1527 results about "Reporter gene" patented technology

The invention relates to a microfabricated device for the rapid detection of DNA, proteins or other molecules associated with a particular disease. The devices and methods of the invention can be used for the simultaneous diagnosis of multiple diseases by detecting molecules (e.g. amounts of molecules), such as polynucleotides (e.g., DNA) or proteins (e.g., antibodies), by measuring the signal of a detectable reporter associated with hybridized polynucleotides or antigen/antibody complex. In the microfabricated device according to the invention, detection of the presence of molecules (i.e., polynucleotides, proteins, or antigen/antibody complexes) are correlated to a hybridization signal from an optically-detectable (e.g. fluorescent) reporter associated with the bound molecules. These hybridization signals can be detected by any suitable means, for example optical, and can be stored for example in a computer as a representation of the presence of a particular gene. Hybridization probes can be immobilized on a substrate that forms part of or is exposed to a channel or channels of the device that form a closed loop, for circulation of sample to actively contact complementary probes. Universal chips according to the invention can be fabricated not only with DNA but also with other molecules such as RNA, proteins, peptide nucleic acid (PNA) and polyamide molecules.

The instant invention provides stable and novel lineage I WNV reverse genetics systems, and methods for making the reverse genetics systems, specifically, a fully-infectious lineage I WNV cDNA or replicon system engineered with one or more nucleotide sequences each encoding a reporter gene to be used in high throughput cell-based screening assays for the identification of novel antiflaviviral chemotherapeutics and/or vaccines effective to treat and/or immunize against infections by WNV and other emerging flaviviruses, such as, for example, JEV, SLEV, AV, KV, JV, CV, YV, TBEV, DENV-1, DENV-2, DENV-3, DENV-4, YFV and MVEV. The present invention further provides methods of high throughput screening of antiflaviviral compounds or improved derivatives thereof using novel lineage I WNV reverse genetics systems and/or cell lines stably containing the reverse genetics systems. Also, the invention provides novel pharmaceutical compositions comprising an attenuated lineage I WNV that is less virulent but similarly immunogenic as the parent WNV and is capable of providing a protective immune response in a host.

The invention is related to a transducing particle that comprises a bacteriophage coat and a DNA core that comprises plasmid DNA comprising: a) a host-specific bacteriophage packaging site wherein the packaging site is substantially in isolation from sequences naturally occurring adjacent thereto in the bacteriophage genome, b) a reporter gene, c) a bacteria-specific promoter operably linked to said reporter gene, d) a bacteria-specific origin of replication, and optionally e) an antibiotic resistance gene. The invention includes phage transducing particles, methods of making transducing particles, and methods of using the transducing particles in bacterial detection.

Compostions, kits and methods are provided for generating highly diverse libraries of proteins such as antibodies via homologous recombination in vivo, and screening these libraries against protein, peptide and nucleic acid targets using a two-hybrid method in yeast. The method for screening a library of tester proteins against a target protein or peptide comprises: expressing a library of tester proteins in yeast cells, each tester protein being a fusion protein comprised of a first polypeptide subunit whose sequence varies within the library, a second polypeptide subunit whose sequence varies within the library independently of the first polypeptide, and a linker peptide which links the first and second polypeptide subunits; expressing one or more target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion protein to the target fusion protein.

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

The present invention provides methods of screening for a molecule that inhibits the expression or activity of a protein encoded by a target gene which affects the fitness of a cell. The methods are based on a co-culture assay, and entail culturing together two cell populations, each of which is a population of identical cells, of the same species that differs substantially only in the expression or activity of the gene to be targeted or its encoded protein and the presence or absence of a reporter gene. The screen can be applied to cultured cells, unicellular and multicellular organisms. Manipulating the expression or activity of the target gene sensitizes the host to a molecule which inhibits the target gene or its encoded protein such that the cell or organism comprising the manipulated target gene grows at a different rate from the cell or organism comprising the unmanipulated gene in response to exposure to the molecule. The methods of the invention can be used for identifying drugs, proteins or any other molecules that inhibit the function of proteins encoded by target genes.

