Research Tools

Most Recent Inventions

Efficient In Vitro Assay for Antigen-Specific Tolerance

Building on their work, UW–Madison researchers have now developed a T cell-bound cytokine (T-CBC) assay for detecting and quantifying regulatory T cells specific to self-antigens or donor alloantigens. The new method comprises (a) culturing the subject’s T cells for 24 hours in the presence of one or more target antigens and (b) analyzing the cultured T cells for expression of a marker (EBi3; TGFβ/LAP) indicative of antigen-specific immune suppression.

Monomeric Fluorescent Protein-Ligand Complexes with Strong Fluorescence in the Far-Red Region

Research from the University of Wisconsin-Washington County in collaboration with the Institute for Stem Cell Biology and Regenerative Medicine in India, has resulted in the development of monomeric variants of the naturally occurring Sandercyanin Fluorescent Protein (SFP) using site-directed mutagenesis. This work has stemmed from earlier research focused on development of the tetrameric form of SFP, a biliverdin-binding lipocalin protein originally isolated from the mucus of the blue walleye fish, Sander vitreus. Monomeric variants of SFP (mSFPs) have been found to possess the same non-covalent, bili-binding characteristics of the tetramer but are one-quarter the size (~18.6kDa) and do not oligomerize. They are therefore anticipated to be more useful in a host of biotechnology applications. Like the tetrameric form, the mSFPs have a large stokes shift (375nm/675nm) and fluoresce in the far-red or near infrared region, which is advantageous for a wide range of applications including investigation of protein-protein interactions, spatial and temporal gene expression, assessing cell biology distribution and mobility, studying protein activity and protein interactions in vivo, as well as cancer research, immunology, and stem cell research and sub-cellular localization. In addition, the newly developed mSFP’s far-red fluorescence is particularly advantageous for in vivo, deep-tissue imaging.

Enhanced Endotoxin Detection: New Advantages in Liquid Crystal Assays for Gram-Negative Pathogens

UW–Madison researchers have now demonstrated enhanced endotoxin detection in the presence of masking agents in their previous liquid crystal system.

Unlike the LAL assay, the LC-based method does not suffer from LER or any loss of sensitivity due to the presence of cations (e.g., Ca2+ or Mg2+), buffers (e.g., citrate), surfactants (e.g., SDS), chelating agents (e.g., EDTA), proteins or nucleic acids (e.g., DNA or RNA). Thus, the LC-based method provides faster and cheaper detection of endotoxin when compared to existing methods, such as the LAL assay.

Adapted Rhinovirus C for Maximum Virus Yield

Building on their work, the researchers have now developed a mutated RV-C strain that induces strong cytopathic effect and replicates vigorously in the HeLa-E8 cells, yielding more than a log higher level of infectious rhinovirus particles compared to the parental clinical isolate.

DNA “Millichip” Enables Low-Cost, High Throughput Gene Expression Analysis

UW-Madison researchers have developed a DNA “millichip” designed for low-cost, high throughput gene expression analysis in whole genomes.  The millichips consist of 1,000 to 100,000 different oligonucleotides probes immobilized on small solid support arrays with relatively high density.  The probes, which range from 30 to 100 nucleotides long, occupy separate, known sites in the arrays. 

For example, a maskless array synthesizer (MAS) can be used to synthesize about 800,000 70-mer oligonucleotides on a glass microscope slide.  Then the slide is divided into 96 pieces, each containing about 30,000 of the 70-mer DNA sequences.  These small pieces can be used in any experiment that uses standard DNA chips.

Because the millichips are small, less than 10 cubic centimeters, small volumes of solutions can be used for analysis.  In addition, the small substrate size allows the arrays to be visualized using instrumentation readily available in research laboratories.

Most Recent Patents

New Protein Production Strategy for Plants

UW–Madison researchers have identified a new plant viral IRES that can facilitate the efficient expression of multiple proteins from a single mRNA. The researchers discovered the new IRES in the Triticum mosaic virus (TriMV), a wheat virus that expresses 10 proteins from a single mRNA strand.

High-Throughput Genome Editing and Engineering of Industrial Yeast, Other Fungi

UW–Madison researchers have developed expression cassettes that facilitate genome editing and sequence replacement in fungi at an extraordinarily high rate. Their HERP (Haploid Engineering Replacement Protocol) cassettes combine thymidine kinase (TK) enzyme with meganucleases, and permit hundreds of thousands of independent transformations to be obtained in a single experiment.

TK (from human Herpes Simplex Virus) serves as both a selectable and counter-selectable marker. Since the common ancestor of all fungi lacked the gene, the marker is likely of nearly pan-fungal utility. Relevant species should include Saccharomyces cerevisiae, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces uvarum and Neurospora crassa.

Modified E. coli for Enhanced Production of Pyruvate, Ethanol

UW–Madison researchers have developed a variety of new E. coli strains capable of producing pyruvate up to 95 percent of the maximum theoretical yield from renewable sources under aerobic conditions. This exceeds the highest previously reported yields of 78 percent.

The researchers used a genome-scale metabolic model of E. coli to identify multiple gene deletion targets that couple growth rate with pyruvate production. Further engineering of these new strains enabled them to produce ethanol at near maximum theoretical yields.