This Special Advertising Feature is brought to you by AAAS OPMS

DOI: 10.1126/science.opms.p1200061

LIFE SCIENCE TECHNOLOGIES
A New Era For Clinical Models

For PDF versionNew Products

picture

Not long ago, a clinically relevant and genomics-based model of disease meant an animal, but today's models also include cell lines and even computer-based simulations. Still, bioengineered mice and rats make up many of the models, and these genetically modified organisms provide increasingly accurate representations of how drugs treat human diseases. To get the most from genomics-based models for drug research, scientists can now combine information from bioengineered organisms, genomically modified cell lines, and computational models.

By Mike May



Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

The lab mouse represents an icon of clinical research. A PubMed search of mouse model pulls up more than 150,000 articles, which indicates the pervasive use of this clinical tool in modeling diseases. Moreover, groups of scientists keep developing new ways to use mice in basic and medical research.

For example, two consortia—the International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC)—hope to improve bioengineered mouse models. First, the IKMC, based in laboratories in Europe and North America, is making individual mutant embryonic stem cell lines in which one of the mouse's roughly 20,000 protein-coding genes has been knocked out. So far, the IKMC has created more than three-quarters of these cell lines. As a next step, the IMPC—composed of 10 research institutions around the world—will produce knockout animals for each gene and analyze the phenotypic changes. Although the list of phenotypic tests remains under development, it will likely range from basic blood work to behavioral assessments. The IMPC plans to phenotype all 20,000 knockout mice by 2020.

To breed the cohorts of mice needed for this phenotyping project, the IMPC turned to some powerhouses in breeding, including Charles River Laboratories in Wilmington, Massachusetts. "The repository of knockout-mouse lines and the corresponding phenotypic data will lead to a global availability of novel mouse models across many areas of disease," says Iva Morse, corporate vice president, Charles River Genetically Engineered Models and Services. "Because of the IKMC and IMPC's efforts, researchers will be able to search for specific genes, examine the primary phenotype screens, and then get the animals for further testing."

Once all of the knockout mice are phenotyped, researchers will be able to use a database to review some of the fundamental anatomical, biochemical, and physiological impacts of each gene as well as possible disease-related factors. This phenotypic database should free researchers and resources to pursue more complex questions, such as how genes interact with environmental factors.

MAKING MOUSE MODELS MORE DIVERSE

Rather than only working with existing mouse strains, some researchers are developing new ones since many of the existing strains have little genomic variation. "The standard lab mouse is not exactly what we thought in terms of diversity," says John French, leader of the host susceptibility group at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina. He explains that inbreeding has created some identical regions in the genomes of lab mice. For example, rather than having gene variants at a specific locus, inbred mice often all have the same allele in that spot. Consequently, mice from one line can be very similar to each other but extremely different from other mouse lines. For example, French describes using 18 strains of inbred mice to study the physiological impact of benzene exposure on biochemical processes that modify foreign compounds, such as drugs or poisons. Some characteristics—such as the amount of benzene in the blood after the same level of exposure—varied by as much as 36-fold between the strains. French notes that benzene exposure can lead to leukemia in both mice and humans, and this variance between inbred strains may be useful for risk assessment, because some strains may show higher incidence of disease than others.

Other experts also worry about the lack of diversity in traditional mouse lines. "The way rodent models have been used in drug development and toxicity testing only surveys a limited scope of the genetic diversity that's available," says Gary Churchill, a biostatistician at The Jackson Laboratory in Bar Harbor, Maine. "To assess if a drug will have dangerous side effects in a subset of the human population, we must sample broad genetic diversity in the model system."

To meet that need, Churchill and his colleagues created the Diversity Outbred (DO) Mouse population, which represents 45 million single nucleotide polymorphisms (SNPs) and 4 million copy number variations. As Churchill says, "We've captured mouse genetic diversity." In comparison, the most commonly used strains of mice—roughly 100 of them—only include about 12 million SNPs.

When studying a drug's impact, Churchill says, it will probably take 100-200 DO animals to explore the potential for diverse effects. Using this many animals should cover all of the SNPs and copy number variations, which will indicate how the drug would perform when encountering various genotypes.

