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At least 271 records · Page 15Linked to original sources
PubMed1987

Potential risks of tumor virus subgenomes in the production of biologicals.

In this report, an attempt was made to describe what is presently known about the biological activities of tumor virus DNAs, their regulatory regions, and some factors which interplay with their oncogenic potential. In light of current knowledge, it is likely that the gene transfer risks of using continuous cell lines expressing known oncogenes, or cells containing oncogenic virus-recombinant DNA vectors, are minimal.

DNA, Recombinant↗
PubMed1995

Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes.

The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune-mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors.

Adenovirus E4 Proteins↗
PubMed1994

Recombinant Salmonella vectors in vaccine development.

A diversity of means are available for the attenuation of Salmonella which can be used to immunize animals and humans orally to elicit mucosal, humoral and cellular immune responses. Avirulent Salmonellae can be genetically engineered to express foreign antigens and the recombinant avirulent Salmonellae are capable of stable, high-level expression of the foreign antigen in the orally immunized animal or human host. The resulting vaccines are safe, efficacious, and are easy and economical to use.

Administration, Oral↗
PubMed1991

Systemic delivery of human growth hormone by injection of genetically engineered myoblasts.

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.

Animals↗
PubMed1989

Conservation of polyhedrin gene promoter function between Autographa californica and Mamestra brassicae nuclear polyhedrosis viruses.

The DNA sequence of the polyhedrin gene of the Mamestra brassicae multiple nucleocapsid nuclear polyhedrosis virus (MbMNPV) was determined and compared with the polyhedrin genes of Autographa californica (Ac) and Panolis flammea (Pf) MNPVs. Using this information, a transfer vector was constructed based on the EcoRI I fragment of AcMNPV in which the polyhedrin promoter was replaced by the homologous region extending 481 nucleotides upstream from the MbMNPV polyhedrin coding sequence. The Escherichia coli lacZ gene was also included downstream from the putative MbMNPV promoter. Cotransfection of this transfer vector with wild-type AcMNPV DNA produced stable recombinant viruses expressing the lacZ gene under the control of the MbMNPV polyhedrin promoter. The levels of beta-galactosidase produced by these recombinants in infected cells were 30% lower than the expression level obtained from viruses with the authentic AcMNPV promoter in front of the lacZ gene. The MbMNPV promoter has thus been shown to function efficiently in the genetic environment of AcMNPV. The implications of this finding for the release of genetically manipulated baculovirus insecticides and for the construction of baculovirus multiple expression vectors are discussed.

Amino Acid Sequence↗
PubMed2000

High efficiency retroviral vectors that contain no viral coding sequences.

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared with the well-known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

3T3 Cells↗
PubMed1998

Nonsegmented negative-strand RNA viruses: genetics and manipulation of viral genomes.

Protocols to recover negative-stand RNA viruses entirely from cDNA have been established in recent years, opening up this virus group to the detailed analysis of molecular genetics and virus biology. The unique gene-expression strategy of nonsegmented negative-strand RNA viruses, which involves replication of ribonucleoprotein complexes and sequential synthesis of free mRNAs, has also allowed the use of these viruses to express heterologous sequences. There are advantages in terms of easy manipulation of constructs, high capacity for foreign sequences, genetically stable expression, and the possibility of adjusting expression levels. Fascinating prospects for biomedical applications and transient gene therapy are offered by chimeric virus vectors carrying novel envelope protein genes and targeted to defined host cells.

Base Sequence↗
PubMed1989

[New cloning vectors constructed in Bacillus].

Two new plasmids pNQ216 (4.1kp) and pNQ402 (2.8 kb) were constructed by combining the replication origin of the plasmid pNK289, a cryptic plasmid resides in B. pumilus 289, and the cat-86 gene from plasmid pPL601. These two plasmids can be maintained steadily in both B. subtilis and B. pumilus. The penetrance of these plasmids on LB medium with Cm (20 micrograms/ml) is 30% higher than that of pPL600. Thus both of these plasmids can be used as new cloning vectors in Bacillus.

Bacillus↗
PubMed1985

[Construction of a gene library using partial filling of DNA sticky ends].

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.

Animals↗