Drug Discovery : Preclinical testing

Drug Discovery Portfolios


Dramatically More Sensitive Ion Channel Antenna

UW–Madison researchers have developed a new antenna for analyzing ion channel activity. The antenna and circuitry can be used to amplify a signal produced from a capacitance change at a single nanopore.

Specifically, the antenna provides radio frequency measurements of electrical changes (impedance) in cell walls. It has two lobes spaced apart, and is shaped like a variant of the ‘bow-tie’ design to support high gain and broad bandwidth. It is placed around a nanopore made of glass, quartz or other material. A radio frequency signal applied across the antenna lobes determines changes of electrical flow across the cellular membrane when positioned appropriately.

Patch Clamp Providing On-Chip Thermal Control

A UW–Madison researcher has developed a patch clamp chip providing precise, localized temperature control of cell membranes.

The chip’s temperature system uses a Peltier device, which is capable of heating or cooling depending on the direction of current flow. It is a semiconducting membrane and can be etched on a silicon wafer, separated and then bonded to the substrate of the patch clamp. Using techniques previously developed by the researcher, the sandwiched structure is exposed to laser radiation to drill a nanoscale pore.

Laser Drilling Quartz to Make Patch Clamp Plate Arrays

UW–Madison researchers have developed a technique to form pores as small as 200 nanometers in single crystal quartz. The process can employ UV-emitting excimer lasers or other types of lasers.

In the new technique, the quartz substrate is backed by an energy absorbing material like acetone and/or fluorescent immersion oil, which absorbs UV radiation. When the laser is directed into the quartz, it passes through and strikes this absorbing material. The material increases in temperature, melting a small pore or crater in the overlying quartz.

High-Speed On-Chip Patch-Clamping Technique for High Throughput Drug Screening Applications

UW-Madison researchers have developed a high frequency patch-clamp system that quickly and accurately measures changes in ion channels.  The system uses a “tank circuit” to analyze the impedance of the membrane at high frequency.  This circuit design is reliable and is commonly used in high impedance devices.  It improves sensitivity by rapidly detecting small current changes in the ion channels. 

The tank circuit is fabricated on the top of a glass/quartz chip with an array of apertures. A whole cell, bi-lipid membrane or single ion channel within a membrane is positioned over an aperture, and opposing electrodes are placed on opposite sides of the cell membrane.  Then a high frequency electrical signal is applied to the electrodes to provide a connection between the electrodes and the cell membrane. The effects of chemical signals on ion channels may be observed in real-time via the impedance changes across the cell membrane.  These impedance changes can be measured easily by monitoring the resonance of the tank circuit.

Improved Nanoscale Aperture Fabrication Technique for Mass Production of Patch Clamp Plate Arrays

UW-Madison researchers have developed an improved laser ablation technique to manufacture glass plates with an array of nanoscale apertures having smooth contact surfaces.  The method involves backing the plate substrate with a thermally expanding material that may either be a solid or a thin layer of liquid.  A first ablation period is used to burn a crater in the plate substrate that does not extend all the way through the material.  During the final stage of ablation the laser is applied in such a fashion that heat generates a shock wave in the thermally expanding substance, creating a smaller counter-facing concave crater in the plate substrate and completing the aperture.  Specific laser parameters also are controlled during the final stage of ablation such that etching via laser produces a smooth finish around the pore.

The improved method can be used to produce smooth pores less than 500 nanometers in diameter, making it ideal for production of patch clamp plate arrays.  The use of transparent substrates, such as glass, permits the use of both optical and electrical measurements of cell membranes.  With respect to the thin liquid layer method, the volatilizable substance provides a pressure wave to improve aperture formation and adjustment of the liquid layer thickness also permits precise control of the shock wave formation parameters.

In Vitro Differentiation of Neural Stem Cells and Neurons from Human Embryonic Stem Cells

UW-Madison researchers have developed a simple and efficient method of differentiating human embryonic stem cells into neural stem cells and neurons for pharmaceutical screening and potential transplant therapy. The system is easily standardized and completely chemically defined. First, hES cells are aggregated and treated with fibroblast growth factors to induce the cells’ development into early neural stem cells. Different combinations of growth factors are then used to direct these naïve neural stem cells to become progenitors of various types of neurons. The neural progenitors organize into neural tube-like rosettes that can be readily enriched and further differentiated into functional spinal motor neurons, midbrain dopaminergic neurons or forebrain dopaminergic neurons.

Differentiation and Purification of Neural Precursors from Human Embryonic Stem Cells

UW-Madison researchers have developed a simple and efficient method of differentiating human embryonic stem cells into neural precursor cells for pharmaceutical screening and potential transplant therapy. This system is easily standardized. First, hES cells are aggregated into embryoid bodies. Next, the embryoid bodies are treated with fibroblast growth factors to induce the cells’ development into a relatively pure culture of neural precursor cells. The resulting neural precursor cells have been shown to differentiate into neurons and glia, both in vitro and after transplantation into neonatal mouse brains.

Transgenic Mouse Expressing Green Fluorescent Protein in Glial Cells

UW-Madison researchers have developed a transgenic mouse containing a fluorescent reporter gene linked to the glial fibrillary acidic protein promoter. The mouse was engineered by inserting a genetic construct containing a GFAP promoter, a humanized, mutant green fluorescent reporter (GFP) gene and a polyadenylation signal sequence, into the pronucleus of a zygote.