During drug development, testing a new compound for toxicity is vital. However, Alison Harrill, a research investigator at The Hamner Institutes for Health Sciences in Research Triangle Park, North Carolina, says, "Drug safety is becoming a key bottleneck." When taking a new compound from the discovery stage through to regulatory approval, toxicity testing can take so much time and effort that it slows down the entire process. Moreover, some toxicity issues only emerge after a drug has gained regulatory approval. For instance, Harrill says, "Hepatotoxicity issues [have been found to] exist in about one-third of drugs pulled from the market."

In hopes of revealing toxicities at the research stage, Harrill works with DO mice because, she says, they provide a "better estimate of human responses and identify genetic variants that might increase the risk of an adverse drug reaction." She also notes that this strategy can be used to rescue drugs that have failed toxicity testing in previous clinical trials and allow researchers to develop screens that predict which patients would respond safely to the drugs and which ones would not.

MODELS OF METABOLISM

Mouse models of diseases can also help researchers find new drug targets. For diabetes, for instance, scientists at Lexicon Pharmaceuticals in The Woodlands, Texas, searched the company's collection of knockout mice for animals that have altered glucose control. According to Brian Zambrowicz, chief scientific officer, they found that knockout mice lacking the gene for either sodium-dependent glucose transporter 1 (SGLT1) or SGLT2 exhibited a "much improved oral glucose tolerance," or the ability to maintain balanced blood levels of glucose after eating. Because type II diabetes causes glucose levels to rise dangerously high in the blood, Zambrowicz and his colleagues hoped that a compound that blocks SGLT1 and 2 might treat the disease. The Lexicon scientists then developed cell-based assays to screen for compounds that inhibit these transport proteins and discovered LX4211, a compound that inhibits both transporters. One way this compound helps to maintain a balanced level of blood glucose is through its effects on SGLT2, which participates in the absorption of glucose by the kidney. Blocking this protein increases the amount of glucose that gets excreted in urine. In addition, LX4211 triggers the release of glucagon-like peptide-1 and peptide YY in the gastrointestinal track, and this mechanism provides further glucose regulation.

Genomic tools can also be used to reveal how a drug is metabolized. For example, Xavier de Mollerat, senior scientist at Life Technologies in Carlsbad, California, says that a scientist who suspects that a drug's metabolism depends on a specific receptor can use small interfering RNA (siRNA) to knock down, or inhibit, the receptor's expression and measure the drug's distribution before and after treatment. If the measurements differ, the receptor is involved. Researchers can also use this technique to make a transient animal model of a disease. If the lack of one or more genes is known to be involved in a disease, a researcher can use siRNA to knock down the gene(s). For example, Invivofectamine 2.0 from Life Technologies can simultaneously silence up to four genes with just one application of siRNA. As explained by de Mollerat, "This technology's lipid delivery system is designed to use siRNA in vivo. You inject the siRNA, and it knocks down its targets for weeks."

This technology also only requires a few steps. A scientist combines the desired siRNA with the Invivofectamine 2.0 reagent, performs a couple of simple processes, such as an incubation step, and then injects the mixture into the animal's blood stream or to a more specific location if desired. According to de Mollerat, the siRNA will knock down the gene's protein expression by 80-90 percent. "If you know the sequence, you can target it," de Mollerat says.

De Mollerat says that similar technology might work with messenger RNA (mRNA). Then, a researcher could compare knocking down a gene with siRNA to overexpressing it with mRNA. That would provide further information about a gene's function in general or how it participates in a disease.

RESEARCHING NEW DISEASES WITH RATS

Although mice have been the main bioengineered animal used for disease modeling in the past and will continue to be abundant in the future, genomically modified rats will also become an increasingly common tool. The numerous and extensive history of existing mouse lines provide good reasons to stick with this rodent, but the rat model offers upsides of its own. Just the larger size of rats can enhance certain aspects of drug research. For example, when scientists need a large amount of blood, such as for testing a compound's absorption, distribution, metabolism, and excretion (ADME), it is easier to obtain the necessary quantity from bigger animals. In addition, the physiology of liver metabolism in rats resembles that of humans more closely than mice do.

Consequently, bioengineered rat models will likely become more widely available in the next few years. For example, Charles River Laboratories arranged to distribute rat models of cancer related to the genes p53 and BCRP from Transposagen Biopharmaceuticals in Lexington, Kentucky. As Morse explains: "New methods for manipulating the rat genome further advance functional genomics and allow us to make novel models of human disease." Even Pfizer, headquartered in New York, New York, is providing a genetically engineered rat model of diabetes to Charles River for distribution.

