Docstoc

Transgenic mice generation and husbandry

Document Sample
Transgenic mice generation and husbandry Powered By Docstoc
					Transgenic animals and
   knockout animals
3 main ways to do biological research:

1. Do research in test tubes.
2. Do research with cells.
3. Do research directly with animals.
Transgenic animals and knockout animals

Part 1: Transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animals?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animals.
            Transgenic Animal
• Animal has one or more foreign genes inserted into
  chromosome DNA inside its cells artificially.

• After injecting foreign gene into the pronucleus of a
  fertilized egg or blastocyst, foreign gene is inserted in a
  random fashion into chromosome DNA:
   – Randomly (Foreign gene may disrupt an endogenous
     gene important for normal development, and the
     chance is about 10%. )
   – multiple copies
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animal.
ES cell transformation




Injection of gene into fertilized egg
         Method 1: ES cell transformation
  vs. Method 2: Injection of gene into fertilized egg

       1. ES cell transformation works well in mice only.
    Other transgenic animals are produced by egg injection


2. ES cell transformation provides more control of the integration
                (selection of stably transfected ES cells)


    3. Injection of gene into fertilized egg is less reliable
          (viability of eggs, frequency of integration),
             but it helps to avoids chimeric animals
      Injecting fertilized eggs
• The eggs are harvested from mice
  (superovulated or natural matings).
• The DNA is usually injected into the male
  pronucleus.
• The eggs can be transferred in the same
  day (1 cell) or the next day (2-cells) into
  pseudopregnant female oviducts.
    Breeding Transgenic animals
       (transgenic founders)
• Transgenic animals Individually are
  backcrossed to non-transgenic animals.
• DO NOT intercross different founders.
  Each founder results from a separate
  RANDOM transgene integration event.
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animals?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animal.
       Conditional Transgenic mouse


The expression of transgene in transgenic mouse can be induced
Important Considerations for
  Conditional Transgenes

  • Transgenes have low or no expression when
    not induced

  • Large difference between induced and non-
    induced gene expression

  • Transgene expression rapidly turns on or off.

  • Inducer (doxycycline, tamoxifen, cre) is not
    toxic and easily administered
    Tetracycline Controlled
      Transactivator tTA
           “Tet-off”

                tetR   VP16




Doxycycline blocks      tTA binds to tetO to
 tTA DNA binding       activate transcription
Reverse Tetracycline Controlled
      Transactivator tTA
           “Tet-on”

                 rtetR   VP16




 Doxycycline allows      Without doxcycline rtTA
 rtTA to bind to tetO     can not bind to tetO
       Tetracycline Regulation:
              Summary

       No Doxycycline   Doxycycline
tTA        expressed    not expressed
rtTA       not expressed expressed
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animal.
Applications of Transgenic Animals
Transgenic mice are often generated to
  1. characterize the ability of a promoter to direct tissue-specific gene
  expression
      e.g. a promoter can be attached to a reporter gene such as
      LacZ or GFP
   2. examine the effects of overexpressing and misexpressing
      endogenous or foreign genes at specific times and locations in
      the animals
   3 Study gene function
     Many human diseases can be modeled by introducing the same
     mutation into the mouse. Intact animal provides a more complete
     and physiologically relevant picture of a transgene's function
     than in vitro testing.
   4. Drug testing
       Example 1: Transgenic Cattle

• Cloned transgenic cattle produce milk with
  higher levels of beta-caein and k-casein

Published in Nature, Jan, 2003
                   Example 2: Transgenic Mouse
           The growth hormone gene has been engineered to be expressed
                            at high levels in animals.

                       The result: BIG ANIMALS
                                          Mice fed with heavy metals are 2-3 times lar




Metallothionein
   promoter
 regulated by
 heavy metals
     Example 3: Transgenic Mouse




Trangenic mouse embryo in which the promoter for a gene expressed in
neuronal progenitors (neurogenin 1) drives expression of a beta-
galactosidase reporter gene. Neural structures expressing the reporter
transgene are dark blue-green.
Example 4: GFP transgenic mouse (Nagy)




   9.5 day embryos -
                           Tail tip
   GFP and wt
GFP transgenic mouse (Nagy)
Example 5: Wild and domestic trout respond differently
       to overproduction of growth hormone.




      So, GH is not effective to domestic trout.
Example 6: Transgenic mice as tools

• Normal mice can't be infected with polio virus.
  They lack the cell-surface Polio virus receptor.
  But, human has Polio virus receptor.

• Transgenic mice expressing the human gene
  for the Polio receptor can be infected by polio
  virus and even develop paralysis and other
  pathological changes characteristic of the
  disease in humans
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animals.
             knock-out Animal
  One endogenous gene in an animal is
  changed. The gene can not be expressed
  and loses its functions.

