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Name: Adenosine deaminase Symbol: AdaGenetic Map:Mutation: DNA transposon gene trap insertion within intron 7 Associated Human Diseases: • Severe Combined Immunodeficiency (SCID)• Lung Eosinopilia• Priapism

Phenotype: Homozygous Ada mutant rats die prenatally or perinatally from severe respiratory distress. They show defects in purine metabolism, growth retardation and have liver cell degeneration. Ada mutant rats are T-cell and B-cell deficient.

Applications: • Immunodeficient model• Asthma/pulmonary inflammation model• Humanized liver rat model

The Knockout Rat Consortium (KORC) Aron M. Geurts1, 2, 3, Edwin Cuppen4, 5, and Eric M. Ostertag6, 7, 8

1Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI, USA. 2PhysGen Knockout Team, Human and Molecular Genetics Center, Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA. 3Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA. 4Hubrecht Institute, Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, Netherlands. 5Department of Medical Genetics, University Medical Center Utrecht, Utrecht, Netherlands. 6Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA. 7 Department of Pathology & Laboratory Medicine, University of Kentucky Chandler Hospital, Lexington, KY, USA. 8Transposagen Biopharmaceuticals, Inc., Lexington, KY, USA.

Summary

The Knockout Rat Consortium (KORC) is formed by individuals and institutions interested in the goal of creating knockout rats using multiple technologies including transposon-based and chemical mutagenesis. The KORC website is www.knockoutrat.org and contains a database, news items, a discussion form, and links that are useful to researchers interested in rat models. The KORC database lists the majority of knockout rats that are currently available to the research community and can be searched by gene name, ontology, or disease association. The KORC now has over 300 genetically modified rats in its database. Examples of knockout rats available include a SCID model, a p53 knockout, a model of pain (Trpc4), a model of hydrocephalus (Myo9a), and a new model of obesity (Mc4r). The SCID rat contains an insertional mutation within the adenosine deaminase (Ada) gene, which is an underlying cause of many cases of severe combined immunodeficiency (SCID) in humans. SCID rats share similar or identical defects reported in mice and humans with Ada mutations (e.g., athymia, depressed cell counts in the spleen, and severe liver defects). The SCID rat can be used for the transplantation of stem cell populations to create humanized tissues or organs for in vivo analysis. The world’s first p53 knockout rat is also available (orders fulfilled by Charles River Laboratories) and now has extensive phenotype information, including tumor spectrum data. p53 knockout rats display a wide variety of solid tumors, such as rhabdomyosarcoma, hemangiosarcoma, adrenocortical carcinoma and others. The Trpc4 knockout rat is a novel model for pain research and demonstrates greatly reduced sensitivity to visceral pain. A rat knockout of Myo9a enables researchers to study hydrocephalus in an animal model that allows facile surgical manipulation. Melanocortin 4 receptor (Mc4r) mutations are the single most common form of monogenic obesity in humans. The Mc4r knockout rat is obese and gains white adipose tissue rapidly due to hyperphagia. On behalf of the KORC we invite all interested investigators to participate in our efforts to assemble a repository of knockout rats. This will be a tremendous resource for the rat research community.

Goals

Transposon-Mediated Mutagenesis

PiggyBac (PB) and Sleeping Beauty (SB) are the DNA transposons most often used in mutagenesis of the rat. DNA transposons mobilize via a cut-and-paste mechanism whereby a transposase enzyme excises and re-integrates the transposon at other sites within the genome. Mutagenesis can be performed directly in the germ line of transgenic rats or in rat embryonic stem cells or spermatogonial stem cells. The transposase protein recognizes inverted terminal repeats (ITRs) that flank the transposon; it binds to these sequences and catalyzes excision of the transposon. The transposon then integrates into random sites within the genome. There is also a simple requirement of a TTAA (PB) or TA (SB) at the target site. Advantages of the transposon system include high-efficiency and low cost of mutagenesis, ease of identification of the mutations and genome-wide coverage.

