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Lecture 5

BIOC14Winter2013 Lecture 5 Notes.docx

7 Pages
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Department
Biological Sciences
Course Code
BIOC14H3
Professor
Patrick Mc Gowan

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superovulating female mice that have been mated to a ‘stud’ male BIOC14Winter2013 Lecture 5: Mouse Genetic Engineering and Genetic dissection of neural circuits (Article 1 and Article 2) Article 1: Mouse Genetic Engineering The mouse as a model organism o Similar genome size, gene number, genome structure to humans  most human genes have mouse counterparts o Mutations causing diseases (or symptoms) in humans can cause similar disease (or symptoms) in mice o Small, easy to maintain in the lab  short breeding cycle (2 months), 5-9 pups/litter and approximately one liter per month o Transgenic approaches are now highly advanced Generating Transgenic Mice (2 Approaches) I. DNA microinjections into single cell embryos o Produce transgenic mice containing multiple copies of randomly integrated DNA DNA microinjection into single cell embryos o 1. Fertilized once-cell eggs for DNA microinjection are obtained from superovulating female mice that have been 2. Purified experimental DNA mated to stud madeed into the male o 2. Purified experimental DNA is directly injected into the male pronucleus of a fertilized mouse egg using a pronucleus of a fertilized momicroneedle  male pronucleus is the nucleus of the sperm from the male (the sperm is in the egg, but transfer of DNA is not yet complete) o o when multiple copies of DNA are introduced into nuclei, they are integrated into random sites within the mouse genome o DNA copies are integrated as head to tail concatemers due to homologous recombination  copies all point in the same direction (because they are lined up in homologous recombination in the same way and then pieces exchanged) o Suggested that mammalian somatic cells possess enzymes for mediating somatic recombination  at the time, they did not think that somatic cells (cells that are not used in sexual reproduction) would contain machinery for homologous recombination o 3. Following microinjection, 20-30 eggs are impanted into the oviduct of pseudopregnant surrogate female (recently mated to vasectomized male)  female is bread by infertile male, such that all symptoms of pregnancy occur (and egg without embryo develops) o 4. After 19 days, the transgenic mice pups are born: transgenic litter mice are identified by PCR analysis of tail samples performed at 3 weeks of age Application of pronuclei injection methods o transgenic mice are often generated to : o (over)express wild type genes o (over)express mutant genes o (over)express reporter genes  Gren fluorescent protein, Red fluorescent protein, LacZ 9.5 day transgenic embryos – wildtype and GFP Adult transgenic mouse expressing GFP o Cell type specific overexpression of a transgene Cell type specific overexpression of a transgene Cell type specific promoteGene of Interest Cell-specific promoters can be used to direct expression of a gene to specific cell type in transgenic mice. o Pax6-GFP o cell-specific promoters can be used to direct expression of a gene to specific cell type in transgenic mice oCell-specific promoters can be used to" gene for regulating eye, nose, central nervous system and pancreatic direct expression of a gene to specificess the GFP cell type in transgenic mice.s recombination) II. Use of homologous recombination at defined genomic loci o Create gene knockouts (Kos) or replacements (Knock-ins) Genome: 1 2 3 + Targeting vector : Neo R By using homology arms, one can target any area of the mouse genome Generating transgenic mice (By homologous recombination) Genome: 1 Neo R 3 o (targeted) o PositHomologous recombination is achieved in ES cellsnserting a marker gene that makes the cell resistant to antibiotics (example: Neomycin) o Homologous recombination is achieved in ES (embryonic stem) cells using electroporation gene that makes the cell resistant to antibiotics (e.g. Neomycin). Problems with homologous recombination • Unwanted random insertion is much more frequent than homologous recombination events. • Having one selection marker (Neo R) provides no selection against random insertions. o Solution: introducing a negative selection marker o Problems with homologous recombination  unwanted random insertion is much more frequent than homologous recombination events; having one seletion marker (Neo R) provides no selection against random insertions o Solution: introducing a negative selection marker Genome: 1 2 3 + o Targeting vector : Neo R Diphtheria toxin 2 *** Diphtheria toxin: a negative selection cassette outside of the homology arms o Diphtheria toxin: a negative selection cassette outside of the homology arms What can happen in ES cells after electroporation? o No homologous recombination / no random insertion o Random insertion o Homologous recombination/ random insertion o So if we grow the ES cells in neomycin containing medium.. Applications of Homologous recombination o Knock-in  targeted insertion of the transgene at a selected locus in a chromosome o More consistent levels of expression than the pronuclei injection approach o More time consuming (vector assembly, identify ES cells that have undergone recombination) o Knock out  targeted deletion of a gene o While pronuclei injection method and knock-in method are typically used to express a certain gene, knock out is used to eliminate a gene Conventional Knock-out approach o Delete both alleles so that the gene is entirely absent from all cells Conditional knock-out approach o Delete a gene in particular organ, cell type, or at a specific time point (can avoid compensatory effects) o Circumvent lethality (about 15% of knock-outs are lethal) o More powerful in defining gene function in physiology and behavior o There are several different ways of making conditional knock outs o Most widely used method is the Cre-LoxP system genome: Gene of interest Cre If CRE recombinase is expressed… …the gene between loxP sites is removed. o o conditional knock out approach requires 2 components: gene flanked by 2 loxP sites (F
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