Transgenic and knockout mice

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Transgenic and knockout mice

• Dr. Sumbul Fatma

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Mice - Models of Human Diseases

• Although the human is the mammal we are generally
  most interested in learning more about, it is
  also the one animal we cannot use for genetic
  experiments for obvious ethical reasons
• Mice naturally develop conditions that mimic
  human disease, such as cardiovascular disease,
  cancer and diabetes
• Mouse are a favorite model for human disease
  because it has a relatively low cost of
  maintenance and a generation time that measures
  only nine weeks
• Developments in molecular biology and stem cell
  biology have allowed researchers to create
  custom-made mice through gene targeting in mouse
  embryonic stem (ES) cells
• Certain diseases that afflict only humans, such
  as cystic fibrosis and Alzheimer's can also be
  induced by manipulating the mouse genome and
  environment

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ES Cells and Chimeric Mice

• Embryonic stem (ES) cells are pluripotent cell
  lines with the capacity of self-renewal and a
  broad differentiation plasticity
• They are derived from pre-implantation embryos
  and can be propagated as a homogeneous,
  uncommitted cell population for an almost
  unlimited period of time
• Even after extensive genetic manipulation, mouse
  ES cells are able to reintegrate fully into
  viable embryos when injected into a host
  blastocyst
• After these pre-implantation embryos are
  implanted into a surrogate mother, they develop
  into mosaic offspring known as chimeras. The
  tissues of chimeric mice are comprised of a
  mixture of cells that originated from both the
  host embryo and the ES cells.

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Knockout Mice

• A knockout mouse is a genetically engineered
  mouse in which one or more genes have been turned
  off through a gene knockout
• Important animal models for studying the role of
  genes which have been sequenced, but have unknown
  functions
• By causing a specific gene to be inactive in the
  mouse, and observing any differences from normal
  behaviour or condition, researchers can infer its
  probable function.

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Transgenics

• This technique permits the introduction of
  foreign genes or altered forms of an endogenous
  gene into an organism
• Mostly, this technique does not result in
  replacement of the endogenous gene, but rather
  the integration of additional copies of it
• The introduced gene is called transgene and the
  organism carrying it is referred to as transgenic

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Transferring DNA into eukaryotic cells

• Production of both knockout and transgenic
  animals requires the transfer of DNA into
  eukaryotic cells.
• Calcium precipitation
• Liposome delivery
• Microinjection
• Electroporation

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• Calcium phosphate precipitates of DNA form when
  DNA is mixed with calcium chloride. When these
  DNA precipitates are added to animal cells
  growing in culture, the precipitated DNA can be
  taken up by the cells, again transferred to the
  nucleus and expressed
• Liposomes are artificial membranes that can be
  formed in a test tube. DNA can be mixed with the
  liposome preparation under the appropriate
  conditions. This results in the encapsulation of
  DNA into synthetic lipid membranes. When this
  membrane fuses with the cell plasmamembrane, DNA
  is released into the cell and somehow ends up in
  the nucleus.

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DNA microinjection

• DNA can also be injected directly into the nuclei
  of both cultured cells and developing embryo

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Electroporation

• Cells are subjected to a brief electric shock of
  several thousand volts and become transiently
  permeable to DNA. Presumably the shock briefly
  opens holes in the cell membrane allowing the DNA
  to enter the cells before the holes reseal

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DNA incorporation in the cell

• Once the foreign DNA is inside the host cell,
  enzymes that function normally in DNA repair and
  recombination join the fragments of foreign DNA
  into the host cells chromosome
• The new fragment can either replace an endogenous
  gene- homologous recombination or it can remain
  as an independent extrachromosomal DNA molecule
  referred to as an episome

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Identification of transgenic cells

• Since only a relatively small fraction of cells
  take up DNA, a selective technique must be
  available to identify the transgenic cells
• In most cases the exogenous DNA includes two
  additional genes
• The small fraction of cells in which homologous
  recombination takes place can be identified by a
  combination of positive and negative selection

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Positive and Negative selection

• Positive Selection- One of the additional genes
  (neoR) confers neomycin resistance it permits
  positive selection of cells in which either
  homologous (specific) or non-homologous (random)
  recombination has occurred
• Negative selection- The second gene, thymidine
  kinase gene from Herpes Simplex Virus (tkHSV)
  confers sensitivity to gancyclovir(a cytotoxic
  nucleotide analog). This gene permits negative
  selection of ES cells in which non-homologous
  recombination has occurred
• Only ES cells that undergo homologous
  recombination (i.e. gene-targeted specific
  insertion of the DNA construct) can survive this
  selection scheme

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(No Transcript)
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Positive and Negative selection of recombinant
ES cells
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Knockout Mice

• Gene knockout is a technique for selectively
  inactivating a gene by replacing it with a mutant
  allele in an otherwise normal organism (mice)
• Knockout mice are a useful model system for
  studying certain human genetic diseases.

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Making knockout mice

• Mutant alleles are introduced by homologous
  recombination into Embryonic Stem cells
• ES cells containing the knockout mutation are
  introduced into early mouse embryos. The
  resultant mice will be chimeras containing
  tissues derived from both the transplanted ES
  cells and host cells. These cells can contribute
  to both germ cell and somatic cell population
• Chimeric mice are mated to assess whether the
  mutation is incorporated into the germline
• Chimeric mice each heterozygous for the knockout
  mutation are mated to produce homozygous knockout
  mice

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Knockout Mice to study genetic diseases

• Knockout mice make good model systems for
  investigating the nature of genetic diseases and
  the efficacy of different types of treatment and
  for developing effective gene therapies to cure
  these often devastating diseases
• For instance, the knockout mice for CFTR gene
  show symptoms similar to those of humans with
  cystic fibrosis

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Transgenic animals

• Transgenic animals carry cloned genes that have
  integrated randomly into the host genome
• Transgenic technology has numerous experimental
  application and potential therapeutic value
• The frequency of random integration of exogenous
  DNA into mouse genome at non-homologous sites is
  very high, therefore, the production of
  transgenic mice is a highly efficient and
  straightforward process

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• Foreign DNA containing a gene of interest is
  injected into one of the two pronuclei (the male
  and female nuclei contributed by the parent) of a
  fertilized mouse egg before they fuse
• The injected DNA has a good likelihood of being
  randomly integrated into the chromosome of the
  diploid zygote

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• Injected eggs are then transferred to foster
  mothers in which normal cell growth and
  differentiation occurs
• About 10-30 of progeny will contain the foreign
  DNA in equal amounts in all tissues, including
  the germ cells
• Immediate breeding and backcrossing of these mice
  can produce pure transgenic strains homozygous
  for the transgene

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Transgenics and gene therapy

• Once a gene mutation is identified to be the
  cause of a disease, the next step is to cure the
  disease by introducing normal genes( transgene)
  into affected individual
• In experimental animals, some genetic disorders
  have been cured by gene therapy, but in humans,
  numerous technical issues need to be resolved
  before it can be widely used
• a) how to reliably and safely introduce various
  genes into human cells
• b) tissue/ cell specific introduction of genes
• c) large size of genes
• d)how to address the ethical issues