• DNA is introduced first into embryonic stem (ES) cells.
• ES cells that have undergone homologous
  recombination are identified.
• ES cells are injected into a 4 day old mouse embryo: a
  blastocyst.
• Knockout animal is derived from the blastocyst.
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animals.
        Vector design

• Recombinant DNA methods: Simple KO
  – Structural gene desired (e.g. insulin gene)
    to be "knocked out" is replaced partly or
    completely by a positive selection marker
    to knock out the gene functions.
  – Vector DNA to enable the molecules to be
    inserted into host DNA molecules
              KNOCKOUT MICE

         Normal (+) gene X               Isolate gene X
                                    and insert it into vector.
Genome


                                       Inactivate the gene
                                   by inserting a marker gene
                                     that make cell resistant
                     Defective    to antibiotic (e.g. Neomycin)
                        (-)
                      Gene X
                                       Transfer vector
VECTOR                                 with (-) gene X
                                        into ES cells
         MARKER GENE e.g.(NeoR)     (embryonic stem cells)
   Vector and
     genome
 will recombine
via homologous
                                       Genomic gene
   sequences
                      Homologous recombination
                      and gene disrution



                        Grow ES cells in
                  antibiotic containing media;
                   Only cell with marker gene
                  (without normal target gene)
                           will survive
         Problems with homologous
               recombination
      Unwanted random non-homologous recombination
                      is very frequent.
         This method provides no selection against it

         Solution: Replacement vectors
   The knock-out construct contains the 1) NeoR gene
     flanked by 2) two segments of the target gene
                 and 3) the HSVtk gene
         Part of the gene replaced with NeoR

ES cells are selected for integration of NeoR and
against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir
Replacement vectors

                       NeoR
                                    HSVtk         Linearized
                                                  replacement plasmid

     Homologous
     recombination                     Random integration




            NeoR


       NeoR+/ HSVtk-                     NeoR+/ HSVtk+
                              HSVtk will convert gancyclovir into a
                              toxic drug and kill HSVtk+ cells
           Typical KO vector




*tk:thymidine kinase
               Inject ES cells
               with (-) gene X
         into early mouse embryo

  Transfer embryos
to surrogate mothers     Resulting chimaras
                          have some cells
                           with (+) gene X
                           and (-) gene X.
      Mate them with normal mice

                   It is lucky,
         if germline contain (-) gene X

     Screen pups to find -/+ and mate them


      Next generation will split as 3:1
               (Mendelian)
        Embryonic stem cells
• Harvested from the inner cell mass of
  mouse blastocysts
• Grown in culture and retain their full
  potential to produce all the cells of the
  mature animal, including its gametes.
ES cells growing in culture
ES cells are transformed
• Cultured ES cells are exposed to the vector
• Electroporation punched holes in the walls of
  the ES cells
• Vector in solution flows into the ES cells
• The cells that don't die are selected for
  transformation using the positive selection
  marker
• Randomly inserted vectors will be killed by
  gancyclovir
Successfully transformed ES
   cells are injected into
         blastocysts
   Implantation of blastocysts
• The blastocysts injected with transformed
  ES cells are left to rest for a couple of
  hours
• Expanded blastocysts are transferred to
  the uterine horn of a pseudopregnant
  female
• Max. 1/3 of transferred blastocysts will
  develop into healthy pups
Implanting blastocysts




     1             2
Implanting blastocysts




    3            4
         Testing the offspring
• A small piece of tissue - tail or ear - is
  examined for the desired gene
• 10-20% will have it and they will be
  heterozygous for the gene
 Breeding Chimeras (knock-out
           founder)
Chimera - the founder
• germ-line transmission - usually the ES cells are
  derived from a 129 mouse strain (agouti or white
  colour) and the ES cells are injected into
  blastocyst derived from a C57Bl/6 mouse
  (black).
• The more that the ES cells contribute to the
  genome of the knockout mouse, the more the
  coat colour will be agouti. The chimera mouse is
  usually “tiger” striped.
 Breeding Chimeras (knock-out
           founder)
• Males that are 40% to 100% based on
  agouti coat colour should be bred
• Females should not be bred (low
  incidence of success).
• Breed aggressively- rotate females
  through male's cage. If the male produces
  more than 6 litters without transmitting
  knockout gene, the knockout gene will not
  likely go to germline and should not be
  used for more breeding.
Littermates

              Black mouse -
              no apparent ES cell
              contribution

              Chimeric founder -
              strong ES cell
              contribution

              Chimeric founder -
              weaker ES cell
              contribution
Chimeric mouse
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animal.
   Conditional knock-out animals
     How to make FLOXed gene
       Gene of interest


                             NeoR          TK
                                                Electroporate targeting vector
                                                into ES cells, followed
loxP                                            by +/- selection
                                loxP




                                                  NeoR+/ HSVtk-
  loxP                              loxP          cells selected
        Gene flanked by loxP sites (floxed)

 Make mice and breed floxed allele to homozygousity.
         Mate FLOXed mice with mice
           carrying a Cre transgene
                             Marker gene



Promoter elements   Cre   IRES   GFP            SV40 p(A)
                                       intron


                      Crucial element. Recombinase
                    would be expressed in accordance
                     with specificity of your promoter.