Scientists are continually expanding the catalog of diseases modeled in rats. One of the most exciting new advances arose from a collaboration between scientists at Sigma Life Science in St. Louis, Missouri, and Autism Speaks, an advocacy group. As Edward Weinstein, director of Sigma Advanced Genetic Engineering Labs, explains, "We thought that we could use rats to make some impact on autism." So he and his colleagues worked with Autism Speaks to help them "connect to the community and understand what the best models to make would be," Weinstein says.

Through this collaboration, Sigma scientists learned that a range of genes—including MECP2, FMR1, NLGN3, and others—contribute to autism. Consequently, Weinstein and his colleagues developed knockout rats for many of these genes.

"We hope that using a higher level organism—a rat instead of a mouse—will help unravel some of [the genomic complexity of autism]," explains Weinstein.

BEYOND THE ANIMAL MODEL

Creating models for clinical research does not solely depend on animal use these days. When conducting drug research, scientists often turn to cells for modeling as soon as possible, and Sigma Life Science offers a range of disease-model cell lines.

In particular, Sigma makes many cell lines for cancer research. Brad Keller, product manager at Sigma Life Science, says, "Genetically engineered cell lines for breast cancer are our biggest offering." He adds that they also have cell lines for colorectal and lung cancer. "Each cell line is specific," says Keller. For example, a cancer cell line will include a mutated gene discovered in patients with the disease. Moreover, these cell lines are created from human cells, which makes any experimental results more relevant to human disease.

Beyond cell lines, some projects involve computer simulations. For instance, the Broad Institute in Cambridge, Massachusetts, developed The Connectivity Map (cmap), which uses computer algorithms and genome-wide expression data to explore the connection between diseases, genes, and drugs. To expand cmap, researchers developed the L1000 assay, which runs on the FLEXMAP 3-D system from Luminex in Austin, Texas. This assay can screen about 1,000 genes per sample. "The assay makes genome-wide transcriptional profiling of compound treatments possible at library scale for the first time," says Matt Grow, Luminex's manager of strategic development.

In addition, Colin Hill, chief executive officer at GNS Healthcare in Cambridge, Massachusetts, points out that computer models "are being seen more and more as an important alternative to animal models." As an example, the National Cancer Institute of the U.S. National Institutes of Health came to Hill's company for the analysis of data from genetically modified mouse models of nonsmall cell lung cancer. In short, Hill's machine learning technology is searching the data—from imaging, genes, pathology, and more—for connections that could reveal mechanisms behind this cancer.

By combining animal models, cell lines, and simulations, researchers are now able to develop a wide ranging toolkit for modeling diseases. This breadth of models then allows for more in-depth testing of new drugs in a wider range of genomic variations.


Featured Participants



Autism Speaks
www.autismspeaks.org

Broad Institute
www.broadinstitute.org


www.criver.com

GNS Healthcare
www.gnshealthcare.com

Lexicon Pharmaceuticals
www.lexgen.com

Life Technologies
www.lifetechnologies.com

Luminex
www.luminexcorp.com





National Cancer Institute
www.cancer.gov

National Institute of Environmental Health Sciences
www.niehs.nih.gov

Pfizer
www.pfizer.com

Sigma Life Sciences
www.sigmaaldrich.com/life-science.html

The Hamner Institute for Health
www.thehamner.org

The Jackson Laboratory
www.jax.org

Transposagen Biopharmaceuticals
www.transposagenbio.com




Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Mike May is a freelance writer and editor for science and technology.

DOI: 10.1126/science.opms.p1200061



This article was published as a special advertising feature in the 24 February 2012 issue of Science magazine.


New Products: Genomics

For PDF version


PERSONAL UV-VIS SPECTROPHOTOMETER

picture

The NanoDrop Lite is a compact ultraviolet-visible microvolume spectrophotometer. The new instrument is small enough to fit in a drawer, but powerful enough to help accelerate life science workflows related to sequencing, polymerase chain reaction (PCR)/real-time PCR, protein isolation, antibody production, HLA typing, and other applications. While NanoDrop Lite is designed with fewer features than the 2000 or 8000 series, it delivers where it counts: rapid, accurate, and reproducible microvolume measurements without the need for dilutions. It uses the same sample retention technology that has become a hallmark of NanoDrop instruments and surfaces can simply be wiped clean between samples. Features include local control and an optional docking printer that prints freezer-compatible, adhesive labels, offering even more convenience in the lab. The NanoDrop Lite can measure nucleic acid and protein concentration in sample sizes between 1.0 and 2.0 μL and can measure 260/280 ratios for nucleic acids.

