A knockout rat is a
genetically engineered
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
rat
Rats are various medium-sized, long-tailed rodents. Species of rats are found throughout the order Rodentia, but stereotypical rats are found in the genus ''Rattus''. Other rat genera include ''Neotoma'' ( pack rats), ''Bandicota'' (bandicoot ...
with a single
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
turned off through a targeted mutation (
gene trapping Gene trapping is a high-throughput approach that is used to introduce insertional mutations across an organism's genome.
Method
Trapping is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a prom ...
) used for academic and pharmaceutical
research
Research is "creativity, creative and systematic work undertaken to increase the stock of knowledge". It involves the collection, organization and analysis of evidence to increase understanding of a topic, characterized by a particular att ...
. Knockout rats can mimic human diseases and are important tools for studying gene function (
functional genomics) and for
drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered.
Historically, drugs were discovered by identifying the active ingredient from traditional remedies or by ...
and development. The production of knockout rats was not economically or technically feasible until 2008.
Technology developed through funding from the
National Institutes of Health
The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the late ...
(NIH) and work accomplished by the members of the Knock Out Rat Consortium (KORC) led to cost-effective methods to create knockout rats. The importance of developing the rat as a more versatile tool for human health research is evidenced by the $120 million investment made by the NIH via the Rat Genome Sequencing Project Consortium, resulting in the draft sequence of a laboratory strain of the
brown or Norway rat (''Rattus norvegicus'').
[Rat Genome Sequencing Project Consortium, Genome sequence of the brown rat yields insights into mammalian evolution. Nature, 2004. 428(6982): p. 493-521.] Additional developments with
zinc finger nuclease
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc ...
technology in 2009 led to the first knockout rat with targeted, germline-transmitted mutations.
[Guerts, A.M., et. al, Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases. Science. Vol 325: 433 (24 July 2009) ] Knockout rat disease models for
Parkinson's
Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...
,
Alzheimer's
Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As t ...
,
hypertension
Hypertension (HTN or HT), also known as high blood pressure (HBP), is a long-term medical condition in which the blood pressure in the arteries is persistently elevated. High blood pressure usually does not cause symptoms. Long-term high bl ...
, and
diabetes
Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ap ...
using zinc-finger nuclease technology are being commercialized by SAGE Labs.
Research use
Mice, rats, and humans share all but approximately 1% of each other's genes
making rodents good model organisms for studying human gene function. Both mice and rats are relatively small, easily handled, have a short generation time, and are genetically inbred. While mice have proven to be a useful rodent model and techniques have been developed for routine disruption of their genes, in many circumstances rats are considered a superior laboratory animal for studying and modeling human disease.
Rats are physiologically more similar to humans than are mice. For example, rats have a heart rate more similar to that of humans, while mice have a heart rate five to ten times as fast. It is widely believed that the rat is a better model than the mouse for human
cardiovascular disease
Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels. CVD includes coronary artery diseases (CAD) such as angina and myocardial infarction (commonly known as a heart attack). Other CVDs include stroke, h ...
, diabetes,
arthritis
Arthritis is a term often used to mean any disorder that affects joints. Symptoms generally include joint pain and stiffness. Other symptoms may include redness, warmth, swelling, and decreased range of motion of the affected joints. In som ...
, and many
autoimmune,
neurological
Neurology (from el, νεῦρον (neûron), "string, nerve" and the suffix -logia, "study of") is the branch of medicine dealing with the diagnosis and treatment of all categories of conditions and disease involving the brain, the spinal ...
, behavioral, and addiction disorders. In addition, rat models are superior to mouse models for testing the
pharmacodynamics
Pharmacodynamics (PD) is the study of the biochemical and physiologic effects of drugs (especially pharmaceutical drugs). The effects can include those manifested within animals (including humans), microorganisms, or combinations of organisms (fo ...
and toxicity of potential therapeutic compounds, partially because the number and type of many of their detoxifying enzymes are very similar to those in humans. Their larger size makes rats more conducive to study by instrumentation, and also facilitates manipulation such as blood sampling, nerve conduction, and performing surgeries.