The activity of multiple proteins in a single living cell, portion of a cell or in a group of cells is simultaneously measured by introducing reporter molecules. The reporter(s) is chemically modified by the enzyme of interest. In some cases the enzyme(s) is affected by the addition of a stimulus or a pharmaceutical compound to the cell. The reactions between the enzymes and the reporters are diminished or terminated, and the reporter and modified reporter are removed. The activity of the enzyme(s) is determined by measuring the amount of reporter remaining, the amount of altered reporter produced, or by comparing the amount of reporter to the amount of altered reporter. A database is compiled of the activities of the different proteins. By performing a series of experiments at different time points, conditions, and varieties of cell types, a database is developed for molecular cellular mechanisms in health and disease states. By exposing cells to a variety of compounds data for drug development and screening is provided.

The invention provides an exogenous gene knocking-in and integrating system on the basis of CRISPR/Cas9, a method for establishing the exogenous gene knocking-in and integrating system and application thereof. The exogenous gene knocking-in and integrating system comprises vectors with report/donor functions and Cas9 expression vectors. Each report/donor vector comprises two target gent homologous arms and an exogenous sequence fragment positioned between the two target gene homologous arms; homologous sequences, which are positioned on a target gene, of the two target gene homologous arms of each report/donor vector are respectively positioned on two sides of a target sequence of the target gene and are connected with the target sequence of the target gene; the exogenous sequence fragments comprise promoters, resistant genes, shorn peptide sequences, report genes and polyA tails which are sequentially arrayed, two SSA repair homologous sequences of each resistant gene are inserted into the resistant gene, and the target sequence of each target gene is inserted in a space between the two corresponding SSA repair homologous sequences. The exogenous gene knocking-in and integrating system, the method and the application have the advantages that exogenous genes can be integrated with endogenous gene sequences in an efficient site-directed and accurately targeted manner, and double-chromosome allelic gene double-knocking-in can be efficiently carried out.

This invention relates in general to a method for molecular fingerprinting. The method can be used for forensic identification (e.g. DNA fingerprinting, especially by VNTR), bacterial typing, and human/animal pathogen diagnosis. More particularly, molecules such as polynucleotides (e.g. DNA) can be assessed or sorted by size in a microfabricated device that analyzes the polynucleotides according to restriction fragment length polymorphism. In a microfabricated device according to the invention, DNA fragments or other molecules can be rapidly and accurately typed using relatively small samples, by measuring for example the signal of an optically-detectable (e.g., fluorescent) reporter associated with the polynucleotide fragments.

The present invention relates to methods for identifying compounds that modulate untranslated region-dependent expression of a target gene. The invention particularly relates to using untranslated regions of a target gene or fragments thereof linked to a reporter gene to identify compounds that modulate untranslated region-dependent expression of a target gene. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads.

Methods for agents useful for treating asthma are disclosed. The methods include screening for agents that inhibit the production of a PKC-θ protein, as well as for agents that inhibit the kinase activity of a PKC-θ protein, or a functional fragment thereof, wherein such agents are useful for treating asthma. The methods also include screening for agents that inhibit the production of a reporter gene product encoded by a nucleic acid sequence operably linked to a PKC-θ promoter. Also disclosed are methods of treating asthma that include administering an agent that inhibits the production of a functional PKC-θ protein or the kinase activity of a PKC-θ protein or a functional fragment thereof. An isolated mast cell lacking expression of endogenous PKC-θ is also disclosed.

The invention discloses a CRISPR-Cas9 system used for assembling DNA and a DNA assembly method. The CRISPR-Cas9 system includes the following parts: a plasmid used for expressing Cas9 gene, a first guide RNA, and/or a plasmid used for expressing the first guide RNA, wherein the first guide RNA has a CRISPR site. The CRISPR site is combined with a first reporter gene carried by a semisynthetic chromosome used for assembling according to base complementation pairing rule. The CRISPR-Cas9 system has advantages of high replacement success rate of homologous recombination, less species of guide RNA which needs to design and use, less harmful effect subjected by the genomic sequence, and low off-target rate.

