Knockout rat
Encyclopedia
A knockout rat is a genetically engineered
rat
with a single gene
turned off through a targeted mutation (gene trapping
) 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 discovery
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
(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). Additional developments with zinc finger nuclease
technology in 2009 led to the first knockout rat with targeted, germline-transmitted mutations. Knockout rat disease models for Parkinson's, Alzheimer's, hypertension
, and diabetes using zinc-finger nuclease technology are being commercialized by SAGE Labs.
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. In addition, rat models are superior to mouse models for testing the pharmacodynamics
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 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 60% of mouse genes, a large majority of common human diseases do not have a knockout mouse
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 phenotypes.
ation, autoimmune disorders (rheumatoid arthritis
), cancer
, and wound & bone healing. While the completion of the rat genome
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 (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.
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
). 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%. 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, 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.
s (ZFNs) and Transcription Activator-Like Effector Nuclease
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.
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
in a tissue specific manner. Knock-ins can be produced by recombination mediated cassette exchange.
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.
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.
-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. L1 insertions at genomic sites are also irreversible, and thus any mutagenic event caused by an L1 insertion is “tagged” by L1 sequences.
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
rat
Rat
Rats are various medium-sized, long-tailed rodents of the superfamily Muroidea. "True rats" are members of the genus Rattus, the most important of which to humans are the black rat, Rattus rattus, and the brown rat, Rattus norvegicus...
with a single gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
turned off through a targeted mutation (gene trapping
Gene trapping
Gene trapping is a high-throughput approach that is used to introduce insertional mutations across the mammalian genome. It is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a promoterless reporter gene and/or selectable genetic marker flanked by...
) used for academic and pharmaceutical research
Animal testing
Animal testing, also known as animal experimentation, animal research, and in vivo testing, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million...
. Knockout rats can mimic human diseases and are important tools for studying gene function (functional genomics
Functional genomics
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
) and for drug discovery
Drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
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
National Institutes of Health
The National Institutes of Health are an agency of the United States Department of Health and Human Services and are the primary agency of the United States government responsible for biomedical and health-related research. Its science and engineering counterpart is the National Science Foundation...
(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
Brown Rat
The brown rat, common rat, sewer rat, Hanover rat, Norway rat, Brown Norway rat, Norwegian rat, or wharf rat is one of the best known and most common rats....
(Rattus norvegicus). Additional developments with zinc finger nuclease
Zinc finger nuclease
Zinc-finger nucleases 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 desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...
technology in 2009 led to the first knockout rat with targeted, germline-transmitted mutations. Knockout rat disease models for Parkinson's, Alzheimer's, hypertension
Hypertension
Hypertension or high blood pressure is a cardiac chronic medical condition in which the systemic arterial blood pressure is elevated. What that means is that the heart is having to work harder than it should to pump the blood around the body. Blood pressure involves two measurements, systolic and...
, and diabetes using zinc-finger nuclease technology are being commercialized by SAGE Labs.
Research use
Mice, rats, and humans share all but approximately 1% of each others’ 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
Heart disease or cardiovascular disease are the class of diseases that involve the heart or blood vessels . While the term technically refers to any disease that affects the cardiovascular system , it is usually used to refer to those related to atherosclerosis...
, diabetes, arthritis
Arthritis
Arthritis is a form of joint disorder that involves inflammation of one or more joints....
, and many autoimmune, neurological, behavioral, and addiction disorders. In addition, rat models are superior to mouse models for testing the pharmacodynamics
Pharmacodynamics
Pharmacodynamics is the study of the biochemical and physiological effects of drugs on the body or on microorganisms or parasites within or on the body and the mechanisms of drug action and the relationship between drug concentration and effect...
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 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 60% of mouse genes, a large majority of common human diseases do not have a knockout mouse
Knockout mouse
A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA...
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 phenotypes.
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, diabetes, transplantOrgan transplant
Organ transplantation is the moving of an organ from one body to another or from a donor site on the patient's own body, for the purpose of replacing the recipient's damaged or absent organ. The emerging field of regenerative medicine is allowing scientists and engineers to create organs to be...
ation, autoimmune disorders (rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...
), cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, and wound & bone healing. While the completion of the rat genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
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 (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 DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
and chemical mutagenesis using N-ethyl-N-nitrosourea (ENU
ENU
ENU, also known as N-ethyl-N-nitrosourea , 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....
). Although cloning by somatic cell nuclear transfer
Somatic cell nuclear transfer
In genetics and developmental biology, somatic-cell nuclear transfer is a laboratory technique for creating a clonal embryo, using an ovum with a donor nucleus . It can be used in embryonic stem cell research, or, potentially, in regenerative medicine where it is sometimes referred to as...
(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.
Gamete
A gamete is a cell that fuses with another cell during fertilization in organisms that reproduce sexually...
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, 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
Medical College of Wisconsin
Medical College of Wisconsin is a private, freestanding medical school and graduate school located in Milwaukee, Wisconsin, United States. It was formerly affiliated with Marquette University....
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 nucleaseZinc finger nuclease
Zinc-finger nucleases 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 desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...
s (ZFNs) and Transcription Activator-Like Effector Nuclease
Transcription Activator-Like Effector Nuclease
Transcription Activator-Like Effector Nucleases are artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain.-Use:...
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 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 (spermSperm
The term sperm is derived from the Greek word sperma and refers to the male reproductive cells. In the types of sexual reproduction known as anisogamy and oogamy, there is a marked difference in the size of the gametes with the smaller one being termed the "male" or sperm cell...
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
Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...
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 an 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 toover-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 RNARNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
-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. 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 ratBiobreeding ratBiobreeding rat also known as the BB or BBDP rat is an inbred laboratory rat strain that spontaneously develops autoimmune Type 1 Diabetes. Like the NOD mice, BB rats are used as an animal model for Type 1 diabetes...
- GeneticsGeneticsGenetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
- Gene knockoutGene knockoutA gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
- Lab animals
- Transgenic animal