Knock Out Technology (Final)

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Knock out technology

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<p>Gene knock out technology and animal models for human genetic disorders</p> <p>Submitted by: Dr. Vijayata M.V.Sc Scholar</p> <p>Gene knock out technology</p> <p> Knock outs can be produced by removing the gene or inducing a mutation that disables its expression.</p> <p> The elimination of a single gene product from the genome canyield important clues as to the function of that gene through the phenotypic analysis of the resulting mutant.</p> <p>Biotechnology 101, Science 101, ISSN 19313950 by Brian Robert Shmaefsky, First published in 2006 , GREENWOOD PRESS Westport, Connecticut London, pp138</p> <p>Researchers who developed the technology for the creation ofknockout mice won Nobel Prize in the year 2007 The Nobel Prize in Physiology or Medicine 2007 was awarded</p> <p>jointly to Mario R. Capecchi, Sir Martin J. Evans and OliverSmithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic</p> <p>stem cells".</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>The basic method for Gene knock out technology A targeting vector is created by flanking a mutated DNA sequence (the gene of interest) with the DNA sequence homologous to the endogenous gene. This vector is then introduced into mouse embryonic stem (ES)</p> <p>cells where the mutant DNA replaces the native gene viahomologous recombination. The recombinant ES cells are then introduced into a fresh blastocyst, where they mix with the cells of the inner cell mass.Transgenic and Gene-Knockout Rodents as Research tools for Cardiovascular Disorders, by Kapil Kapoor* &amp; Madhu Dikshit, Scand. J. Lab. Anim. Sci. No. 2. 2005. Vol. 32Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398</p> <p>Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 398</p> <p>ES cells are harvested from the inner cell mass of a blastocyst and cultured in vitro. Here they can be genetically modified before being returned to a fresh blastocyst</p> <p> The blastocyst is then implanted into the uterus of apseudopregnant female and pups produced. Since the implanted blastocyst contains two different types of ES cell (normal and recombinant), the resulting offspring will be chimeric some cells will contain the transgene, while</p> <p>other will not. The chimeric pups are then crossed with wild type animals to generate true heterozygotes, which can then subsequently be</p> <p>inbred to create a homozygote.Transgenic and Gene-Knockout Rodents as Research tools for Cardiovascular Disorders, by Kapil Kapoor* &amp; Madhu Dikshit, Scand. J. Lab. Anim. Sci. No. 2. 2005. Vol. 32Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398</p> <p> As the mutant gene encodes a major deletion or missense mutation, mice homozygous for the targeted allele do not</p> <p>express the native gene product and can be used to study theeffect of a total lack of a given protein. Breeding of various heterozygous and homozygous knockout animals can be used to combine alterations in the expression of multiple genes and to develop animal models of polygenic</p> <p>diseases (Mauvais-Jarvis and Kahn, 2000).Transgenic and Gene-Knockout Rodents as Research tools for Cardiovascular Disorders, by Kapil Kapoor* &amp; Madhu Dikshit, Scand. J. Lab. Anim. Sci. No. 2. 2005. Vol. 32Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398</p> <p>Generation of Knockout Mouse Gene targeting by homologous recombination in embryonicstem cells is a multi-step process. It begins with the generation of the targeting vector, which is transferred by electroporation into the ES cells. The ES cells are cultured and analysed for the presence of the homologously recombined DNA sequence; the targeted ES cells are then injected into blastocyst stage embryos.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society</p> <p>Embryonic Stem Cell Culture Embryonic stem (ES) cells are undifferentiated cells isolated from the inner cell mass of a blastocyst (Evans and Kaufman, 1981).</p> <p> The crucially important factor about the progenitor cells ofthese early embryos is that they are pluripotent they have the potential to differentiate into any cell type, including the germ cells, of the subsequent embryo.Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398</p> <p> ES cells in culture remain undifferentiated provided that theyare grown well separated from each other. It has been found that the presence of the cytokine leukaemia</p> <p>inhibitory factor (LIF) is essential to ensure that ES cells donot differentiate in vitro. For this reason, ES cells are generally grown on a feeder layer of fibroblasts which secrete LIF into the culture medium. Most ES cells lines currently in use have been derived from</p> <p>the 129 strain of mouse which has an agouti coat colourgenotype ; this is useful when identifying chimeric mice.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>ES cell colonies growing on a layer of fibroblast feeder cells. Healthy, undifferentiated ES cells.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>Step 1. Generation of a Targeting Vector When designing and constructing a targeting vector, a number of factors must be considered which will influence the type of mutation to be introduced, the efficiency of targeting and the ease with which successful targeting can be detected.</p> <p>DNA homologous with the chromosomal/gene site of interest For successful and efficient targeting, the vector must contain at least 510 kb of isogenic DNA homologous with the sequence to be targeted.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> This homologous sequence is divided between the short arm of homology (11.5 kb) and a long arm of homology (48 kb); this permits easy screening of the ES clones. It is ideal to identify gene targeted colonies by PCR designed to span the short arm of homology. It is known that the efficiency of homologous recombination is</p> <p>decreased when there are base pair differences between the donorand recipient DNA. For this reason, it is now common practice for the DNA used to</p> <p>construct the targeting vector to originate from the same mousestrain as the ES cells (i.e. isogenic DNA).Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>Positive and negative selection cassettes Since gene targeting by homologous recombination occurs at low frequencies (typically 105106 of ES cells treated with construct DNA) and the targeting construct is much more likely to insert randomly into the genome, it is essential to be</p> <p>able to screen ES cell colonies quickly and efficiently forsuccessful targeting. For this reason, most targeting vectors will be designed to insert a positive selection cassette into the gene of interest.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The most common positive selection marker is the neomycinphosphotransferase (neor) gene, which when expressed in the ES cell genome will render the cells resistant to treatment with the antibiotic neomycin sulfate (G418). A negative selection marker, the HSV thymidine kinase (HSV-</p> <p>tk) gene can also be used to enrich for gene targeted colonies.</p> <p>Positive selection cassette</p> <p>Negative selection cassette</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The negative selection marker is cloned outside of the homologous sequence in the targeting vector and will therefore not insert into the genome when homologous recombination occurs. For example, the herpes simplex virus thymidine kinase gene</p> <p>(HSVtk) when expressed in ES cells will produce a toxic product inthe presence of gancyclovir (a thymidine analogue), killing ES cells expressing this gene.</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>A schematic of a targeting vector:</p> <p>Targeting vector</p> <p>Overview: Generation of Gene Knockout Mice, Bradford Hall1, Advait Limaye1, and Ashok B Kulkarni1,1 Curr Protoc Cell Biol. 2009 September ; CHAPTER: Unit 19.1217. doi:10.1002/0471143030.cb191 2s44.</p> <p>Two homology arms flank a positive drug selection marker (neor). A negative selection marker (HSV-tk) is placed adjacent to one of the targeting arms. A unique restriction enzyme site is located between the vector backbone and the homology arm. When linearized for gene targeting, the vector backbone will then protect the HSV-tk from nucleases.</p> <p>Step 2. ES Cell Transfection The most efficient method for introducing the targeting vector into the ES cells is by electroporation. The linearised vector DNA is electroporated into a large number of ES cells in a single cell suspension; the cells are</p> <p>then plated on to fresh feeder cells. Then, 24 h after electroporation, the selection process can begin, which will kill cells which have not incorporated the targeting vector by homologous recombination.</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The ES cells are cultured in media containing the drugs used forselection for 710 days; this will enrich the population with cells that have undergone homologous recombination; however, it must be noted that this process is not 100% efficient. Gene targeting by Homologous Recombination</p> <p> Homologous recombination is a DNA repair mechanism that isemployed in gene targeting to insert a designed mutation into the homologous genetic locus.</p> <p> Targeted homologous recombination can be performed inmurine ES cells through electroporation of a targeting construct.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The technique of gene targeting by homologous allows for the</p> <p>introduction of engineered genetic mutations into a mouse at adetermined genomic locus. (generating mouse strains with defined mutations in their genome)</p> <p> The most common application of gene targeting is to produceknockout mice, where a drug resistance marker replaces an essential coding region in a genetic locus.</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>Targeting Vector</p> <p>Genomic locus Mutated locus</p> <p>Homologous recombination results in the transfer of only the neomycin resistance gene to the host cell.Analysis of Genes and Genomes, Richard J. Reece, John Wiley &amp; Sons, Ltd. 2004. Chapter 13 Engineering animals p 379 - 398</p> <p>Step 3. Identification of ES Cells Targeted by HomologousRecombination</p> <p> To identify the ES cells that have undergone gene targeting byhomologous recombination, discrete colonies are identified and picked. The colonies are dissociated into single cells by treatment with trypsin, divided between two wells on duplicate microtitre plates and cultured.</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The purpose of dividing the cells between duplicate plates is toallow one plate of cells to be used to prepare DNA to identify targeted ES cells and the cells from the second plate can be used to inject into blastocysts. Genomic DNA is prepared from each ES cell clone, which is then</p> <p>screened by PCR to identify clones in which homologousrecombination has occurred. Positive clones must then be further analysed, usually by Southern</p> <p>blotting and DNA sequencing, to verify that all regions of thetargeting vector have undergone the desired recombination event.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p>Step 4. Injection of ES cells into Blastocysts Blastocysts, which are 3.5 day old embryos, are collected from the uterus of the donor female. It is usual when using ES cells from the 129 strain of mouse to collect blastocysts from a C57Bl/6 mother; this mouse line has a black coat colour . ES cells carrying the desired mutation are treated to give a single cell suspension.</p> <p>Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J. CARTWRIGHT AND XIN WANG, p 390-414</p> <p> The ES cells are drawn up into the injection pipette by gentlesuction and the blastocyst to be injected is held by suction on the holding pipette. The injection pipette is advanced into the cavity of the blastocyst, which is known as the blastocoel, an 1015 ES cells are released . After injection, the embryos are cultured for a few hours to</p> <p>allow them to re-expand slowly before being transferred to theuterus of a pseudo-pregnant foster mother.Molecular Biology and Biotechnology 5th Edition Edited by John M Walker and Ralph Raply. ISBN: 978-0-85404-125-1,2009, Royal Society of Chemistry 2009, CHAPTER 14 Transgenesis ELIZABETH J....</p>