One of the impediments to the treatment of some human brain tumors (e.g. gliomas) has been the degree to which they expand, migrate widely, and infiltrate normal tissue. We demonstrate that a clone of multipotent neural progenitor stem cells, when implanted into an experimental glioma, will migrate along with and distribute themselves throughout the tumor in juxtaposition to widely expanding and aggressively advancing tumor cells, while continuing to express a foreign reporter gene. Furthermore, drawn somewhat by the degenerative environment created just beyond the infiltrating tumor edge, the neural progenitor cells migrate slightly beyond and surround the invading tumor border. When implanted at a distant sight from the tumor bed (e.g., into normal tissue, into the contralateral hemisphere, into the lateral ventricles) the donor neural progenitor/stem cells will migrate through normal tissue and specifically target the tumor cells. These results suggest the adjunctive use of neural progenitor/stem cells as a novel, effective delivery vehicle for helping to target therapeutic genes and vectors to invasive brain tumors that have been refractory to treatment.

Methods and composition of induction of pluripotent stem cells are disclosed. For example, in certain aspects methods for generating induced pluripotent stem cells using reporter genes are described. Furthermore, the invention provides novel reprogramming vectors employing reporter genes.

The present invention is directed to protocells for specific targeting of hepatocellular and other cancer cells which comprise a nanoporous silica core with a supported lipid bilayer; at least one agent which facilitates cancer cell death (such as a traditional small molecule, a macromolecular cargo (e.g. siRNA or a protein toxin such as ricin toxin A-chain or diphtheria toxin A-chain) and/or a histone-packaged plasmid DNA disposed within the nanoporous silica core (preferably supercoiled in order to more efficiently package the DNA into protocells) which is optionally modified with a nuclear localization sequence to assist in localizing protocells within the nucleus of the cancer cell and the ability to express peptides involved in therapy (apoptosis/cell death) of the cancer cell or as a reporter, a targeting peptide which targets cancer cells in tissue to be treated such that binding of the protocell to the targeted cells is specific and enhanced and a fusogenic peptide that promotes endosomal escape of protocells and encapsulated DNA. Protocells according to the present invention may be used to treat cancer, especially including hepatocellular (liver) cancer using novel binding peptides (c-MET peptides) which selectively bind to hepatocellular tissue or to function in diagnosis of cancer, including cancer treatment and drug discovery.

A system and method for developing and utilizing particle-based n-multiplexed assays that include three or more reporters utilizes n particle sets that are associated with particle identification images or labels (IDs) that differ between sets. The encoded particles for a given set are coated with a specific binding member, or in the case of the sandwich assay with coupled capture and detector binding pair members, to form particle types. The sets of particle types are then pooled, and aliquots of the particle types are removed to assay vessels. Next, samples with three or four reporter molecules are supplied to the respective vessels. After one or more incubation periods, the particles are supplied to a reader system, which determines the particle IDs to identify the particle types and also detects the reporter signals. The reader system includes multiple excitation lasers that excite the various reporters in sequence or in parallel, to supply associated signals to one or more detectors. Emission filters and wavelength discriminators are included such that a given detector receives at a given time the signals associated with a single assay binding label. The system further develops greater capacity sandwich assays by assigning subsets of capture and detector antibody pairings to the three or four reporters, respectively. The system performs greater numbers of differential RNA expressions based on the use of the three or more reporters, with one or more reporters assigned to the reference sample and the other reporters assigned to respective test samples. The system and method are also capable of performing greater numbers of SNPs utilizing primer extension reactions, by assigning different color reporters to the respective nucleotides or terminators.

The invention provides assays and methods for determining whether a compound inhibits gamma secretase in a substrate specific manner. The invention provides an isolated cell wherein the cell stably expresses APP and at least one gamma secretase substrate other than APP. The invention provides assays and methods comprising contacting a cell with gamma secretase and detecting production of Abeta, detecting production of intracellular domain (ICD), and detecting a signal from a reporter gene under transcriptional control of the ICD. The invention also provides compounds that inhibit gamma secretase, pharmaceutical compositions comprising such compounds, and methods of treating Alzheimer's disease using such compounds.

The present invention relates to panels of reporter expression cassettes and the generation of transgenic non-human animals, wherein said reporter expression cassettes have selected control elements operable linked to reporter genes. The invention includes methods of use thereof for the identification and characterization of the effects of compounds administered to the live transgenic non-human animals.