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Premium member Presentation Transcript KNOCKOUT ANIMAL Presented by- Moriyom Akhter Department of pharmacy World University of Bangladesh firstname.lastname@example.org, : KNOCKOUT ANIMAL Presented by- Moriyom Akhter Department of pharmacy World University of Bangladesh email@example.com , What is knockout animal?: What is knockout animal? A 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. The term also refers to the process of creating such an organism, as in "knocking out" a gene. Knockout is often abbreviated as KO. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO). History: History In 1974 Rudolf Jaenisch created the first genetically modified animal by inserting a DNA virus into an early-stage mouse embryo and showing that the inserted genes were present in every cell. Causes of producing knockout animal To study the gene identity and gene function relationship. Manipulation of gene expression by genetic manipulation creates animal models. Loss of Function-model: Gene deletion by replacement knockout animal. In learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Gene knockout : Gene knockout Knockout mouse Knockout rat knockout moss A knockout mouse is a laboratory mouse in which researchers have inactivat - ed , or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. Causes changes in a mouse's phenotype which includes appearance, behavior and other observable physical and biochemical characteristics. 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. A knockout moss is a moss plant in which one or more specific genes are deleted or inactivated by gene targeting. After deletion of a gene, the knockout moss has lost the trait encoded by this gene. Knockout-mosses are relevant for basic research in biology as well as in biotechnology. Knockout mouse & process of producing knockout mice: Knockout mouse & process of producing knockout mice Knockout mice: Knockout mice Knockout mice are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behavior or physiology, researchers can infer its probable function. Examples of research in which knockout mice have been useful include Strains: There are several thousand different strains of knockout mice. Many mouse models are named after the gene that has been inactivated. For example, the p53 knockout mouse is named after the p53 gene which codes for a protein that normally suppresses the growth of tumors by arresting cell division Process : Process There are several variations to the procedure of producing knockout mice; the following is a typical example. The gene to be knocked out is isolated from a mouse gene library . ↓ Then a new DNA sequence is engineered which is very similar to the original gene and its immediate neighbor sequence ↓ Usually, the new sequence is also given a marker gene , a gene that normal mice don't have and that confers resistance to a certain toxic agent or that produces an observable change (e.g. color or fluorescence). ↓ Process : Process Stem cells are isolated from a mouse blastocyst (a very young embryo ) and grown in vitro . For this example, we will take stem cells from a white mouse. ↓ Then the new sequence is introduced into the stem cells from step 2 by electroporation . ↓ The electroporated stem cells will incorporate the new sequence with the knocked-out gene into their chromosomes in place of the original gene and this process is called homologous recombination ↓ Altered cells will have the new sequence in only one of the two relevant chromosomes - they are said to be heterozygous . Process : Process The stem cells that incorporated the knocked-out gene are isolated from the unaltered cells using the marker gene ↓ The knocked-out stem cells are inserted into a mouse blastocyst . For this example, we use blastocyst from a grey mouse. The blastocyst now contain two types of stem cells: the original ones (from the grey mouse), and the knocked-out cells (from the white mouse). ↓ These blastocysts are then implanted into the uterus of female mice, where they develop. ↓ The newborn mice will therefore be chimeras : some parts of their bodies result from the original stem cells, other parts from the knocked-out stem cells. Their fur will show patches of white and grey, with white patches derived from the knocked-out stem cells and grey patches from the recipient blastocyst. Process: Process Blastocyst containing cells, that are both wild type and knockout cells, are injected into the uterus of a foster mother. This produces offspring that are either wild type and colored the same color as the blastocyst donor (grey) or chimera (mixed) and partially knocked out. The chimera mice are crossed with a normal wild type mouse (grey). This produces offspring that are either white and heterozygous for the knocked out gene or grey and wild type. Methods of preparing gene knockout: Methods of preparing gene knockout Knockout mice are created from embryonic stem cell (ES cells) by harvesting them approximately 4 days after fertilization. The reason for using the ES cells so early on is because the swapping of gene sequences can be properly passed on to the rest of the cells during division and develop along with the all the other adult cells. This process is completed in one of two methods: 1. Gene Targeting 2. Gene Trapping Gene targeting: Gene targeting A particular gene is manipulated within the nucleus of the ES cells of the mouse through homologous recombination ↓ Next is to make a new DNA sequence that is needed to be inserted into a chromosome. ↓ That chromosome is going to take the place of a wild-type allele ↓ The artificial inactive DNA sequence is introduced This artificial sequence flanks the DNA sequence in both directions on