Antibodies

High-level over-expression of commercially important proteins in B. subtilis has been difficult to achieve.  While there are several different types of B. subtilis plasmids that have been used such as pUB110, pE194, pMTLBS72, or pSM.beta.1, these plasmids are unstable and don’t segregate well during cell growth, making them relatively difficult to use for gene expression.  During large scale fermentation without antibiotic selection, a significant number of cells (50-99.9%) lose their plasmids.  Even under antibiotic selection, the bacteria may lose their plasmids unless they have this stable segregation system.

UCSD investigators have now discovered a sequence that allows a Bacillus subtilis plasmid vector to be stably maintained.  It can be used to stably express heterologous proteins in B. subtilis, which was not achievable through plasmid expression before.  This new vector contains a plasmid stability determinant functional in B. subtilis that makes it significantly more stable than any other vector available.  The plasmid contains a novel type of DNA segregation system that segregates newly replicated plasmids prior to cell division, even without antibiotic selection.  The plasmid vector can replicate in both E. coli and B. subtilis.

COMMERCIAL POTENTIAL:  The worldwide market for enzymes produced by strains of Bacillus is estimated to be greater than $1 billion, especially for high-level production of cellulases used to produce biofuels or for other industrial processes.

This technology is available for licensing.

Patent pending; worldwide rights available

SD2008-278

UCSD researchers have developed a novel zebrafish model useful for studying mechanisms of accumulation of lipid in blood vessel walls and associated vascular inflammation. This model uses a special diet enriched with cholesterol and/or knockdown of zebrafish ApoE (homologous to human and mouse ApoE) to trigger lipid accumulation in the blood vessels and atherosclerosis The vessels and lipids are easily visualized in the transparent zebrafish due to fluorescent (green) vasculature and fluorescently (red) labeled cholesterol. The model has been tested with a cholesterol lowering drug and validated for reducing cholesterol accumulation and vascular damage when the drug is present.

ADVANTAGES

• Optical transparency for 30 days allowing real time imaging of lipid accumulation and inflammatory processes in the live animal
• New drug candidates are easily administered through water or diet
• Increased throughput and more economical compared to mice or rabbits

for anti-giantin: Lesa GM, Seemann J, Shorter J, Vandekerckhove J, Warren G. The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115.
J Biol Chem. 2000 Jan 28;275(4):2831-6.

for anti-Golgi integral membrane protein: Stanley H, Botas J, Malhotra V. The mechanism of Golgi segregation during mitosis is cell type-specific.
Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14467-70.

Reference: Tyler JK, Collins KA, Prasad-Sinha J, Amiott E, Bulger M, Harte PJ, Kobayashi R, Kadonaga JT. Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors.
Mol Cell Biol. 2001 Oct;21(19):6574-84.

SD2001-224

and

SD2001-225

Both monoclonal and polyclonal antibodies have been prepared which bind to the unconventional myosin, myosin VIIa, an actin-bindin protein, which has been shown to be involved in auditory signal transmission and may be useful for the biochemical and immunohistochemical research on deafness, blindness, and phagocytic events.

SD2001-224 (Monoclonal) and SD2001-225 (Polyclonal)

Navarro, L. and David, M. Journal of Biological Chemistry Vol. 274, No. 50 (1999), 35535-38

UCSD investigators have improved a version of the reverse two hybrid screen designed for the purpose of screening large chemical libraries of inhibitors of a given protein-protein interaction. They have created a small molecule permeable reporter yeast strain, a positive control activator and a panel of selective inhibitors which serve as positive controls. These improvements may significantly increase the probability of identifying a small molecule inhibitor of any protein-protein interaction that can be detected by two-hybrid analysis.

ref: Viriyakosol, S., Tobias, P.S., Kitchens, R.L. and Kirkland., T.N. (2001) J.Biol.Chem. 276: 38044-38051

       - / - (PS1 Deficient)
      + / - (PS1)
      + / - (PS2)
      + / - (APP)
                                                                 (reference)

Invention: This invention is  a transgenic mouse which carries the entire full-length human CYP1A1 gene plus the regulatory or flanking DNA. When the animals are exposed to certain environmental toxicants, the gene is induced through activation of the Aryl hydrocarbon (Ah) or dioxin receptor. Detection of gene expression can then be demonstrated by usual laboratory techniques such as enzymatic expression or Western blot. These mice are unique in that an entire human CYP1A1 gene is in a mouse.


Commercial Applications: 

1. These mice can be used to identify agents that induce the gene or activate the transcriptional regulator of the gene, i.e. toxicants.
2. To identify drugs that are potential targets for the Ah receptor.
3. An excellent model system to identify environmental toxicants.
4. Can be used to "humanize" mice for the CYP1A1 gene.
5. Can be used to explore the developmental impact of agents that might induce the CYP1A1 gene during development.
6. Can be used to understand signal transduction and events controlling the Ah receptor.

Also see SD2004-144, for transgenic mice containing  the CYP1A1 gene linked to a luciferase reporter gene.

UCSD researchers have developed a transgenic mouse that carries a reporter gene, CYP1A1,  constructed from the human gene P450 which is linked through its promoter and regulatory regions to a luciferase reporter gene. Previously, only mouse or rat CYP1A1 genes were used, this is the first example of a human CYP1A1 gene integrated into the mouse genome. Activation of the gene is conducted through the dioxin or Ah receptor. Environmental toxicants, such as polycyclic aromatic hydrocarbons or heterocyclic aromatic hydrocarbons will induce the gene; this is then followed by the increase of luciferase activity in the mice.The luciferase activity can be measured in the usual way by either extracting liver tissue or using a CDD camera.

