Transcript 12 transgenic mice

The strategy of controlled interference is the basis for using
mutants to understand development
Controlled interference: modify just one part of a complex system and
examine how the modification effects development.
In the past, embryologists used controlled interference classically to
remove certain parts of the embryo to show their importance in development
(Spemann’s organizer).
Modern developmental biology focuses on individual genes. The genes are
altered by inducing mutations or through recombinant methods, and effects
on development are studied.
Genetic analysis provides much finer understanding than does surgery,
however, there are still problems.
Pleiotropy: one gene (genetic change) may alter several genetic traits.
Polygenic traits: many traits may be influenced by more than one gene
Genetic analyses have been effective and represent the future of
developmental biology. Mutations are developed by 2 general methods:
1.
2.
saturation mutagenesis to create mutations randomly
use of recombinant DNA to purposefully create a specific mutant.
Saturation mutagenesis
Saturation mutagenesis: method for developing mutations in all genes that
effect a specific trait. Large numbers of organisms are mutagenized and then
bred. Offspring are carefully screened for changes in a particular trait.
Loss of function mutants: lessen the function of a gene (most common)
Null mutants: completely lose function
Gain of function mutants: activate gene in the wrong place or the wrong time
Features that are necessary for saturation mutagenesis are small numbers of
genes, organisms that are cheap and easy to breed and maintain, short
gestation, tolerant of mutagenesis. Drosophila and C. elegans have been used
frequently for this work. Mice are not appropriate due to the longer gestation
time, expense, smaller litter size, and larger genome size.
Drosophila have been used to study pattern
formation. Pattern formation: array of
elements in a distinct sequence. Drosophila
has an intricate body pattern with segments
and numerous appendages. They are useful
for saturation mutagenesis because they are
resistant to X-rays, they have a short
gestation, and they are cheap to keep. A large
number of Drosophila mutants exist.
Estimates are that 200 different genes control
overall body pattern in flies.
C. elegans: a small nematode (1mm long),
that is used to study cell lineage. The most
interesting fact about C. elegans is that they
always contain the same number of body
cells (959) plus about 2000 gametes. This
makes it easy to follow a cell to understand
its lineage. The are excellent subjects for
saturation mutagenesis because they are
easy to keep, they mature in 3 days, and they
have a tiny genome. They are also
transparent so one can see the inside.
Arabidopsis thaliana: mouse ear cress, is the
model for plant mutagenesis and
development. It has a very small genome and
it can reproduce in test tubes. It is possible
to use saturation mutagenesis with this
plant.
Zebrafish: Danio rerio, is the best suited to
study development in vertebrates. It is small
and cheap to grow. It is tolerant to saturation
mutagenesis. The embryo is free living and
clear, so it is possible to see developmental
changes on the inside.
The house mouse, Mus musculus, is an excellent genetic model of vertebrate
development. Mouse and human development are very similar, and many
genetic diseases in humans have a counter part in mice. Unfortunately,
saturation screens are not possible due to the expense of maintaining
animals, smaller litters, longer maturation time, and their larger genome.
White spotting
Piebaldism
However, more direct methods have been used with great success to study
mouse development. Genes can be specifically mutated in the lab and
directly introduced into mice. This results in a transgenic mouse that
contains the transgene in all cells of the body. Alternately, genes can be
knocked out to see what happens to development when they are missing.
How can specific genes be altered in mice?
Strategies for overexpression, interference,
and knockout of specific genes
Loss of function, null, or gain of function
mutations in many genes can be created
very specifically using molecular biology.
The first step is to isolate the gene and
clone it into an expression vector.
The gene can be modified several ways:
1. Overexpression (gain of function): the
normal gene can be attached to a strong
promoter so that high levels of protein are
produced. Expression can also be
directed to any tissue by cloning it into a
tissue specific promoter (ie, express a
muscle cell gene in liver).
The gene is then introduced into an
appropriate cell. An embryonic stem cell
to study in vivo function or any cell line to
study function in vitro.
Overexpression of growth hormone produces giant mice
An example of this technology was a early experiment performed with with
growth hormone gene cloned into the metallothionine promoter. This promoter
targets expression to the liver or any cell in the presence of heavy metal (zinc).
The normal promoter was cut off and the metallothionine promoter was
inserted in front of the growth hormone gene.
When the transgenic mice were weaned, they were fed water supplemented
with zinc. This turned on the promoter in all cells of the body and resulted in a
giant mouse, about twice as large as normal.
Why do these mice have stubby tails?
2. Antisense RNA (similar to a loss of
function mutation): Just flip the transgene
so that the complementary strand is
transcribed. When the flipped gene is
expressed, it naturally hybridizes to RNA
from the normal gene. This inhibits
translation of the RNA into protein. The
complex of dsRNA is also unstable and
rapidly degraded. Less of the gene is
expressed.
3. Dominant negative mutants (also similar
to loss of function): Engineer a gene so
that it makes a defective peptide. When the
peptide is expressed, it will interfere with
the function of the normal protein. For
example, if you are studying a protein that
dimerizes to function, you might modify
one part of the sequence so that it
encodes a protein that cannot dimerize, or
dimerizes incorrectly. Less of the function
is present.
