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Next-Generation CRISPR: tunable and reversible, one-step gene expression control

Accurate control of gene expression has applications in biotechnology, bench science, and clinical medicine. The accuracy and utility of CRISPR-(d)Cas9 has caused significant interest in many fields utilizing advanced biotechnology techniques. However, the system suffers from some lengthy preparatory steps, and concerns regarding toxicity potentially caused by constitutive expression of Cas9. Current methods may therefore be inadequate for the broad range of desired uses.

Antibodies targeting mammalian Sterol Regulatory Element Binding Proteins (SREBP) 1 and 2

Sterol Regulatory Element Binding Proteins (SREBP) are important factors that control lipid homeostasis in mammals. Researchers at UCI have prepared antibodies that have good affinity and specificity for human SREBP1/2 for use as research tools. These antibodies have application in genetic and immunotherapeutic research areas.

Enhanced Cell/Bead Encapsulation Via Acoustic Focusing

The invention consists of a multi-channel, droplet-generating microfluidic device with a strategically placed feature. The feature vibrates in order to counteract particle-trapping micro-vortices formed in the device. Counteracting these vortices allows for single particle encapsulation in the droplets formed by the device and makes this technology a good candidate for use in single cell diagnostics and drug delivery systems.

Massively-Parallel Genetic Screening Tool

Most high throughput genetic screening technology relies on phenotypes that can be coupled to an easily-detectable phenotype, such as fluorescent cell sorting, cell imaging, or cell death. However, many genetic variants may result in a phenotype that is much more subtle and cannot be easily detected by existing screening technologies. It is currently difficult for researchers to examine a variety of different outcomes in one setting, with many of those parameters not being possible to quantify. Being able to assess the functional outcomes on a larger number of individual cells may substantially improve the efficiency of screening.

New label-free method for direct RNase activity detection in biological samples

Researchers at the University of California, Davis have developed a new and simple, label-free method to detect milligram levels of RNase activity in undiluted biological samples that is selective, accurate and scalable

Continuous, enhanced detection of droplet contents in electrical impedance spectroscopy

The inventors at UCI have developed a method and system to make enhanced electrical impedance spectroscopy measurements in a continuously flowing train of microfluidic droplets. The technique increases the sensitivity of the electrical impedance spectroscopy measurements, lowering detection limits and increasing the frequency of continuous measurements.

Controllable Emulsification And Point-Of-Care Assays Driven By Magnetic Induced Movement Of The Fluid

UCLA researchers in the department of Bioengineering have developed a novel microfluidic droplet generation technique, where instead of pumps, only magnetic force is used for controllable emulsification of ferrofluid containing solutions. 

Directed Evolution Of AAV Vectors That Undergo Retrograde Axonal Transport

Brain functions such as perception, cognition, and the control of movement depend on the coordinated action of large-scale neuronal networks, which are composed of local circuit modules that are linked together by long-range connections.  Such long­ range connections are formed by specialized projection neurons that often comprise several intermingled classes, each projecting to a different downstream target within the network. Projection neurons have also been implicated in the spread of several neurodegenerative diseases. Selective targeting of projection neurons for transgene delivery is important both for gaining insights into brain function and for therapeutic intervention in neurodegenerative diseases.   Viral vectors constitute an important class of tools for introducing transgenes into specific neuronal populations, but their potential for biological investigation and gene therapy is hampered by excessive virulence.  Other viruses can infect neurons when administered directly to the nervous system, with "pseudorabies", adenoviruses and lentiviruses used most commonly in animal research. However, these viruses mediate only modest levels of transgene expression, have potential for toxicity, and are currently not easily scalable for clinical or large animal studies.  Recombinant adeno-associated viruses (rAAVs) are an effective platform for in vivo gene therapy, as they mediate high-level transgene expression, are non-toxic, and evoke minimal immune responses.  rAAVs have allowed retrograde access to projection neurons, but their natural propensity for retrograde transport is low, hampering efforts to address the role of projection neurons in circuit computations or disease progression.    UCB and HHMI researchers have produced a new rAAV variant (rAAV2-retro) that permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde labeling reagents.  The rAAV2-retro gene delivery system can be used on its own or in conjunction with Cre recombinase driver lines to achieve long-term, high-level transgene expression that is sufficient for effective functional interrogation of neural circuit function, as well as for CRISPR/Cas9-mediated and other genome editing in targeted neuronal populations.  As such, this designer variant of adeno-associated virus allows for efficient mapping, monitoring, and manipulation of projection neurons.

Sensitive, Specific Ratiometric Fluorescence-based DNA Detection

Fluorescent silver nanoclusters for nucleic acid detection. 