the chromosome. ↓ The cell recognizes the identical stretches of DNA, and "trades" the existing gene with the artificial DNA. ↓ The artificial DNA is inactive, the function of the existing gene has now been "knocked out" by gene targeting. The new cells will keep growing and dividing with the new gene inside of it. Gene trapping: Gene trapping Gene trapping is done by using a sequence of artificial DNA which holds a "reporter gene" that is made to insert into any gene at random. ↓ The artificial DNA prevents RNA splicing in the cell, thus preventing the existing gene from synthesizing its assigned protein and eliminating its function. ↓ Now the activity of the artificial "reporter gene" can be observed and studied, to determine the existing gene's normal function in the mouse. For both of these methods, a DNA vector is used to carry the artificial DNA into the embryonic stem cells of the mice. Once the DNA is injected, the cells are cultured in-vitro, and then injected into mouse embryos. These embryos are given planted into female mice, which then give birth to mice with the knocked out genes. Knockout rat: Knockout rat Knockout rats can mimic human diseases and are important tools 1. For studying gene function ( functional genomics ) and for drug discovery and development. 2. 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. Additional developments with zinc finger nuclease technology in 2009 led to the first knockout rat with targeted, germ line-transmitted mutations. 3. Knockout rat disease models for Parkinson's , Alzheimer's , hypertension , and diabetes using zinc-finger nuclease technology are being commercialized by SAGE Labs. Difference between knockout mouse and rat : Difference between knockout mouse and rat 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. 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. Knockout moss: Knockout moss For altering moss genes in a targeted way, the DNA-construct needs to be incubated together with moss protoplasts and with polyethylene glycol (PEG). As mosses are haploid organisms, the regenerating moss filaments ( protonemata ) can be directly assayed for gene targeting within 6 weeks utilizing PCR-methods. For example: Chloroplast division: The first scientific publication about identification of the function of a hitherto unknown gene utilizing knockout moss appeared 1998 and was authored by Ralf Reski and coworkers. They deleted the ftsZ -gene and thus functionally identified the first gene pivotal for the division of an organelle in any eukaryote. Protein modifications: By multiple gene knockout Physcomitrella plants were engineered, that lack the plant-specific glycosylation of proteins, an important post-translational modification. These knockout-mosses are used to produce complex biopharmaceuticals in the field of molecular farming. Mutant collection: In co-operation with the chemical company BASF Ralf Reski and coworkers established a collection of knockout mosses that is used for gene identification. More advanced types of knockouts: More advanced types of knockouts Recently, newer technologies have been developed which allow for conditional knockouts or tissue-specific gene targeting. The goal of conventional knockout technology is to knock out both alleles so that the gene is entirely absent from all cells. The purpose of conditional knockouts, in contrast, is to delete a gene in a particular organ, cell type, or stage of development. Researchers can use the technique to knock out certain portions of specific genes at particular times when those genes are important. Some limitation of Knockout gene: Some limitation of Knockout gene Knockout mice are extremely useful in studying gene function, producing custom knockout mice is very expensive. The knockout animals, as they require special transgenic core facilities where staff can assist researchers with housing and taking proper measures to keep these knockout animals at optimal conditions for research purposes. The cost of equipping and maintaining such a facility is usually very high. The mouse depend on the gene of interest for other important bodily functions; if gene was disrupted, the mouse might die or stop functioning correctly in unexpected ways. The gene that is knocked out in the mouse may not even produce an observable change in any of the mouse's characteristics. Gene knockouts in mice embryos may sometimes inhibit the mice from growing into adult mice. This problem is often overcome through the use of conditional mutations . Application: Application This laboratory technique has been used in various types of research: Cancer Research. Cystic Fibrosis. Lung, Heart, Blood, and Parkinson Diseases Aging. Anxiety. Arthritis. Diabetes. Obesity. Neural Pathway Functions. Substance Abuse. A specific gene studied from the knockout mouse can also be useful in studying how different recreational drugs affect the animal, which can be used to test therapies for drug abuse in humans. Advantages:: Advantages: Produce animals in which a specific gene has been deactivated in all its body cells Can produce model systems for studying inherited human diseases Powerful tools for investigating the nature of genetic diseases & the efficacy of different types of treatment. Latest advances: A team of researchers from UT Southwestern Medical Center have successfully knocked out a specific gene in mouse brain thought to be involved in the onset of Alzheimer's disease which codes for the enzyme cyclin-dependent kinase 5 (Cdk5). Such genetically-engineered mice were found to be 'smarter' than normal mice and were able to handle complex tasks more intelligently compared to 'normal' mice bred in the laboratory. THANK YOU: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.