Techniques for genetic manipulation are available in the mouse, which is commonly used to model human disease. Although published knockouts exist for approximately 60% of mouse genes, a large majority of common human diseases do not have a
knockout mouse
A knockout mouse, or knock-out mouse, is a genetically modified mouse (''Mus musculus'') in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are importan ...
model. Knockout rat models are an alternative to mice that may enable the creation of new gene disruptions that are unavailable in the mouse. Knockout rat models can also complement existing transgenic mouse models. Comparing mouse and rat mutants can facilitate the distinction between rodent-specific and general
mammalian
Mammals () are a group of vertebrate animals constituting the class (biology), class Mammalia (), characterized by the presence of mammary glands which in Female#Mammalian female, females produce milk for feeding (nursing) their young, a ...
phenotypes
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
.
Production challenges
Rat models have been used to advance many areas of medical research, including cardiovascular disease, psychiatric disorders (studies of behavioral intervention and addiction),
neural regeneration
Neuroregeneration refers to the regrowth or repair of nervous tissues, cell (biology), cells or cell products. Such mechanisms may include generation of new neurons, glia, axons, myelin, or synapses. Neuroregeneration differs between the periphera ...
, diabetes,
transplant
Transplant or Transplantation may refer to:
Sciences
*Transplanting a plant from one location to another
*Organ transplantation, moving an organ from one body to another
*Transplant thought experiment, an experiment similar to Trolley problem
*Tra ...
ation, autoimmune disorders (
rheumatoid arthritis
Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are involv ...
),
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
, and wound & bone healing. While the completion of the rat
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
sequence provides very key information, how these diseases relate to gene function requires an efficient method to create knockout rat models in which specific genomic sequences are manipulated.
Most techniques for genetic manipulation, including random mutagenesis with a
gene trap Gene trapping is a High-throughput screening, high-throughput approach that is used to introduce insertional mutations across an organism's genome.
Method
Trapping is performed with gene trap vectors whose principal element is a gene trapping cas ...
(retroviral-based and non-retroviral-based), gene knock-outs/knock-ins, and conditional mutations, depend upon the culture and manipulation of embryonic stem (ES) cells. Rat ES cells were only recently isolated and no demonstration of gene modification in them has been reported. Consequently, many genetic manipulation techniques widely used in the mouse are not possible in the rat.
Early methods
Until the commercial development of mobile
DNA technology in 2007 and zinc-finger nuclease technology in 2009, there were only two technologies that could be used to produce rat models of human disease:
cloning and chemical mutagenesis using N-ethyl-N-nitrosourea (
ENU
ENU, also known as ''N''-ethyl-''N''-nitrosourea (chemical formula C3H7N3O2), is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. It is also toxic at high doses.
The chemical is an alkylatin ...
). Although cloning by
somatic cell nuclear transfer
In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an enucleated oocyte (egg cell) and implanting a ...
(SCNT) could theoretically be used to create rats with specific mutations by mutating somatic cells, and then using these cells for SCNT, this approach has not been used successfully to create knockout rats. One problem with this strategy is that SCNT is extremely inefficient. The first published attempt had a success rate of less than 1%. Alternatively, ENU mutagenesis is a common random mutagenesis gene knockout strategy in the mouse that can also be used in the rat. ENU mutagenesis involves using a chemical, N-ethyl-N-nitrosourea (ENU), to create single base changes in the genome. ENU transfers its ethyl group to oxygen or nitrogen radicals in DNA, resulting in mis-pairing and base pair substitution. Mutant animals can be produced by injecting a male mouse with ENU, and breeding with a wild type female to produce mutant offspring. ENU mutagenesis creates a high frequency of random mutations, with approximately one base pair change in any given gene in every 200-700
gametes. Despite its high mutagenicity, the physical penetration of ENU is limited and only about 500 genes are mutated for each male and a very small number of the total mutations have an observable phenotype. Thousands of mutations typically need to be created in a single animal in order to generate one novel phenotype.