ENU-Mediated Mutagenesis

N-ethyl-N-nitrosourea (ENU) transfers its ethyl group to oxygen or nitrogen radicals present on the DNA. Subsequent replication of damaged DNA can result in mispairing and produce single base substitutions. To generate mutant rats, males are treated with ENU to cause alkylation of DNA followed by point mutations in rapidly dividing spermatogonial stem cells. Every affected cell will contain a random and unique set of induced mutations. Thus, one treated male will have a genetically heterogeneous sperm population contributing to offspring with distinct mutations. Offspring from a treated male and wildtype female will result progeny that are screened for heterozygous mutations identified using high-throughput mutation discovery methods. Mutants of interest can then be bred to homozygosity. Advantages of ENU mutagenesis include a very high frequency of mutation and the ability to create hypomorphic mutations.

Featured NEW Knockout Rat Models

p53

Trpc4

Contact information

• The mission of KORC is to create rats with a single gene disruption causing a knockout mutation by any available technology.

• Our goal is to generate at least one rat with a null mutation for every gene in the rat genome.

• Knockout lines will be stored as cryopreserved sperm or stem cells and made available to the research community upon request.

On behalf of the KORC we invite all interested investigators to participate in our efforts to assemble a repository of knockout rats. This will be a tremendous resource for the rat research community. For more information or to browse our current list of knockout models, please visit the KORC website at www.knockoutrat.org. If you wish to initiate your own project of rat mutagenesis please contact KORC at info@knockoutrat.org.

Name: Tumor proteinSymbol: p53, Tp53Genetic Map:Mutation: ENU-mediated premature stop codon (nonsense mutation) at amino acid position C273 in the middle of the DNA binding domain Associated Human Diseases: • Tumors (Medulloblastoma, Neurofibromatosis)• Cancer (Bladder, Breast, Endometrial, Lung, Mesothelioma, Ovarian,

Rhabdomyosarcoma, Squamous Cell Carcinoma)Phenotype: Mutation affects cell cycle regulation and apoptosis. Homozygous p53 mutant rats show high incidence of early-onset tumors (3-4 months), whereas heterozygous mutants have a delayed onset of tumor development (9 months). Heterozygous mutants can develop loss of heterozygosity tumors.

Applications: • Tumorgenesis model• Carcinogenicity and toxicology studies• Aging, antioxidant, inflammatory, and insulin resistance studies

Mc4r

Myo9a

SCID

Several high-value knockout rat models have recently been added to KORC:

ENU

x

High-throughput Mutation Discovery

Screen

Name: Melanocortin 4 receptorSymbol: Mc4rGenetic Map:Mutation: ENU-mediated premature stop codon (nonsense mutation) at amino acid position K314 producing a truncated protein that is unable to localize to the plasma membrane and results in a complete loss of function phenotype Associated Human Diseases: • Obesity

Phenotype: Homozygous Mc4r mutant rats are severely obese, overeating, hyperinsulinemia and elevated leptin.

Applications: • Obesity model• Diabetes model

Name: Myosin IXaSymbol: Myo9aGenetic Map:Mutation: DNA transposon gene trap insertion within intron 8Associated Human Diseases: • Hydrocephalus

Phenotype: Homozygous mutant Myo9a rats have an abnormal accumulation of cerebrospinal fluid in the brain causing seizures and hydrocephalus

Applications: • Hydrocephalous model

Name: Transient receptor potential cation channel, subfamily C, member 4Symbol: Trpc4Genetic Map:Mutation DNA transposon gene trap insertion within intron 1Associated Human Diseases: • Hypertension• Cardiovascular disease

Phenotype: Homozygous Trpc4 mutant rats show an increase in pain tolerance. Heterozygous mutant rats also show increase pain tolerance at approximately 50% of homozygous mutant rats. Hypertensive or vascular inflammation phenotypes have not yet been fully characterized .

Applications: • Pain and nociception studies• Hypertension/vascular inflammation• Cardiac defects

Abnormal liver histology

Full knockout

Athymic

Macroscopic view of primary tumor and metastasis to lungs Microscopic view lung metastasis H&E stained

In Situ hybridization reveals a full knockout phenotype

KO WT Heterozygous Homozygous KO

Extreme Dilation of the Lateral Ventricle Cerebral Aqueduct Stenosis