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At least 199 records · Page 11Linked to original sources
PubMed1999

Rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics.

RSV is a major cause of pediatric respiratory tract disease worldwide, but a vaccine is not yet available. It is now possible to prepare live infectious RSV completely from cDNA. This provides a method for introducing defined mutations into infectious virus, making possible the rational design of a live-attenuated vaccine virus for intranasal administration. This is particularly important for RSV, for which achieving the appropriate balance between attenuation and immunogenicity by conventional methods has proven elusive. We took advantage of the existence of a panel of biologically derived vaccine candidate viruses that were incompletely attenuated but well characterized biologically. The mutations in these viruses were identified by sequence analysis and characterized by insertion into recombinant virus, thereby providing a menu of known attenuating mutations. These included a series of amino acid point mutations, mostly in the L polymerase, and a nucleotide substitution in a transcription gene-start signal, a cis-acting RNA element. The second source of mutations was from experimental mutational analysis of recombinant virus and involves deletion of the NS1, NS2, or SH gene. We have reconstructed a previously tested, biologically derived attenuated virus, cpts248/404, in recombinant form and are now proceeding to introduce additional mutations from the menu to achieve stepwise increases in attenuation. The ability to modify the attenuation phenotype incrementally in a directed manner should result in an appropriate vaccine virus.

DNA, Complementary↗
PubMed1996

Temperature sensitive mutants of influenza A virus generated by reverse genetics and clustered charged to alanine mutagenesis.

Temperature sensitive (ts) mutants of influenza A virus have the potential to serve as live attenuated (att) virus vaccines. Previously, ts mutants were isolated by chemical mutagenesis or arose spontaneously, and most likely contained point mutations in one or more genes. While sufficiently attenuated, even the most genetically stable of these viruses was found to revert to a more virulent form in a seronegative vaccinee. Recently developed technology, however, allows the introduction of engineered mutations into the genome of influenza A and B viruses, permitting the rational design of attenuated mutants with the potential for increased genetic stability. To accomplish this goal, we have introduced ts mutations into the PB2 gene of A/Los Angeles/2/87 (H3N2) and rescued the mutated genes into infectious viruses. We have used clustered charged to alanine mutagenesis (substitution of alanine for charged amino acid residues which are present in clusters) of the PB2 gene to generate novel ts mutants. Viruses containing such ts PB2 genes were attenuated in mice and ferrets. This approach has thus yielded several vaccine candidates with ts and attenuated characteristics in animal models. Combination of these mutations with each other or with other ts mutations may lead to a high level of genetic stability.

Alanine↗
PubMed2002

Reverse genetics.

Explore the source record for details and available documents.

Influenza A virus↗
PubMed2003

Development of a vaccine for immunization against classical as well as variant strains of infectious bursal disease virus using reverse genetics.

Available IBDV vaccines based on classical IBDV strains do not induce full protection in the presence of maternally derived antibodies (MDA) against variant strains. The aim of this study was the generation of an IBDV chimera between both classical and variant strains. Characterization of the generated chimeric IBDV (D78-Chim) was performed in both cell culture and susceptible chickens using the classical vaccine strain D78 for comparison. Growth kinetics on cell culture showed that the D78-Chim replicates comparable to the live vaccine strain D78. Administration of the D78-Chim to SPF chickens resulted in equally high virus neutralizing antibodies titres against both, classical and variant IBDV strains. In vivo experiments in commercial chickens revealed that D78-Chim and D78 induced comparable bursal damage in presence of maternally derived antibodies. Both D78-Chim and D78 viruses were able to breakthrough a similar level of MDA.

Animals↗
PubMed2003

Androgen receptor functions from reverse genetic models.

The androgen receptor (AR) is a ligand-dependent transcription factor involved in the regulation of many different physiological processes. AR dysfunction causes a diverse range of clinical conditions, including testicular feminization mutation (Tfm) syndrome, prostate cancer, and motor neuron disease (Kennedy's disease). However, due to lack of genetic models, the molecular basis of the AR in these disorders remains largely unknown. Using a conditional targeting technique based on the Cre-loxP system, we successfully generated null AR mutant (ARKO) mice. ARKO males exhibited normal healthy growth, but showed typical Tfm abnormalities. Hormonal assay of ARKO males revealed that while serum androgen levels were very low, estrogen levels were normal. Another hallmark of ARKO males was late-onset obesity, with marked accumulation of white adipose tissue. To clarify the role of human AR (hAR) mutants with expanded polyQ stretches as observed in neurodegenerative disease, we also established a Drosophila model in which either wild-type or polyQ-expanded hAR were ectopically expressed. Although no overt phenotype was detected in adult fly-eye neurons expressing mutant hAR, the ingestion of androgen caused marked neurodegeneration.

Animals↗
PubMed2001

A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans.

BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.

Actinomycetales↗
PubMed2002

Reverse genetics of essential genes in tissue-culture cells: 'dead cells talking'.

In the 'post-genomic' era, cDNA and genomic sequences are now available that encode huge numbers of proteins. Assigning functions to these proteins is a daunting task. Cell biologists have traditionally approached this problem by disrupting protein function with dominant-negative or structural mutants. Here, we describe several alternative approaches whereby cells or cell lines lacking particular gene products can be generated from genomic sequences for use in functional studies. These include gene targeting in mouse, human and chicken DT40 cells, and recent advances in double-stranded RNA-mediated interference (RNAi).

Animals↗
PubMed2006

[Generation high yield vaccine strain wholly derived from avian influenza viruses by reverse genetics].

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the hemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 was generated by cells transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with hemagglutination unit of 1:2048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce hemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and maximum geometric mean HI-titer were observed 4 approximately 5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1 infected- and H5N3 vaccinated animals.

Animals↗
PubMed2003

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

AGAMOUS Protein, Arabidopsis↗
PubMed2006

Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis.

Genome sequencing, in combination with various computational and empirical approaches to sequence annotation, has made possible the identification of more than 30,000 genes in Arabidopsis thaliana. Increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of these genes gives rise to a complex organism. The combination of classical forward genetics with recently developed genome-wide, gene-indexed mutant collections is beginning to revolutionize the way in which gene functions are studied in plants. High-throughput screens using these mutant populations should provide a means to analyse plant gene functions--the phenome--on a genomic scale.

Arabidopsis↗
PubMed2004

RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age.

Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes. The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy.

Animals↗