Despite recent improvements in ENU technology,
[Chen, Y., D. Yee, K. Dains, A. Chatterjee, J. Cavalcoli, E. Schneider, J. Om, R.P. Woychik, and T. Magnuson, Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nature Genetics, 2000. 24(3): p. 314-7.] mapping mutations responsible for a particular phenotype is typically difficult and time-consuming. Neutral mutations must be separated from causative mutations, via extensive breeding. ENU and cloning methods are simply inefficient for creating and mapping gene knockouts in rats for the creation of new models of human disease. Through 2007, the largest rat ENU mutagenesis project to date run by the
Medical College of Wisconsin was able to produce only 9 knockout rat lines in a period of five years at an average cost of $200,000 per knockout line. Although some companies are still pursuing this strategy, the Medical College of Wisconsin has switched to a more efficient and commercially viable method using mobile DNA and CompoZr ZFN technology.
Zinc-finger and TALE nuclease technology
Zinc finger nucleases (ZFNs) and
Transcription Activator-Like Effector Nuclease
Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA ...
s (TALENs) are engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. Double strand breaks are important for site-specific mutagenesis in that they stimulate the cell's natural DNA-repair processes, namely homologous recombination and non-homologous end joining. When the cell uses the non-homologous end joining pathway to repair the double-strand break, the inherent inaccuracy of the repair often generates precisely targeted mutations. This results in embryos with targeted gene knockout.
Standard microinjection techniques allow this technology to make knockout rats in 4–6 months. A major advantage of ZFN- and TALEN-mediated gene knockout relative to the use of mobile DNA is that a particular gene can be uniquely and specifically targeted for knockout. In contrast, knockouts made using mobile DNA technology are random and are therefore unlikely to target the gene of interest.
Mobile DNA technology
Mobile DNA (jumping gene) technology uses
retrotransposons and
transposons
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transpo ...
for the production of knockout rat models. This platform technology meets all of the criteria for a successful gene knockout approach in mammals by permitting random mutagenesis directly in the
germ cells (
sperm
Sperm is the male reproductive cell, or gamete, in anisogamous forms of sexual reproduction (forms in which there is a larger, female reproductive cell and a smaller, male one). Animals produce motile sperm with a tail known as a flagellum, whi ...
and
oocytes) of mammalian model organisms, including rats. Using this technology, genes are disrupted completely and in a stable manner, are knocked out at a high frequency, and are randomly disrupted throughout the entire genome. The genomic location of mutations can be easily mapped, creating a library of knockout rats for later use. Once the random knockout mutations are created, more refined mutations such as conditional mutations can be created by breeding knockout lines with rat lines expressing
CRE recombinase
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site ...
in a tissue specific manner. Knock-ins can be produced by recombination mediated cassette exchange.
''piggyBac'' (PB) DNA transposons
''piggyBac'' (PB) DNA transposons mobilize via a "cut-and-paste" mechanism whereby a transposase enzyme (PB transposase), encoded by the transposon itself, excises and re-integrates the transposon at other sites within the genome. PB transposase specifically recognizes PB inverted terminal repeats (ITRs) that flank the transposon; it binds to these sequences and catalyzes excision of the transposon. PB then integrates at TTAA sites throughout the genome, in a relatively random fashion. For the creation of gene trap mutations (or adapted for generating transgenic animals), the transposase is supplied in trans on one plasmid and is co-transfected with a plasmid containing donor transposon, a recombinant transposon comprising a gene trap flanked by the binding sites for the transposase (ITRs). The transposase will catalyze the excision of the transposon from the plasmid and subsequent integration into the genome. Integration within a coding region will capture the elements necessary for gene trap expression. PB possesses several ideal properties: (1) it preferentially inserts within genes (50 to 67% of insertions hit genes) (2) it exhibits no local hopping (widespread genomic coverage) (3) it is not sensitive to
over-production inhibition in which elevated levels of the transposase cause decreased transposition 4) it excises cleanly from a donor site, leaving no “footprint,” unlike Sleeping Beauty.