The mouse can be used in a non-invasive assay of neurotoxicity. In the assay, a mouse is exposed to a suspected neurotoxic agent. The presence of green fluorescence signal in glial cells, such as astrocytes, Mueller cells or Schwann cells, indicates the GFAP promoter has been up-regulated in response to chemical or physical injury to cells, confirming the neurotoxicity of the substance.

Transgenic Mouse for Testing Chemotherapeutic Agents

UW-Madison researchers have developed transgenic mice that are conditional knockouts for mdm2. In these mice, exons 7-9 of the mdm2 gene are flanked with a particular sequence. When exposed to a specific recombinase, these exons should be deleted, rendering the gene non-functional.

Mouse Expressed Sequence Tags Related to Liver, Skin, Kidney, Thymus, Lung and Palate Toxicology

UW-Madison researchers have developed tools for monitoring changes in gene expression associated with exposure to various tissue-specific toxicants in the mouse model. Their approach involves determining the frequency of expressed sequence tags (ESTs), which are 200 to 500 base pair sequences of genomic DNA generated from cDNA.

The researchers first constructed cDNA libraries from mRNA extracted from various tissues of control mice and the livers of mice exposed to certain toxicants; sequenced the ends of the cDNA clones to create ESTs; and then organized the clones and counted them. Comparing EST libraries of control and toxin-exposed livers allows researchers to determine how toxicant exposure affects gene expression.

The EST frequency approach also generates a reagent for use in cDNA microarrays, allowing more rapid comparison of expression patterns between samples. Thus, this technology includes three reagents: EST clone sets from various mouse tissues, an organized version of this information and arrays of cDNAs in either liquid culture or as spots in microarrays.

Targeted Disruption of the Murine NADPH Cytochrome P-450 Oxidoreductase Gene

The UW-Madison researchers have created a mouse line in which one or both alleles of the CYPOR gene have been disrupted. The knockout of both alleles is lethal, while heterozygotes show only 60 to 75 percent of the enzyme activity of controls and therefore display increased sensitivity to factors affecting cytochrome P450- and other CYPOR-dependent metabolic pathways. These animal models can be used to directly assess the potential risks of new, xenobiotic compounds. 

Cell Line Stably Expressing KvLQT1 and minK

UW-Madison researchers have developed an HEK 293 cell line that stably expresses KvLQT1 and minK. Since unintended block of potassium channel activity by drugs can cause an acquired form of long QT syndrome, which leads to potentially fatal arrhythmias, this system provides an important screening tool for drugs in development.

Cell Line Stably Expressing the Human Heart Sodium Channel Beta1 Subunit

UW-Madison researchers have developed a stably transfected cell line expressing the beta1 subunit of the human heart sodium channel. The beta1 subunit was first cloned in the early 1990s. The researchers have now re-cloned this subunit and created a cell line that permanently expresses it.

Human Heart Sodium Channel Beta1 Subunit (SCNB1)

The beta1 subunit of the human heart sodium channel is now available as a biomaterial through WARF. SCNB1 was first cloned in the early 1990s. UW-Madison researchers have now re-cloned this subunit and created a cell line that stably expresses it.

HERG-1 Transfected HEK 293 Cell Line B

UW-Madison researchers have cloned the full-length cDNA of the HERG-1 cardiac potassium channel gene into human embryonic kidney (HEK 293) cells, allowing expression of these channels in an experimental system. Since unintended block of HERG channel activity by drugs can lead to potentially fatal arrhythmias, this system provides an important screening tool for drugs in development.

Controllable Murine Models of First Pass Metabolism

UW-Madison researchers have developed mutant mouse lines that display an open (patent) ductus venosus. They also developed a way to control the closure of the ductus venosus so that some mice have first pass clearance while others do not. 

The researchers used embryonic stem cell targeting to create alleles of the transcriptional regulators AHR and ARNT with reduced gene expression. Because AHR and ARNT play a role in vascular development in the fetus, mutations affecting these genes alter blood vessel formation. The particular mutations developed by the researchers result in an open ductus venosus.

HERG CDNA with Native PolyA Tail

UW-Madison researchers have developed the full-length cDNA of HERG1a. This cDNA has been cloned and expressed in Xenopus oocytes, human embryonic kidney cells (HEK293) and Chinese hamster ovary cells. It is similar to a previously cloned cDNA, except that a polyadenylation signal has been removed from its vector sequence. The removal of this sequence does not affect HERG expression levels in heterologous systems.

Cell Line Stably Expressing Human Cardiac Ion Channel Common Polymorphism, K897T

UW-Madison researchers have developed a new human embryonic kidney (HEK 293) cell line that stably expresses the K897T polymorphism of the HERG potassium channel. The K897T polymorphism is the most common HERG polymorphism and is present in 25 to 30 percent of humans. Experiments suggest that carriers of this polymorphism may have a subtle but discernable phenotype that is distinct from the wild-type phenotype.

Method to Screen for Novel Antibiotics

UW-Madison researchers have developed an indicator strain of Bacillus subtilis, which can detect potential antibiotic compounds that inhibit cell wall biosynthesis. This reporter strain includes the vancomycin-inducible VanA operon, which is responsible for a major form of inducible resistance to glycopeptide antibiotics, operably linked to a reporter gene.

To screen compounds for efficacy as antibiotics, a test compound is exposed to the strain. The effects of the compound on the indicator strain’s growth, as well as the presence or absence of the reporter gene product are observed. If the reporter gene product is present and growth of the indicator bacterial strain is inhibited or reduced, then the compound likely inhibits bacterial wall synthesis and is a potential, new antibiotic compound.