Download Transgenic and knockout mice

Dr. Sumbul Fatma
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
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.
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.
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
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
 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.
DNA microinjection
 DNA can also be injected
directly into the nuclei of
both cultured cells and
developing embryo
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
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
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
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 nonhomologous (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 nonhomologous 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
Positive and Negative selection of
recombinant ES cells
Recombinats with
random insertion
Nonrecombinat cell
Recombinats with genetargeted insertion
Treat with neomycin
(positive selection)
Treat with gancyclovir
(negative selection)
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.
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
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
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
•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
 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
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