                      Promoter could be regulated !!!
                          artificailly or naturally
Conditional knock-out animals
       inactivate a gene only in specific tissues
    and at certain times during development and life.

                              Your gene of interest
                             is flanked by 34 bp loxP sites (floxed).

                                     If CRE recombinase expressed

                                Gene between loxP sites is removed
Transgenic animals and knockout animals

Part 1: transgenic animals:
•   Introduction to transgenic animals.
•   How to make transgenic animals?
•   How to make conditional transgenic animal?
•   Applications of transgenic animals.

Part 2: Knockout animals
•   Introduction to knockout animals.
•   How to make knockout animals?
•   How to make conditional knockout animals?
•   Applications of knockout animal.
Applications of Knock-out animals


    – Find out if the gene is indispensable
      (suprisingly many are not!)
    – Check the phenotypes of knockout animals
    – Determine the functions of knockout gene.
    Health Monitoring Programs
• Costly
• Monitor health status of colony
• Long-term savings: time, effort, money
• Inform investigator (collaborators) of
  pathogen status
• Prevent entry of pathogens
• Promptly detect and deal/eliminate
  pathogen entry
  Health Monitoring Programs
• Months of research data may have to be
  thrown out because of undetected
  infection:
  – Unfit for research
  – Data unreliable
                Pathogens
• Viral, bacterial, parasitic, and fungal
  – Sometimes no overt signs
  – Many alter host physiology - host unsuitable
    for many experimental uses
• Cures can be bad too!
       Pathogens:
Some common pathogens and
       their effects
  • Sendai virus
    – Mouse, rat, hamsters
    – One of the most important mouse
      pathogens
    – Transmission - contact, aerosol - very
      contagious
    – Clinical signs - generally asymptomatic;
      minor effects on reproduction and growth
      of pups
     Pathogens (cont):
Some common pathogens and
        their effects
– Infected shortly after birth
– stop breeding
– Altered physiology: as the virus travels down
  the respiratory tract -necrosis of airway
  epithelium, pneumonia in lungs, lesions.
– 129/J and DBA, aged and immunodeficient
  mice most susceptible; SJL/J and C57Bl/6
  most resistant
      Pathogens (cont):
 Some common pathogens and
         their effects
• Reported effects
  – Interference with early embryonic
    development and fetal growth
  – Alterations of macrophage, natural killer (NK)
    cell, and T- and B-cell function
  – Pulmonary hypersensitivity
  – Wound healing
     Pathogens (cont):
Some common pathogens and
        their effects
• MHV
 – Probably most important pathogen of
   laboratory mice
 – Extremely contagious; aerosol, direct contact;
 – No carrier state
 – Clinic state: varies dependent upon MHV and
   mouse strains
      Pathogens (cont.):
   Some common pathogens
       and their effects
– Diarrhea, poor growth, death
– Immunodeficient (e.g. nu/nu) wasting
  syndrome -eventual death
– Reported effects: necrotic changes in several
  organs, including liver, lungs, spleen,
  intestine, brain, lymph nodes, and bone
  marrow; differentiation of cells bearing T-
  lymphocyte markers; altered enzyme
  activities, enhanced phagocytic activity of
  macrophages, rejection of xenograft tumors
  etc.
     Pathogens (cont.):
 Some common pathogens and
        their effects
• Helicobacter spp
  – H. Hepaticus (mice) most prominent
  – Transmission: direct fecal-oral
  – Clinical signs absent in immunocompetent
    mice
  – In immunodeficient mice- rectal prolapse
  – Pathological changes: chronic, active
    hepatitis, enterocolitis, hepatocellular
    neoplasms
     Pathogens (cont.):
 Some common pathogens and
        their effects
• Oxyuriasis (Pinworms)
  – Mouse pinworms (Syphacia obvelata) has
    been reported to infect humans
  – Eggs excreted in faeces, can aerosolize -
    wide spread environmental contamination
  – Infection rate high; infection usually sub
    clinical
  – Athymic (nu/nu) mice are more susceptible
     Pathogens (cont.):
 Some common pathogens and
        their effects
  – Few reports documenting the effects of
    pinworms on research, many consider
    irrelevant
• Acariasis (mites)
  – Hairless mice not susceptible
  – Transmission - direct contact
  – Eradication very labour-intensive
    Pathogens (cont.):
Some common pathogens and
       their effects
  • Reported to have caused:
    – altered behaviour
    – selective increases in immunoglobulin G1
      (IgG1), IgE, and IgA levels and depletion in
      IgM and IgG3 levels in serum
    – Lymphocytopenia
    – Granulocytosis
    – Increased production of IL-4; decreased
      production of IL-2
The End and Good bye!

				
DOCUMENT INFO
Shared By:
Categories:
Stats:
views:77
posted:5/26/2011
language:English
pages:70