The commercial applications of this invention are numerous:
1. Can be used to identify agents that induce CYP1A1 or activate the controlling transcriptional regulator of this gene, the dioxin or Ah receptor.
2. To identify drugs that are potential targets for the Ah receptor.
3. Can be used to explore the developmental impact of agents that might induce the CYP1A1 gene during development.
4. The animals can be cross-bred with other mice that might be deficient in specific pathways that directly impact Ah receptor control.
5. May be an excellent model system for the identification of environmental toxicants.

Transgenic mice expressing hBACEI have been generated and characterized behaviorally.

This transgenic mouse model exists in several lines, each demonstrating an extensive neurological phenotype at early stages of development. Reversal of the phenotype with a known inhibitor has already been shown thus confirming the utility of this model for drug screening and validation.

Reference: SD2003-069-JBC

Collagen-induced arthritis has been used for many years as a model to evaluate potential therapeutic agents in arthritis. We have developed a strain of mice with more severe arthritis than standard collagen-induced arthritis. This strain has defective apoptosis in the joints and increased production of proteases and cytokines.

POTENTIAL COMMERCIAL APPLICATIONS: This mouse model will be useful for identifying and developing therapeutic agents that induce apoptosis in arthritis, thereby overcoming the defect. In addition, it provides a more stringent test of anti-inflammatory agents and protease inhibitors because the disease is more severe and destructive than in presently available strains.

UCSD researchers have constructed transgenic mice with a germline deletion of the mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectiin (mMGL).  This lectin is normally expressed in monocytes, macrophages, and dendritic cells.  The deletion is maintained in C57BL/6 mice.  These transgenic animals are useful for research on tumoricidal activity and innate immunity to certain pathogens.   (reference)

Commercial Potential:

Agriculture companies may be interested in obtaining the plasmid for genetic manipulation of their plants to enhance leaf greening and growth, and regulation of stomatal opening and closure. In addition, basic researchers might want antibodies made against the specific plant nitric oxide synthase.

COMMERCIAL POTENTIAL: As a research tool in organisms from yeast to mice, or in cell lines, this method may have a modulating effect upon gene expression rather than a complete “on or off” switch. The present approach allows multiple discrete allele strengths to be obtained from a single transgene conditional upon a second unlinked transgene, a chromosomal locus or an expressed protein.

Because the subcloning step is eliminated, the generation of specific dsRNA can be accomplished within a single tube, and can be fully automated, thereby generating dsRNA from any gene or subsets of genes within a very short time.

COMMERCIAL APPLICATIONS: RNAi transfected into cells results in the down-regulation of its corresponding gene, thus enabling one to assay the gene's function. When fully automated, as in a robotic system, this research tool can be used as a high-throughput screen to identify gene targets for drug development. This rapid analysis would have been impossible previously if one had to rely on the subcloning of specific DNA sequences into a specialized vector.

These transgenes can be used in the generation of transgenic mice to direct macrophage specific expression of foreign genes. (reference)

*Use of this plasmid to generate transgenic mice requires the recipient to have a license with DuPont.

*Use of this plasmid to generate transgenic mice requires the recipient to have a license with DuPont.

NOVEL RECOMBINANT ADENOVIRUS VECTORS FOR TISSUE SPECIFIC GENE EXPRESSION IN HEART

Selected Territories Available

*Use of this plasmid to generate transgenic mice requires the recipient to have a license with DuPont.

Technology: By studying pathways that play a crucial role in cell survival, plasticity and regeneration of neurons, UCSD researchers have identified a new use for a composition of matter that can promote nerve regeneration and protect neurons from apoptosis. The effectiveness of this compound is much better than neurotrophic factors, including BDNF or bFGF. In addition, the inventors have developed a novel delivery method to control the concentration of compound to achieve the maximal effect. The proof of concept has been demonstrated in animal model studies. This technology could be equally useful for the treatment of CNS or peripheral nerve injuries.

Description: UCSD investigators have designed in vitro system which measures changing fluorescence intensity during the translocation step of protein biosynthesis with exquisite sensitivity. The system is an excellent research tool for the study of microbial translocation and translation mechanisms. The sensitivity, adaptability and ease of the assay is superior to other standard assays for monitoring translocation of tRNA-mRNA on ribosomes, such as puromycin or toeprinting assays.

DESCRIPTION: A glucuronic C5 epimerase suitable for use in production chemistry of complex polysaccharides has been sequenced and made available through its recombinant form. Cells transformed with the cDNA for this C5 epimerase are able to convert D-glucuronic acid to L-iduronic acid. Heparin and similar molecules in the cell are modified to iduronioc acid analogs, yielding modified glycans with differing properties.

ADVANTAGES: The C-5 transformation of heparin and related molecules has not previously been practical to conduct in practical scale.

STAGE OF DEVELOPMENT: Preclinical.

PATENT STATUS:Pending

REFERENCES: On request

ONE SELECTION -STEP, ZERO BACKGROUND SITE-DIRECTED MUTAGENESIS BY TOXIC PROTEIN

• Time Saving: Requires only 14 hours (including incubation) and employs only one round of transformation.
• High Efficiency: 100% efficiency demonstrated with low to zero background.
• Cost-Effective: Eliminates the need for complicated series of enzymatic steps used in other methods to select against parental strand.
• Simple: Only requires T4 DNA polymerase and ligase. No need for PCR or other enzymes.
• Versatile: Suitable for long target fragments. Only one subcloning of target and one selection primer needed to generate different point mutations.

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