Thermo Fisher Scientific
For info: 877-724-7690
www.thermoscientific.com/nanodrop


NEXT GENERATION SEQUENCING SYSTEM

The HiSeq 2500 is a next generation sequencing system that enables researchers and clinicians to sequence an entire genome in approximately 24 hours. The HiSeq 2500 offers: Unprecedented speed and flexibility with two modes allowing researchers to generate 120 gigabases (Gb) of data in 27 hours, or 600 Gb in a standard HiSeq run; high-quality data with a system that uses proven SBS chemistry that has made both the HiSeq 2000 and the MiSeq systems the most accurate next generation sequencers; expanded applications enabling researchers to sequence a human genome or 20 exomes in a day, or 30 RNA sequencing samples in as little as five hours; industry-leading simplicity and ease-of-use with an integrated cluster generation process that enables a simplified workflow; and a simple, field-based upgrade for the HiSeq 2000.

Illumina
For info: 800-809-4566
www.illumina.com


NGS LIBRARY PREP MODULES

The NEXTflex DNA, ChIP-Seq, and PCR-Free Modules offer increased flexibility to next generation sequencing (NGS) library preparation. Modules are available for each step in the library preparation protocol including end repair, adenylation, ligation, and polymerase chain reaction (PCR). The modules are suitable for the library preparation from genomic or ChIP DNA for sequencing using Illumina's GAIIx, HiSeq, and MiSeq instruments. They provide substantial cost savings for scientists who will be preparing 100 or more samples for sequencing. The master mix modules streamline the workflow and in combination with up to 96 NEXTflex Barcodes, these modules are ideally suited for high throughput library preparation. The ligation modules feature the proprietary "Enhanced Adapter Ligation Technology" which results in library preps with a larger number of unique sequencing reads. Every NEXTflex Module passes rigorous enzymatic quality control and is functionally validated by sequencing on an Illumina platform.

Bioo Scientific
For info: 888-208-2246
www.biooscientific.com


GENE FRAGMENTS

gBlocks Gene Fragments are double-stranded, sequence-verified genomic blocks up to 500 base pairs. Their high sequence fidelity and rapid delivery time makes gBlocks Gene Fragments ideal for a range of biology applications, including easy assembly of multiple gene fragments to reliably generate larger gene constructs. gBlocks Gene Fragments significantly reduce the cost for synthetic gene synthesis to less than US$0.20 per base pair. gBlocks Gene Fragments are provided as linear double-stranded DNA rather than already cloned into a vector, meaning that they can be easily and quickly utilized for a wide range of applications including custom protein synthesis, microRNA analysis, and in vitro transcription. For this reason, they are available with or without 5' phosphate modification depending on the required application. Each order is supplied as 200 ng of dried DNA, ensuring maximal stability prior to use, with most orders delivered within 3-4 business days.

Integrated DNA Technologies
For info: 800-328-2661
www.idtdna.com


1-STEP RT-PCR KIT

The 1-Step RT-PCR Kit is designed for optimal convenience in carrying out highly sensitive and specific reverse transcription polymerase chain reactions (RT-PCR) in a single tube. 1-Step RT-PCR is a variation of the standard two-step RT-PCR, in which all components of the RT and PCR are mixed in one tube prior to starting the reactions so that RT and PCR can be carried out sequentially in one tube. The one-step method offers tremendous convenience when applied to analysis of single targets from multiple RNA samples and minimizes the possibility for introduction of contaminants into reactions between the RT and PCR steps. 1-Step RT-PCR Kit includes: AMV Reverse Transcriptase (from avian myeloblastosis virus), an optimized enzyme Taq DNA Polymerase (from Thermus aquaticus), a unique 10x concentrated RT-PCR buffer, our dNTP mixture, ribonuclease inhibitor, and DEPC-treated water.

G-Biosciences
For info: 800-628-7730
www.gbiosciences.com


Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.xhtml for more information.


Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are featured in this space. Emphasis is given to purpose, chief characteristics, and availabilty of products and materials. Endorsement by Science or AAAS of any products or materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.


Look for these Upcoming Articles

Toxicology: Animal-free Techniques — March 2
Polymer Science: Creating Synthetic Materials — March 16
Innovation in Japan — April 13