Sleeping beauty (SB) transposons
The sleeping beauty (SB) transposon is a derivative of the Tc1/mariner superfamily of DNA transposons prevalent among both vertebrate and invertebrate genomes. However, endogenous DNA transposons from this family are completely inactive in vertebrate genomes. An active Tc1/mariner transposon, synthesized from alignment of inactive transposons from the salmonid subfamily of elements, was “awoken” to form the transposon named Sleeping Beauty. SB, like other DNA transposons, mobilizes itself via a cut-and-paste mechanism whereby a transposase enzyme, encoded by the transposon itself, excises and re-integrates the transposon at other sites within the genome. The 340 amino acid SB protein recognizes inverted terminal repeats (ITRs) that flank the transposon; it binds to these sequences and catalyzes excision of the transposon. SB then integrates into random sites within the genome, although some studies report very slight preferences for transcriptional units. There is also a simple requirement of a TA-dinucleotide at the target site, like all Tc1/mariner transposons.
The SB transposon is a powerful tool for insertional mutagenesis in many vertebrate species. It recently exhibited especial utility for germ line mutagenesis in both mice and rats. There are several advantages that make SB a highly attractive mutagen geared toward gene discovery: 1) it has little bias for inserting within particular genomic regions or within specific recognition sequences, 2) de novo insertions of the transposon provide a “tagged” sequence marker for rapid identification of the specific mutation by simple PCR cloning methods, 3) in vivo SB insertional mutagenesis allows multiple mutations to be quickly and easily generated in a single animal, and in a single tissue, such as an adenomatous polyp.
LINE1 (L1) retrotransposons
Transposons and retrotransposons are valuable tools for unbiased gene discovery as mobile pieces of DNA used for gene disruption. Retrotransposons, such as LINEs (long interspersed nuclear elements), mobilize via a “copy and paste” mechanism and are abundant in many eukaryotic species. Several L1 retrotransposons have remained active in mice and humans. L1s contain a small internal promoter within a 5’ untranslated region to drive expression, two
open reading frames (ORFs), and a 3’ untranslated region containing sequences for polyadenylation. The two ORFs encode proteins necessary for autonomous retrotransposition; ORF1 encodes an
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
-binding protein while ORF2 encodes a protein containing endonuclease (EN) and reverse transcriptase (RT) activity, which nick a site in DNA, then produce a copy via RT. These proteins exhibit an overwhelming specificity for binding to and acting on the transcript that encodes them, enabling near exclusive mobilization of the parental L1 RNA. Using the RT activity of the ORF2 protein, the transcribed L1 RNA is copied into DNA by a process termed target primed reverse transcription (TPRT), and integrated into the genome. Integration occurs with little bias for any particular genomic region, requiring a simple consensus sequence, 5’TTTT’A-3’ (along with minor variations of this sequence). Integrated L1 sequences are often truncated at the 5’ end, with an average total size of 1 Kb, many containing only 3’ terminal sequences.
The nature of retrotransposition endows the L1 with some unique advantages; L1 retrotransposons have an essentially unlimited supply of the insertional mutagen since it is continually transcribed from a promoter, which would be useful for applications where large numbers of mutations are needed in a single cell. L1 elements also demonstrate widespread genomic coverage, with a largely random distribution of insertions.
[Cost, G.J. and J.D. Boeke, Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure. Biochemistry, 1998. 37(51): p. 18081-93.] L1 insertions at genomic sites are also irreversible, and thus any mutagenic event caused by an L1 insertion is “tagged” by L1 sequences.
See also
*
Biobreeding rat
*
Genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
*
Gene knockout
A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene kno ...
*
Lab animals
Animals used by laboratories for testing purposes are largely supplied by dealers who specialize in selling them to universities, medical and veterinary schools, and companies that provide contract animal-testing services. It is comparatively rare ...
*
Transgenic animal
Genetically modified animals are animals that have been genetically modified for a variety of purposes including producing drugs, enhancing yields, increasing resistance to disease, etc. The vast majority of genetically modified animals are at th ...
References
External links
NIH Rat Genome & GeneticsScientists Compare Rat Genome With Human, MouseKnock Out Rat Consortium (KORC) websiteNational Institutes of Health websiteTransposagen: Producers of Knockout Rat ModelsKnockout Rats - Horizon Discovery (formerly SAGE Labs)
{{Genetic engineering, state=collapsed
Genetically modified organisms
Laboratory rat strains
Animal testing on rodents