C2c2 - A Dual Function Programmable RNA Endoribonuclease

Bacterial adaptive immune systems employ CRISPRs and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although generally targeted to DNA substrates, the Type VI CRISPR system directs interference complexes against single-stranded RNA substrates and in Type VI CRISPR systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease.   UC Berkeley researchers have discovered that the CRISPR-C2c2 has two distinct RNase activities that enable both single stranded target RNA detection and multiplexed guide-RNA processing.  These dual RNase functions were found to be chemically and mechanistically different from each other and from the CRISPR RNA processing behavior of the evolutionarily unrelated CRISPR enzyme Cpf1.  Methods for detecting the single stranded target RNA were also discovered using a C2c2 guide RNA and a C2c2 protein in a sample have a plurality of RNAs as well as methods of cleaving a precursor C2c2 guide RNA into two or more C2c2 guide RNAs.  

Methods For Obtaining A Synthetic Long Sequencing Read Using Short Read Sequencing

UCLA researchers in the Department of Chemistry and Biochemistry have developed a method to increase the functional read length of existing short read next-generation sequencing (NGS) technologies through a novel library preparation that maintains contiguous coverage of long sequences. 

Versatile Cas9-Mediated Integration Technology

Many advancements to the Cas9 system (both the Cas9 nuclease and the sgRNA sequence) have been made to increase and optimize its efficiency and specificity.  Since many diseases and traits in humans have a complex genetic basis, multiple genomic targets must be simultaneously edited in order for diseases to be cured or for traits to be impacted.  Thus in order for CRISPR/Cas9 to be an effective gene therapeutic technology, huge swathes of the genome must be edited simultaneously, efficiently, and accurately. To address many of these issues, UC Berkeley researchers have developed a system method to rapidly manipulate multiple loci. This system allows for either sequential (maintaining inducible Cas9 present in the genome) or simultaneous (scarless excision) manipulation of Cas9 itself and can be applied to any organism currently utilizing the CRISPR technology.  The system can also be applied conveniently to create genomic libraries, artificial genome sequences, and highly programmable strains or cell lines that can be rapidly (and repeatedly) manipulated at multiple loci with extremely high efficiency.  

Genotyping Bacteria to Predict Social Contact and Structure

Social connectivity has been increasingly recognized as an essential determinant of disease. How individuals form groups and interact is directly linked to the distribution of diseases in the population, a fact highlighted by the recent Ebola epidemic and reemergence of vaccine preventable diseases in the United States. Notably, this applies not only to infectious diseases, where the spread of diseases is sensitive to the structure of the contact network, but also to chronic diseases, where individuals share common exposures.  Unfortunately, the importance of knowing the structure of social networks is matched by the difficulty in accurately measuring them.    UC Berkeley researchers have developed a method to determine the contact network structure of a population by using the microevolution and transmission of commensal bacteria.  As a result, investigators will be able to track chains of transmission more accurately and quickly during epidemic. 

CRISPR genome editing of Zygotes (CRISPR-EZ)

0 0 1 214 1224 UC Berkeley 10 2 1436 14.0 Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} Easily accessible and efficient methodologies to edit the genomes of organisms are an immense resource to the biological and biomedical research community. Traditionally, engineering of the mammalian genome is achieved by homologous recombination (HR)-mediated sequence substitution in embryonic stem cells (ESCs), a time consuming process that occurs at low frequency. Taking genetically engineering in mice for example, after extensive screening for ESC colonies with the desired genetic modifications, ESCs are microinjected into mouse blastocysts to generate chimeras capable of germline transmission. Such chimera mice are then crossed to wild-type mice to generate heterozygous offspring (F1), which are then intercrossed to yield homozygous mutant mice (F2) that can be subjected to phenotypic analyses. Despite the wide use of this technology to generate transgenic mice, the low efficiency of HR in ESCs, the laborious process of screening, the technical difficulty of microinjection, and the nature of the mouse life cycle make this approach a lengthy and costly process.   UC Berkeley researchers developed methods for modifying the genome of a mammalian zygote by introducing a ribonucleoprotein complex (RNP) to the zygote via electroporation.  Suitable genome editing nucleases were found to be CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a type II, type V, or type VI CRISPR/Cas endonucleases.  

