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Results for “vector engineering”

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At least 91 records · Page 5Linked to original sources
PubMed1990

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin.

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.

Animals↗
PubMed1991

Reconstituted viral envelopes--'Trojan horses' for drug delivery and gene therapy?

Reconstituted viral envelopes (RVEs) are formed by solubilizing intact virus in detergent and reassembling the envelope on removal of detergent. RVEs can be formed in the presence of agents that become encapsulated and can then be utilized in vitro and in vivo for drug delivery, cell destruction, transfer of membrane components, and as vectors for genetic engineering. The problems with biotechnological applications of RVEs and possible strategies for overcoming them are discussed in this article.

Animals↗
PubMed2026

Development and preclinical evaluation of a decoy DLL4-encoding oncolytic HSV-1 for high-grade glioma.

Preclinical and clinical investigation of oncolytic HSV-1 (oHSV) treatment for cancer has indicated increased Notch signaling in tumors after treatment. Since Notch activation often heralds cancer cell stemness, angiogenesis, and invasion, the induction of this pathway after oHSV virotherapy can support tumor growth and limit response to virotherapy. Here, we evaluated the impact of blocking DLL4, a Notch ligand, on virotherapy. Matched tumor biopsies pre- and post-oHSV (CAN-3110, NCT03152318) treatment revealed an induction of DLL4 post-therapy. We observed that expression of a recombinant soluble decoy DLL4 (sDLL4) could block Notch activation in tumor cells. Thus, we engineered an oHSV vector designed to encode soluble DLL4 (OVsDLL4) to block ligand-mediated Notch signaling. RNA sequencing and gene set enrichment analysis revealed that, relative to control oHSV, OVsDLL4 blocked Notch and sprouting angiogenesis pathways after treatment. Despite slower virus replication in vitro, OVsDLL4 cytotoxicity remained effective against tumor cells. Transcriptome profiling also indicated a significant dysregulation of metabolic pathways related to oxidative phosphorylation and glutathione metabolism, in accordance with increased oxygen consumption observed by Seahorse analysis in cells expressing sDLL4. OVsDLL4-treated cells further showed increased reactive oxygen species relative to control oHSV-treated cells. Co-culture of infected tumor cells with immune cells revealed that OVsDLL4 treatment polarized them toward an inflammatory phenotype. In vivo, the therapeutic efficacy of OVsDLL4 was underscored, as treatment of glioma-bearing mice resulted in reduced tumor burden and prolonged survival.

Journal Article↗
PubMed1980

The interaction of Agrobacterium Ti-plasmid DNA and plant cells.

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted 'opines'). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Ti-plasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.

Amino Acids↗
PubMed1987

Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit.

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.

Animals↗
PubMed1982

T-DNA of the Agrobacterium Ti and Ri plasmids.

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.

Arginine↗
PubMed1990

Large-scale propagation of insect cells.

Cultured insect cells have many uses in agriculture and medicine. They can be used in the diagnosis and isolation of a number of viruses infecting both animals and plants and for the laboratory study of these viruses. Large-volume culture of insect cells has been envisioned as a way of producing viruses for use in controlling insect pests and for the production of viral antigens for vaccine preparations. Recently they have become a potentially valuable way of producing a variety of proteins for human and veterinary medicine using the genetically engineered baculovirus expression vectors. The development of satisfactory cell lines and culture methods has proceeded at a slow, irregular pace, inhibited by the lack of knowledge of the physiology of the insect, its small size, and often by the lack of consistent, adequate support for the necessary developmental research. However, now that the basic culture systems are available, cell lines have been developed or can easily be developed for most needs. Suitable media are available and recent developments in refining existing media formations have resulted in low-cost media containing little protein to interfere with down-stream processing of cellular metabolites. Future developments are likely to further improve the media formulations and lower the cost. Technical problems relating to oxygen demand and cell fragility that inhibited the continued development of large-volume culture systems beyond the laboratory a few years ago now appear to be solved or at least are solvable. The successful culture of the Spodoptera cells in bioreactors of 40-liter capacity indicates that means of producing insect cells or their metabolic products on a commercial scale can be made economically feasible.

Animals↗
PubMed1995

Retrovirus-mediated transduction of an engineered intron-containing purine nucleoside phosphorylase gene.

We constructed and tested several retroviral vectors containing abbreviated purine nucleoside phosphorylase (PNP) genes in the reverse orientation, a strategy compatible with transduction of intron-containing genes. We observed two types of deletions in these vectors after one round of replication: (i) Deletions flanked by direct repeats with one copy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) Deletions due to fortuitous splice sites in the PNP complementary strand. Two splice donor sites and three splice acceptor sites were identified in a 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysis of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The putative PNP polyadenylation signal and a truncated 117-bp 3' flank were recovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank. Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor signals in the complementary strand were inactivated. A retroviral vector LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reverse orientation, was transduced intact in 23% (5/22) of clones after one round of replication and in 87% (20/23) of clones after a second round of replication from two primary virus producer clones. Directed mutagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.

Base Sequence↗
PubMed1991

Expression vectors and gene transfer.

The development of DNA cloning techniques, together with the possibility of reintroducing cloned DNA fragments into the genome of a living organism, has led to an extraordinary growth of our knowledge in molecular biology over the past twenty years. In the present paper a brief overview of the vectors and techniques used in transforming different groups of organisms is given. The importance and applications of genetic engineering for each group (yeasts, plants, Drosophila and mammals) will be discussed.

Animals↗