2010 Lab 5

From CellBiology

Cell Knockout Method

Mouse Knockouts
Cre Lox Mouse

This laboratory will be an introduction to genome analysis "knock out" techniques. Tropomyosin, a cytoskeletal protein, will be used as an example in how this technique can be applied to cell biology research. Tropomyosin will also be covered in the lecture 2010 Cytoskeleton 3 Microfilaments.

This laboratory will be presented by a guest expert Dr Galina Schevzov. Galina Schevzov
The guest lecturer will provide their own notes for this laboratory.


--Mark Hill 00:28, 13 April 2010 (UTC) Student, please note that this laboratory will form part of your individual assessment.

Objectives

  • Understand the use of mouse models to study the cytoskeleton
  • Brief understanding of knockout mouse generation
  • Brief understanding of the cytoskeleton
  • Brief understanding of this technique using tropomyosin as an example


Laboratory Slides

2009 Laboratory Slides PDF 1 slide/page view only | 4 slide/page printing | 6 slide/page printing

Laboratory Audio

How to study gene function

  • Gene targeting - replaces normal allele with a mutant allele. Precise incorporation of the gene to a specific site in the genome. Mice are known as knock-out or knock-in mice.
  • Transgenic mouse - overexpression of a gene, normal of mutated.

Why make a KO/KI mouse

  • Create mouse models to study pathophysiology of disease and test therapeutic approaches to disease.
  • Most useful to mimic recessive disorders (loss of function mutations).
    • Traditional transgenics can be used for dominant disorders.


How to make a KO mouse

  • Principle is homologous recombination
    • Homologous recombination is normal when germ cells are formed
  • A fragment of genomic DNA is introduced into a mammalian cell and it can locate and recombine with the endogenous homologous sequences.
    • This type of homologous recombination is also commonly refer to as gene targeting.
  • It occurs in yeast, bacteria and certain viruses however it is a rare event in mammalian cells except germ cells.
  • Transfected DNA most commonly integrates into a random chromosomal site.
  • The relative frequency of targeted to random integration events will determine the success of generating a KO mouse.

Knockout Mouse Requires

  • Pluripotent embryonic stem (ES) cells
    • ES cells are isolated from the Inner Cell Mass of a 3.5 day old mouse embryo.
  • Construction of KO vector by standard cloning procedures
  • Introducing the KO vector into the ES cells by electroporation
  • Selecting for gene targeting events
  • Screening the ES colonies
  • Injecting the KO cells into blastocysts.

Selecting for gene replacement events

  • Targeting vector contains marker genes
  • Positive selectable marker, neomycin phosphotransferase is resistant to the antibiotic neomycin.
  • Negative selectable marker, thymidine kinase from Herpes Simplex virus. The TK gene confers sensitivity to the chemical gancyclovir.

Screening the ES colonies

To identify which ES cells accepted the KO gene

  • DNA is isolated from the ES cells
  • Cut with restriction enzymes
  • Run on a gel and hybridised with radioactively labelled DNA probes
  • This is to test for the organisation of the target gene

Injecting the KO cells into blastocysts

  • The progeny will be a chimera consisting of both KO and wild type cells
    • Hopefully some KO cells will contribute to the germ line.
  • Heterozygous and homozygous progeny for the KO construct can be generated and analysed for phenotypic alterations


Use of genetically modified mice

  • KO/KI mice
    • Cystic fibrosis
    • Familial hypertrophic cardiomyopathy (cardiac muscle disorders)
  • Transgenic mice
    • To study dominantly acting alleles of tumor-causing genes (cancer)


Summary

  • Gene targeting is use to delete or mutate an existing gene.
    • KO and KI. Mice are generated by the injection into a blastocyst of genetically modified ES cells.
    • Chimeric mice are made.
  • Transgenic mice are use to study overexpression of a gene product.
    • Mice are generated by DNA microinjection of fertilized oocytes.
    • Results in random integration of the DNA.
  • Both offer a valuable tool for the study of human disorders. ie. Cystic fibrosis- cause by mutation of 1 or more of 4 genes. Use of knockin mutation approach.

Advances in gene targeting

CreLoxP_mouse
  • Ability to inactivate a gene at a specific time and in a specific tissue.
  • Conditional gene targeting is achieved with the use of the Cre/lox system.
    • Cre recombinase is an enzyme the catalyses sequence specific recombination between two 34 base pair repeats (LoxP sites).
    • The result of this recombination is deletion of the DNA between the LoxP sites.

Tropomyosin Example

Neuron growth cone cytoskeleton
  • Tropomyosin Expression
    • Ubiquitously expressed, found in both muscle and non-muscle cells
    • Muscle expresses 5 Tm isoforms the remaining isoforms are found in non-muscle cells
    • A restricted repertoire of Tm isoforms is expressed by different cell types
  • Tropomyosin Function
    • In muscle, Tm plays a very important role in muscle contraction
    • In non-muscle cells the role of Tm is not well understood

Tropomyosin Transgenics

In order to evaluate the role of different tropomyosin isoforms in determining the morphology of neurons, tropomyosin Transgenic mice were generated

  • Tm3 was chosen as an isoform that is not present in neurons
  • Tm5NM1 was chosen as an isoform that is present in neurons and found in the growth cone

Overexpression Results

  • Overexpression of Tm5 results in enlarged growth cones
  • Overexpression of Tm3 and Tm5 leads to distinct neuronal morphogenesis
  • Tm3 impacts on dendrites and Tm5 effects both dendrites and axonal branching

Proposed model for the increase in axonal branching in the Tm5 neurons

We propose that the overexpression of Tm5 within the growth cone leads to enlarge growth cones. The motility of these large growth cones is now altered, they may pause for longer allowing for new branches to be formed.