Mammals can be transformed by injection of a transgene
directly into the egg pronucleus
This method allows the scientist to modify
any gene, to introduce it into all cells of
the mouse, and then examine the effect
on development in a species closely
related to humans.
The fertilized egg is removed and a
transgene is injected into the nucleus.
The gene may recombine with host DNA.
The developing embryo is transplanted to
a surrogate mother = poor efficiency.
Somatic and germ cells of offspring are
tested for the presence of the transgene.
This is done using Southern analysis with
a transgene-specific probe.
The transgenic mice can be bred to form a
colony. If 2 transgenics are crossed, you
may obtain mice with 2 copies of the
transgene.
Transgenic or knock out mice can be made
from embryonic stem cells
An alternate method for producing transgenic mice involves culturing
embryonic stem cells from the inner cell mass of the blastocyst.
A transgene is introduced into the stem cell and it is transplanted back
into the inner cell mass of the blastocyst from another mouse (often a
different species with different color). The cell forms a chimeric mouse.
Chimeric mouse: a mouse composed of cells of 2 genotypes. If one
species is pigmented, it is easy to see which offspring are chimeric.
This technology is used to generate knock out mice, where all copies of
a specific gene are knocked out or made non functional.
This method is more efficient than injection into pronuclei .
How is this done?
3. Homologous recombination to create a
knockout (null mutation): a cloned gene is
prepared with one or two exons replaced
by an antibiotic resistance gene (neo). The
flanking homologous sequences are
retained. When introduced into cultured
stem cells, the transgene recombines with
the normal gene and knocks out its
function. This occurs in only a few cells
but these can be selected by growing the
culture in antibiotic.
The purified stem cells are injected into
the inner cell mass of another mouse, and
the embryo is transplanted to a surrogate
mother. Some of the resulting chimeric
mice should express the transgene in the
germ line. Since only one allele is knocked
out, you cross 2 heterozygotes and hope
for 25% homozygous offspring. In practice,
you often get none because the knocked
out gene was important for embryonic
development.
Cre lox recombination system
A new technology can overcome the problem of blocked development. It
allows one to study what happens when the gene is suddenly knocked out in
adult cells (similar to a mutation that might contribute to cancer). It is useful
for studying many important genes that are required for embryonic
development.
Cre: a recombinase isolated from bacteriophage lambda. It cleaves at all sites
in a DNA sequence that have a specific lox site. Construct a cre gene driven by
an inducible promoter such as metallothionine.
Lox: genetically engineer the gene to be knocked out such that it is flanked by
lox sites.
Make one transgenic mouse for cre and another for lox.
Breed the mice so that one pair is homozygous for lox and also has a cre
gene. When these mice are treated with zinc, the metallothionine promoter is
turned on and the cre recombinase that is produced in all cells cuts out the
transgene flanked by lox sites.
This creates a knockout in the adult mouse that would not be possible with
conventional methods.
Cre lox recombination system
Promoter trapping
Knockout mice are constructed by homologous recombination, targeted
introduction of the transgene to a specific DNA sequence.
However, most transgenic mice are constructed by nonhomologous
recombination. The transgene can insert anywhere in the genome. Sometimes
this creates a problem if the transgene integrates into and disrupts an
important gene.
Promoter trapping is a variation of transgenic mice developed by
nonhomologous recombination. It is designed to identify and isolate genes
that are expressed in specific tissues during development.
Promoter: a regulatory sequence that precedes all genes. It binds specific
transcription factors and turns genes on or off during cell differentiation.
In promoter trapping, the transgene consists of a reporter gene (lacZ) that has
no promoter and can not be expressed. When this gene is transfected into a
stem cell, it integrates randomly into different areas of DNA. If one of these
areas is a promoter for a tissue specific gene, the embryo will be colored blue
in a selected area (its promoter drives the lacZ transgene).
If the transgene is also tagged with a small piece of non mouse DNA, the
interrupted gene can be isolated and cloned.
Embryos stained with b-galactosidase indicate tissues
where the enhancer is active
enhancer for muscle
cells in mouse
enhancers for mesoderm
cells in Drosophila
Transgenic mice in basic and applied science
Analysis of gene regulatory regions: transgenic mice are often made with
certain transcription factors or promoter sequences knocked out. This allows
one to determine unequivocally that this DNA sequence is involved in tissue
specific gene expression.
Role of a specific gene in cancer: transgenic or knockout mice are constructed
that overexpress or lack specific oncogenes or tumor suppressor genes,
respectively. These mice are good tools to identify whether the genetically
engineered mice are more sensitive to cancer.
A model for human genetic diseases: When certain genes are knocked out in
mice, the resulting phenotype often resembles a human inherited disease (i.e.
loss of steel factor receptor in mice leads to a phenotype similar to piebaldism
in humans). These mice are an excellent model to study how gene therapy can
be used to restore the normal gene and correct the disease.