=Knockout rats=A knockout rat is a
ratwith a single genedisruption used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function ( functional genomics) and for drug discoveryand development. The production of knockout rats was not economically or technically feasible until 2008. New technology developed through funding from the National Institutes of Health (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 the Brown Norway laboratory rat (Rattus norvegicus)Rat Genome Sequencing Project Consortium, Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature, 2004. 428(6982): p. 493-521. ] .
Mice, rats, and humans share all but approximately 1% of each others’ genesRat Genome Sequencing Project Consortium, Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature, 2004. 428(6982): p. 493-521.] [International Human Genome Sequencing Consortium, Initial sequencing and analysis of the human genome. Nature, 2001. 409(6822): p. 860-921.] [Mouse Genome Sequencing Consortium, Initial sequencing and comparative analysis of the mouse genome. Nature, 2002. 420(6915): p. 520-62.] 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, diabetes, arthritis, and many autoimmune, neurological, behavioral, and addiction disorders. [Abbott, A., Laboratory animals: the Renaissance rat. Nature, 2004. 428(6982): p. 464-6.] In addition, rat models are superior to mouse models for testing the pharmacodynamicsand toxicity of potential therapeutic compounds, partially because the number and type of many of their detoxifying enzymes are very similar to those in humans. [Lindblad-Toh, K., Genome sequencing: three's company. Nature, 2004. 428(6982): p. 475-6.] Their larger size make 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 10% of mouse genes, a large majority of common human diseases do not have a
knockout mousemodel. 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 phenotypes.
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, diabetes, transplantation, autoimmune disorders ( rheumatoid arthritis), cancer, and wound & bone healing. While the completion of the rat genomesequence 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(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. [Cohen-Tannoudji, M. and C. Babinet, Beyond 'knock-out' mice: new perspectives for the programmed modification of the mammalian genome. Molecular Human Reproduction, 1998. 4(10): p. 929-38.] However, for unknown reasons rat ES cells cannot be isolated and used to create a viable organism4. Consequently, many genetic manipulation techniques widely used in the mouse are not possible in the rat.
Until the commercial development of mobile
DNAtechnology in 2007, there were only two technologies that could be used to produce rat models of human disease: cloningand chemical mutagenesis using N-ethyl-N-nitrosourea ( ENU). Although cloning by somatic cell nuclear transfer(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%. [Zhou, Q., J.P. Renard, G. Le Friec, V. Brochard, N. Beaujean, Y. Cherifi, A. Fraichard, and J. Cozzi, Generation of fertile cloned rats by regulating oocyte activation. Science, 2003. 302(5648): p. 1179.] 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. [Hitotsumachi, S., D.A. Carpenter, and W.L. Russell, Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia. Proceedings of the National Academy of Sciences of the United States of America, 1985. 82(19): p. 6619-21.] 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, [Brown, S.D. and R.E. Hardisty, Mutagenesis strategies for identifying novel loci associated with disease phenotypes. Seminars in Cell & Developmental Biology, 2003. 14(1): p. 19-24.] [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.] [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.] [Zan, Y., J.D. Haag, K.S. Chen, L.A. Shepel, D. Wigington, Y.R. Wang, R. Hu, C.C. Lopez-Guajardo, H.L. Brose, K.I. Porter, R.A. Leonard, A.A. Hitt, S.L. Schommer, A.F. Elegbede, and M.N. Gould, Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nature Biotechnology, 2003. 21(6): p. 645-51.] 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 Wisconsinwas 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 technology.
Mobile DNA technology
Mobile DNA (jumping gene) technology uses
retrotransposonsand transposonsfor 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( spermand 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 resulting knockout rats contain disrupted genes that can be efficiently created, the genomic location of mutations can be easily mapped, and the mutagen has minimal toxicity. In addition, this mutagenesis system is adaptable to all mammals and, 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 in a tissue specific manner. Knock-ins can be produced by recombination mediated cassette exchange.
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-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), [Luan, D.D., M.H. Korman, J.L. Jakubczak, and T.H. Eickbush, Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell, 1993. 72(4): p. 595-605.] 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. [Ostertag, E.M., et al., A mouse model of human L1 retrotransposition. Nat Genet, 2002. 32(4): p. 655-60.] [Babushok, D.V., et al., L1 integration in a transgenic mouse model. Genome Res, 2006. 16(2): p. 240-50.] [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.
leeping 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. [Ivics, Z., et al., Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell, 1997. 91(4): p. 501-10.] 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. [Vigdal, T.J., et al., Common physical properties of DNA affecting target site selection of sleeping beauty and other Tc1/mariner transposable elements. J Mol Biol, 2002. 323(3): p. 441-52.] [Yant, S.R., et al., High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol, 2005. 25(6): p. 2085-94.] There is also a simple requirement of a TA-dinucleotide at the target site, like all Tc1/mariner transposons. [Plasterk, R.H., Z. Izsvak, and Z. Ivics, Resident aliens: the Tc1/mariner superfamily of transposable elements. Trends Genet, 1999. 15(8): p. 326-32.]
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. [Geurts, A.M., et al., Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet, 2006. 2(9): p. e156.] [Horie, K., et al., Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol Cell Biol, 2003. 23(24): p. 9189-207.] [Keng, V.W., et al., Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods, 2005. 2(10): p. 763-9.] [Kitada, K., et al., Transposon-tagged mutagenesis in the rat. Nat Methods, 2007. 4(2): p. 131-3.] [Geurts, A.M., et al., Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol, 2006. 6: p. 30.] [Dupuy, A.J., S. Fritz, and D.A. Largaespada, Transposition and gene disruption in the male germline of the mouse. Genesis, 2001. 30(2): p. 82-8.] [Dupuy, A.J., et al., Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci U S A, 2002. 99(7): p. 4495-9.] 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.
* [http://www.nih.gov/science/models/rat/ NIH Rat Genome & Genetics]
* [http://www.genome.gov/11511308 Scientists Compare Rat Genome With Human, Mouse]
* [http://www.knockoutrat.org Knock Out Rat Consortium (KORC) website]
* [http://www.nih.gov/ National Institutes of Health website]
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