Generation of Tm5 knockout mice

Tropomyosin Key points

  • Multiple Tm isoforms are present within a cell
  • Tms sort to distinct subcellular compartments
  • The distinct sorting of Tms leads to the generation of distinct population of actin filaments with functionally distinct properties
  • The expression of Tm isoforms is important for the establishment of neuronal cell structure and viability

Knockout Basic Steps

  1. Subcloning and characterization of the locus of interest
  2. Knock out / knockout vector construction (targeting vector)
  3. Homologous recombination in Embryonic Stem cells (ES cell)
  4. Blastocyst injection and chimera generation
  5. Breeding to the F1 and F2 generation


Honours Projects - Dr Galina Shevzov

Please discuss potential honours projects with Dr Galina Shevzov during the class laboratory period.

References

Textbooks

Essential Cell Biology

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

Human Molecular Genetics 2

Strachan, Tom and Read, Andrew P. New York and London: Garland Science; c1999

Search Online Textbooks

Books

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

Reviews

  • Kerkhofs S, Denayer S, Haelens A, Claessens F. Androgen receptor knockout and knock-in mouse models. J Mol Endocrinol. 2009 Jan;42(1):11-7. Epub 2008 Oct 15. Review. PMID: 18923000
  • Kos CH. Cre/loxP system for generating tissue-specific knockout mouse models. Nutr Rev. 2004 Jun;62(6 Pt 1):243-6. Review. PMID: 15291397
  • Mercurio AM. Lessons from the alpha2 integrin knockout mouse. Am J Pathol. 2002 Jul;161(1):3-6. Review. No abstract available. PMID: 12107082
  • Deng CX, Brodie SG. Knockout mouse models and mammary tumorigenesis. Semin Cancer Biol. 2001 Oct;11(5):387-94. Review. PMID: 11562181
  • Liu JL, Yakar S, LeRoith D. Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system. Proc Soc Exp Biol Med. 2000 Apr;223(4):344-51. Review. PMID: 10721003
  • Thomas KR. The knockout mouse: six years old and growing stronger. Am J Respir Cell Mol Biol. 1995 May;12(5):461-3. Review. PMID: 7742010

Articles

  • Beck JA, Lloyd S, Hafezparast M, Lennon-Pierce M, Eppig JT, Festing MF, Fisher EM. Genealogies of mouse inbred strains. Nat Genet. 2000 Jan;24(1):23-5. PMID: 10615122
  • Bogue MA, Grubb SC, Maddatu TP, Bult CJ. Mouse Phenome Database (MPD). Nucleic Acids Res. 2007 Jan;35(Database issue):D643-9. Epub 2006 Dec 6. PMID: 17151079
  • Blake JA, Eppig JT, Richardson JE, Bult CJ, Kadin JA. The Mouse Genome Database (MGD): integration nexus for the laboratory mouse. Nucleic Acids Res. 2001 Jan 1;29(1):91-4. PMID: 11125058
  • Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev. 1998 Aug;76(1-2):79-90. PMID: 9867352
  • Yanira Riffo Vasquez and Domenico Spina What have transgenic and knockout animals taught us about respiratory disease? Respir Res. 2000; 1(2): 82–86. Published online 2000 August 3. doi: 10.1186/rr17. PMCID: PMC59547
  • C. Ronald Kahn Knockout Mice Challenge our Concepts of Glucose Homeostasis and the Pathogenesis of Diabetes Exp Diabesity Res. 2003; 4(3): 169–182. doi: 10.1155/EDR.2003.169. PMCID: PMC2478605
  • Ozlem Topaloglu, Paula J. Hurley, Ozlem Yildirim, Curt I. Civin, and Fred Bunz Improved methods for the generation of human gene knockout and knockin cell lines Nucleic Acids Res. 2005; 33(18): e158. Published online 2005 October 7. doi: 10.1093/nar/gni160. PMCID: PMC1255732
  • Danielle R Reed, Maureen P Lawler, and Michael G Tordoff Reduced body weight is a common effect of gene knockout in mice BMC Genet. 2008; 9: 4. Published online 2008 January 8. doi: 10.1186/1471-2156-9-4. PMCID: PMC2263071

Online

  • UNSW Embryology - Mouse Development The mouse (taxon-mus) has always been a good embryological model, easy to generate (litters 8-20) and quick (21d). Mouse embryology really expanded when molecular biologists used mice for gene knockouts, suddenly you had to understand about development in order to understand the effect of knocking out the gene.
  • NCBI Mouse Genome Resources This page is a gateway to mouse resources in and beyond NCBI. NIH Trans-Mouse
  • genome.gov Knockout Mice A knockout mouse is a laboratory mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics.
  • Knockout Mouse Project (KOMP) KOMP is a trans-NIH initiative to generate a public resource of mouse embryonic stem (ES) cells containing a null mutation in every gene in the mouse genome.

Terms

Cre/loxP A genetically engineered site-specific recombination technique based upon Cre (Cyclization Recombination) protein and Lox P (locus of X-over P1).

conditional knockout

knockin mouse A genetically engineered mouse in which genetic information is inserted into a particular locus in the genome. The mouse can now express this inserted gene/protein.

knockout mouse A genetically engineered mouse in which genetic information is deleted from a particular locus in the genome. The mouse can now not express this modified or deleted gene and therefore protein.

KO Acronym for knockout.

2010 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G