similar to that obtained with other selective markers. This plasmid is a useful addition to the relatively small number of dominant selectable markers available for mammalian cells.

Animals↗
PubMed1990

Expression of gag precursor protein and secretion of virus-like gag particles of HIV-2 from recombinant baculovirus-infected insect cells.

A recombinant baculovirus carrying the gag gene but lacking the protease coding sequences of human immunodeficiency virus type 2 (HIV-2) has been constructed. When this recombinant baculovirus is used to infect insect cells, a high level of gag precursor protein, gag pr41, is expressed. Electron microscopy showed that the majority of gag pr41 was budding through the plasma membrane and being released into the culture medium in spherical virus-like particles with a diameter of approximately 100 nm. Metabolic labeling demonstrates that gag pr41 is myristylated. Our results demonstrated that HIV-2 gag pr41 can be assembled into virus-like particles in the absence of other HIV proteins. Rabbits immunized with purified gag pr41 particles produced high-titer antibody and Western blot analysis showed that anti-gag pr41 rabbit sera recognize p17, p24, and p55 gag proteins of HIV-1. These results show that gag pr41 particles are highly immunogenic and that gag proteins of HIV-1 and HIV-2 have similar antigenic epitopes.

Animals↗