Lecture 24
Transgenes and Gene Targeting in Mice II
In the last lecture we discussed sickle cell disease (SCD) in humans, and I
told you the first part of a rather long, but interesting, story describing how a
mouse model for this human disease has been generated. I only got half way
through the story…we will cover the rest today. In the last lecture we
discussed how the human β-globin gene with the sickle mutation (βSH) was
introduced as a transgene in mice, in the hope that it would cause the
precipitation of hemoglobin and the sickling of mouse red blood cells (RBCs);
had this happened this would have generated an animal model for SCD. If you
recall, the transgenic mouse did not have sickling RBCs, and to try to fix this,
the human α-globin gene was also introduced into the mouse genome…but still
the doubly transgenic mouse did not have sickling RBCs. The solution to this
was to inactivate the endogenous mouse α-globin and β-globin genes, and
that’s what we will cover today. BUT, before then, I want to share with you
some great questions that I got after the last lecture, and some responses to
those questions.
βM
αM
the last lecture
αM
αH
βSH
βM
Great Questions from students after
αH
βSH
αM
αM
PROBLEM: These mice still do not have RBCs that sickle very well.
The mouse still has mouse α and β globin molecules and their
presence is enough to prevent the human hemoglobins from forming
fibers, in much the same way that humans heterozygous for the
sickle mutation do not normally have RBCs that sickle.
SOLUTION: Need to get rid of the endogenous mouse α and β
globin genes by targeted homologous recombination to generate
“Knock-out” mice
Inject foreign DNA into
one of the pronuclei
•
How do you know it didn’t integrate into an
important gene?
•
Can’t the phenotype (if you get one) be
because of the disruption of an endogenous
gene?
•
How do you know that the human globin
proteins were expressed?
•
Why didn’t the human βS-globin gene
recombine with the mouse β-globin gene?
•
Could one inject the w.t. human β-globin
gene into a human embryo to correct the
deficiency?
Pronuclei
Fertilized mouse egg prior
to fusion of male and female
pronuclei
Transfer injected eggs
into foster mother.
About 10-30% of offspring
will contain foreign DNA in
chromosomes of all their
tissues and germ line
Breed mice expressing
foreign DNA to propagate
DNA in germ line
Figure by MIT OCW.
So…how do we “get rid of” the endogenous mouse α-globin and β-globin
genes? Just like making transgenic
mice this involves some
manipulations of the mouse
embryo…but this is a much more
Images removed due to copyright reasons.
complex process, and some
background about the
preimplantation mouse embryo is
needed. For about 4-5 days after
fertilization, the mouse embryo is
freefloating (and therefore accessible) and all of the cells that will eventually
form the mouse remain totipotent, meaning that they have the potential to
differentaite into any, and every, mouse cell type. This has been shown in
various dramatic ways. For instance, if the four-cell embryo is dissected and
each cell implanted into a different foster mother, four identical mice will be
born. More interestingly, if cells from two genetically different pre-implantation
embryos (e.g., embryos destined to produce mice with different fur colors) are
simply mixed together (they are sticky) and implanted into a foster mother, a
single chimeric mouse will be born.
Essentially the two types of
Early findings
totipotent cells mix together and
revealed that
the
produce an animal that has a
preimplantation micture two types of cells in its
mouse embryo
body. This animal has four genetic
Images removed due to
is remarkably
parents!!
copyright reasons.
malleable, and
The ability of these genetically
that cells in the different totipotent cells to mix
the
together in the preimplantation
preimplantation embryo is crucial for the mouse
embryo are
gene knock-out technology.
TOTIPOTENT
In order to make a directed genetic change in a specific mouse gene we exploit
homologous recombination just as we have discussed for E. coli and S.
cerevisiae. However, this is much harder to do in mammalian cells than
bacteria and yeast. In yeast, when a linear
In yeast
DNA duplex is introduced into the cell,
about 90% of the time that that DNA is
integrated into the yeast genome it is done
Yeast genomic DNA
by the homologous recombination
machinery such that incoming DNA
In yeast homologous recombination to replace an
endogenous gene with the transfected DNA fragment
fragment is swapped for the endogenous
occurs >90% of the time
gene. In mammalian cells the DNA that is
In mammalian cells such homologous recombination
between genome and transfected DNA fragment is very
integrated into the genome is almost always
rare (<0.01% of the time)
at a non-homologous site, and the
Have to have clever selection schemes to get the rare cells
frequency of homologous replacement of an that integrated a transfected DNA fragment by targeted
homologous recombination
endogenous sequence is about 10-3 to 10-5.