Salmonella-Based Gene Delivery Vectors and their Preparation

Nucleic acid-based gene interference technologies, including ribozymes and small interfering RNAs (siRNAs), represent promising gene-targeting strategies for specific inhibition of mRNA sequences of choice. A fundamental challenge to use nucleic acid-based gene interfering approaches for gene therapy is to deliver the gene interfering agents to appropriate cells in a way that is tissue/cell specific, efficient and safe. Many of the currently used vectors are based on attenuated or modified viruses, or synthetic vectors in which complexes of DNA, proteins, and/or lipids are formed in particles, and tissue-specific vectors have been only partially obtained by using carriers that specifically target certain cell types. As such, efficient and targeted delivery of M1GS sequences to specific cell types and tissues in vivo is central to developing this technology for gene targeting applications. Invasive bacteria, such as Salmonella, possess the ability to enter and transfer genetic material to human cells, leading to the efficient expression of transferred genes. Attenuated Salmonella strains have earlier been shown to function as a carrier system for delivery of nucleic acid-based vaccines and anti-tumor transgenes. Salmonella-based vectors are low cost and easy to prepare. Furthermore, they can be administrated orally in vivo, a non-invasive delivery route with significant advantage. Thus, Salmonella may represent a promising gene delivery agent for gene therapy. Scientists at UC Berkeley have developed a novel attenuated strain of Salmonella, SL101, which exhibited high gene transfer activity and low cytotoxicity/pathogenicity while efficiently delivering ribozymes, for expression in animals. Using MCMV infection of mice as the model, they demonstrated that oral inoculation of SL101 in animals efficiently delivered RNase P-based ribozyme sequence into specific organs, leading to substantial expression of ribozyme and effective inhibition of viral infection and pathogenesis. This strategy could easily be adopted deliver other gene targeting technologies.

Diagnostic and Screening Methods for Atopic Dermatitis

Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine Thymic Stromal Lymphopoietin (TSLP) is thought to drive AD and the atopic march. TSLP is up regulated in atopic dermatitis patients and is thought to act on immune cells to trigger atopic dermatitis. Scientists at UC Berkeley discovered that TSLP also activates a subset of sensory neurons to signal itch by acting on TSLPR, which signals to TRPA1. They demonstrated that sensory neurons that transmit itch signals in AD are the only instance of signaling between TSLPR and TRPA1 in the same cell type. Therefore, blocking the signaling between TSLPR and TRPA1 is a novel and specific target for therapeutics for itch in atopic dermatitis. They also discovered that the Orai I/Stim I pathway triggers expression and secretion of TSLP. This pathway has never been directly demonstrated in human primary keratinocytes and has never before been linked to TSLP. Decreasing expression of Orai I or stim I using siRNA, or the downstream transcription factor, NFATc I, significantly attenuates TSLP secretion, as proven in mice studies. Thus inhibition of Orai I/Stim I/NFATc I signaling pathway is a novel target for therapeutics for itch in atopic dermatitis.

Fluorescent Biosensor for Cyclic GMP-AMP (cGAMP)

The cGAS-cGAMP-STING pathway is an important immune surveillance pathway which gets activated in presence of cytoplasmic DNA either due to a microbial infection or a patho-physiological condition, including cancer and autoimmune disorders. Sensing 2’3’ cGAMP level is important in diagnostics perspective as well as in basic understanding of their regulation.  Small molecule activators of this pathway have also been shown to activate an anti-cancer immune response and thus an important use for pharmaceutical applications. However, a high throughput method to screen for such potential drugs is still not available. UC Berkeley researchers have designed a RNA-based fluorescent biosensor for directly detecting 2’3’ cGAMP. The biosensor was able to detect 2’3’ cGAMP and assay cGAS activity in vitro and thus would be useful for high throughput screening of small molecule modulators of cGAS activity.  The biosensor was sensitive enough to quantify 2’3’ cGAMP in dsDNA- stimulated mammalian cell extracts. 

Monoclonal Antibody Against Cer164 (Clone 11)

Mouse monoclonal antibody against the human centrosomal protein 164kDa (Cep164). This antibody binds to the phosphorylation site of Cep164 and has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibody Against ATR-IP (Clone 5)

Mouse monoclonal antibody against the human ATR-interacting protein (ATR-IP). This antibody has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibody Against Cer164 (Clone 26)

Mouse monoclonal antibody against the human centrosomal protein 164kDa (Cep164). This antibody binds to the phosphorylation site of Cep164 and has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibody Against PNPase (Clone 4C11)

Mouse monoclonal antibody against the human mitochondrial polyribonucleotide nucleotidyltransferase 1 (PNPase). This antibody has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibody Against Pnpase (Clone 2A2)

Mouse monoclonal antibody against the human mitochondrial polyribonucleotide nucleotidyltransferase 1 (PNPase). This antibody has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibodies Against Spc24/25 (Clone 2A10)

Mouse hybridoma cell line secret antibody against the human Kinetochore protein Spc24 (SPC24) and Kinetochore protein Spc25 (SPC25). This antibody has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

Monoclonal Antibodies Against Spc24/25 (Clone 2C8)

Mouse hybridoma cell line secret antibody against the human Kinetochore protein Spc24 (SPC24) and Kinetochore protein Spc25 (SPC25). This antibody has been tested for use in immunocytochemistry/immunofluorescence, immunoprecipitation, and western blot.

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