What this means is that we have to allow thousands of integration events to
take place, and to be able to identify the integration event we want…namely an
integration even that took place by homologous recombination.
Tn7TR
lacZ
URA3
tet
Tn7TR
The first crucial development for this technology was being able to grow the
totipotent cells from preimplantation embryos in culture in the lab; these
are called mouse embryonic stem cells (ES cells); the crucial development
was to devise a clever way to select integrated a DNA construct by
homologous recombination.
Cells from the inner cells mass of a preimplantation embryo at the
blastocyst stage could be removed and cultured in the lab without the cells
losing their totipotency; i.e., even after being cultured in the lab for many
years these cells can still be introduced back into a preimplantation embryo and
go on to make all the tissues of a mouse. What this means, is that the cells
can be genetically manipulated whilst in culture…and then put back into a
mouse preimplantation embryo!!
Preimplantation blastocyst from an
embryo that would produce a mouse
with GREY FUR
neor
Specifically replace your gene
of interest (α or β-globin genes)
with a mutated version of that
gene in cultured ES cells
Construct
tkHSV
Gene X replacement construct
ES
cells
Nonhomologous
recombination
ES
cells
Homologous
recombination
ES-cell DNA
Other genes
Random
insertion
Can remove totipotent
EMBRYONIC STEM CELLS
(ES cells) and culture in vitro
Select for the genetically altered
cells you want
ES-cell DNA
Gene X
Gene-targeted
insertion
No mutation in gene X
Mutation in gene X
Cells are resistant to G-418
but sensitive to ganciclovir
Cells are resistant to G-418
and ganciclovir
Formation of ES Cells Carrying a Knockout Mutation
Targeting Construct
R
N
eo
V
HS
TK
Select for the NeoR gene
and against the TKHSV gene
The only cells to survive
have undergone a targeted
homologous recombination
event at the gene of interest
Now you inject the genetically modified
ES cells (originally from a blastocyst for a
mouse with GREY FUR) and inject into a
new blastocyst that would normally give
rise to a mouse with WHITE FUR
The blastocyst, now containing
two types of totipotent embryonic stem
cells, is implanted into a foster mother;
she will give birth to the chimeric offspring
Select fot the genetically
altered cells you want
Figures by MIT OCW.
Essentially, once you have identified mouse ES cells (originally from a grey
furred mouse) that have been genetically altered the way you wish…these cells
can be used to generate a living animal that contains descendents from these
totipotent ES cells. Lets see how you get from there to a mouse in which
every cell contains that genetic alteration.
Foster
Mom
Some mice are Chimeric
The goal is to have the
GERM CELLS (sperm and
eggs) derived from the
genetically modified ES
cells; if so all the
offspring would have
GREY FUR when mated
with a white mouse grey
fur is a dominant trait
Since the “grey” ES cells were
heterozygous for the KO’d
gene, only half the sperm have
the KO gene, so 50% of the
grey offspring are
heterozygous for the KO.
H eterozygous for
the knocked out
gene
H eterozygous for
the knocked out
gene
α M +/-
α M +/-
α M +/+
α M +/-
α M -/-
25%
50%
25%
H om ozygous m utant
m ice… Viable??
The blastocyts implanted into the foster mother will produce animals with
varying contributions from the “white fur ES cells” and the “grey fur ES cells”,
the latter having been genetically manipulated to have an altered gene, e.g., a
mutated α-globin gene. The crucial step is that the gonads be derived from
the genetically altered “grey fur ES cells”, because then the genetic alteration
can be passed on to an offspring (which will have grey fur) in which every cell
carries the genetic alteration. These offspring can then be crossed to generate
a mouse that is homozygous for the altered gene. This can be done for
generating mice with deletion mutations in the α-globin gene and then again
for deletion mutations in the β-globin gene.
SPERM
αH
βSH
βM
αM
αM
αH
βSH
βM
αM
Μ+βΜ
Μ+
αΜ
αM
Μ+
αΜ+βΜ
SOLUTION: Need to get rid of the endogenous mouse α and β
globin genes by targeted homologous recombination to generate
“Knock-out” mice
neo
NeoR
βSH
αH
neoR
Neo
βSH
αH
neoR
Neo
αH
βSH
αM
βM
neoR
βSH
αM
neoR
Neo
Μ+
αΜ−βΜ
Μ−
αΜ−βΜ
βM
βSH
neo
+/+ +/-
+/+ -/-
+/- +/+ +/- +/-
+/- +/-
+/- -/-
+/- +/+
+/- +/-
-/- +/+
-/- +/-
+/- +/-
+/- -/-
-/- +/-
-/- -/-
Neo R
αM
αH
Neo R
ββSHSH
is essentially
essentially an
an
This is
X AaBb
AaBb cross
cross
AaBb X
the A
A and
and B
B genes
genes
where the
on different
different
lie on
chromosomes and
and are
are
chromosomes
therefore unlinked.
unlinked.
therefore
Human transgenes
transgenes
The Human
homozygous in
in both
both
are homozygous
parents and
and so
so will
will be
be
parents
present in
in all
all offspring.
offspring.
present
offspring have
have the
the
1/16
1/16 offspring
desired genotype
genotype
desired
αHH
α
ββSHSH
αH
αM
+/+ +/+ +/+ +/-
αH
x
neo
Μ−
αΜ+βΜ
Neo
βSH
NeoR
EGGS
neo
βMR
Neo
Μ Μ
Μ+βΜ
Μ− αΜ
−β Μ+ αΜΜ−βΜΜ−
αΜ
NeoRR
Neo
αHH
α
NeoRR
Neo
NeoR
There are many different mating schemes that one could use to generate mice
that are homozygous for deletions in both the mouse α-globin gene and the
mouse β-globin gene, and that also carry the trangenes encoding the
human α-globin gene and the human β-globin gene with the sickle cell
mutation. What I have shown you is just one way to obtain this mouse. It
should be noted that after birth, this mouse ONLY expressed human
hemoglobin, and the mouse is therefore said to be humanized.
The outstanding news is that this mouse
does indeed represent an excellent model of
β
αH
Sickle Cell Disease which is now being
Neo
Neo
β
used to explore therapies for SCD that are
αH
very difficult to carry out on human
Neo
Neo
Sickled Mouse RBCs
SCD mouse has huge
populations. So far, these mice have been
Mouse RBCs Sickle!!
clog the small blood
spleen…working
vessels in tissues
overtime to clear
used to explore the effectiveness of new
defective RBCs
drugs in ameliorating the tendency of RBCs
to sickle. Moreover, the mouse has been
Images removed due to copyright reasons.
used to test out Gene Therapy approaches
to treating the disease. Both of these
SCD
wt
approaches have been successful in the
mouse, paving the way for trying out these treatments in people.
Was it all worth it? Do we have a
Sickle Cell Disease mouse model?
H
S
R
H
S
R
R
R
Circulating RBCs
Images removed due to copyright reasons.
Please see figure 4 in Iyamu, E. W., E. A. Turner, and T. Asakura.
"Niprisan (Nix-0699) Improves the Survival Rates of Transgenic
Sickle Cell Mice Under Acute Severe Hypoxic Conditions."
Br J Haematol. 122, no. 6 (Sep. 2003): 1001-8.
• Isolate mouse
Bone Marrow stem
cells
Images removed due to copyright reasons. • Transfect with
Kidney tissue damage
Human β-globin
gene that produces
a protein that
prevents sickling
• Put the modified
bone marrow back
into a mouse
Lung of control mice
Lung of mice taking Niprisan
Sickle Cell
Disease (SCD)
Mouse
SCD Mouse
After Gene
Therapy
• Monitor
expression of the
transgene and the
health of the mouse