Aplikovaná chemie a biochemie
Přednáška č. 6
In vivo modely
Práce s modelovými organismy:
• modely fyziologických stavů; modely
onemocnění
• transgenní zvířata – induced expression,
knock-out, knock-in; hypomorphic alleles
• tkáňově specifická exprese;
Modelové příklady využití hlodavců:
• genetické modely;
• využití toxických látek;
• virová onemocnění.
Experimentální modely diabetes mellitus:
The best available models for type I diabetes mellitus are the BB rat and the NOD
mouse. These demonstrate many of the autoimmune phenomena characteristic of the
human disease. They have provided the opportunity to evaluate the effects of
manipulating the immune system as well as several environmental factors on the
development of diabetes. Thus, neonatal thymectomy, administration of
immunosuppressive drugs, and antibodies against various lymphocytes have been
effective in preventing diabetes in these models. The BB rat and NOD mouse provide
the opportunity to investigate more precisely targeted forms of immune modulation
that might then be applicable to patients with type I diabetes mellitus. Although these
models are quite useful in studying the etiology of this form of diabetes, the rodents
do not develop the long-term complications that are the major clinical problem in
patients.
Several genetic models for diabetes exist in different strains of mice. These are
relevant to type II diabetes because they are also associated with obesity and do not
have an absolute insulin deficiency. The OB/OB and the db/db mice have been studied
because they exhibit tissue resistance to the action of insulin and do not have severe
insulin deficiency at the onset of diabetes.
In addition to the genetic forms of diabetes in rodents, the disease can be
produced in a wide range of mammals by surgical removal of the pancreas,
administration of drugs such as streptozotocin or alloxan, or overfeeding.
Mouse model of chronic myeloid leukemia –
viral infection:
CML is usually diagnosed by finding a specific
chromosomal abnormality called the Philadelphia
(Ph) chromosome. The Ph chromosome is the
result of a translocation—or exchange of genetic
material—between the long arms of chromosomes
9 and 22 . This exchange brings together two
genes: the BCR (breakpoint cluster region) gene
on chromosome 22 and the proto-oncogene ABL
(Ableson leukemia virus) on chromosome 9. The
resulting hybrid gene BCR-ABL codes for a fusion
protein with tyrosine kinase activity, which
activates signal transduction pathways, leading to
uncontrolled cell growth.
A mouse model has been created that develops
a CML-like disease when given bone marrow
cells infected with a virus containing the BCRABL
gene. In other animal models, the fusion
proteins have been shown to transform normal
blood precursor cells to malignant cells.
Mouse model of hepatocellular carcinoma –
expression of large HBV protein:
Transgenic mice that overproduce the hepatitis
B virus large envelope polypeptide and
accumulate toxic quantities of hepatitis B
surface antigen (HBsAg) within the hepatocyte
develop severe, prolonged hepatocellular injury
that initiates a programmed response within the
liver, characterized by inflammation,
regenerative hyperplasia, transcriptional
deregulation, and aneuploidy. This response
inexorably progresses to neoplasia. The incidence
of hepatocellular carcinoma in this model
corresponds to the frequency, severity, and age
of onset of liver cell injury, which itself
corresponds to the intrahepatic concentration of
HBsAg and is influenced by genetic background
and sex. Thus, the inappropriate expression of a
single structural viral gene is sufficient to cause
malignant transformation in this model.
Chisari et al., 1989
Specifická funkce proteinů:
• transgenní organismy – především myši;
General procedure for producing transgenic mice:
Preparation of embryonic
stem (ES) cells.
Fertilized mouse eggs divide
slowly at first; after 41/2
days, they form the
blastocyst, a hollow
structure composed of about
100 cells surrounding an
inner cavity called the
blastocoel. Only ES cells,
which constitute the inner
cell mass, actually form the
embryo. Other cells form
the trophectoderm, which
gives rise to the membranes
(amnion and placenta) by
which the embryo is
attached to the uterine wall.
Embryonic stem cells can be
removed from the blastocyst
and grown on lethally
irradiated “feeder cells.”
Využití embryonálních kmenových buněk:
When exogenous DNA is introduced into
ES cells, random insertion via
nonhomologous recombination occurs
much more frequently than genetargeted
insertion via homologous
recombination. Recombinant cells in which
one copy of the gene X (orange) is
disrupted can be obtained by using a
recombinant vector that carries gene X
disrupted with neor (light red), a
neomycin-resistance gene, and, outside
the region of homology, tkHSV (purple),
the thymidine kinase gene from herpes
simplex virus. The viral thymidine kinase,
unlike the endogenous mouse enzyme, can
convert the nucleotide analog ganciclovir.
Thus ganciclovir is cytotoxic for
recombinant ES cells carrying the tkHSV
gene. Nonhomologous insertion includes
the tkHSV gene, whereas homologous
insertion doesn't; therefore, only cells
with nonhomologous insertion are
sensitive to ganciclovir.
Isolation of mouse ES cells
with a gene-targeted
disruption by positive and
negative selection.
Recombinant cells are selected by
treatment with neomycin, since
cells that fail to pick up DNA or
integrate it into their genome are
neomycin-sensitive. The surviving
recombinant cells are treated with
ganciclovir. Only cells with a
targeted disruption in gene X, and
therefore lacking the tkHSV gene,
will survive.
Isolation of mouse ES cells with a gene-targeted disruption by
positive and negative selection.
Embryonic stem (ES) cells heterozygous for
a knockout mutation in a gene of interest (X)
and homozygous for a marker gene (here,
black coat color) are transplanted into the
blastocoel cavity of 4.5-day embryos that
are homozygous for an alternate marker
(here, white coat color). The early embryos
then are implanted into a pseudopregnant
female. Some of the resulting progeny are
chimeras, indicated by their black and white
coats. Chimeric mice then are backcrossed
to white mice; black progeny from this
mating have ES-derived cells in their germ
line. By isolating DNA from a small amount
of tail tissue, it is possible to identify black
mice heterozygous for the knockout allele.
Intercrossing of these black mice produces
individuals homozygous for the disrupted
allele, that is, knockout mice.
General procedure for producing
gene-targeted knockout mice.
Příprava AhR -/- myši (Schmidt et al., 1996):
Targeted disruption of the Ahr gene. (A) Schematic of the Ahr targeting
construct pPNTAhr2 aligned with the homologous region of the Ahr structural
gene and the structure of the recombinant allele. Black boxes denote exons,
and probe A used to detect the recombinant allele is indicated. B, BamHI; Bg,
BglII; E, EcoRI; N, NotI; X, XbaI. (B) Southern blot analysis of parent and
targeted ES cell clones.
The targeted Ahr allele produces no AHR protein. (A) Schematic of the
AHR protein and the regions recognized by the antibodies G1295,
BEAR-1, and VG9. (B) Western blots of mouse liver cytosol with the
three AHR antibodies.
KO gen nesmí produkovat protein:
Cytochrome P450IA1 and P450IA2 are not induced by TCDD in Ahr -/- mice. (A)
EROD activity of mouse liver microsomal fractions after control or TCDD
treatment. Error bars represent the high and low values obtained from two
animals. (B and C) P450 Western blotting with specific antisera. B is with a
monoclonal antibody specific for P450IA1 and C is with a polyclonal antibody
recognizing both P450IA1 and P450IA2; the upper band represents P450IA1
and the lower P450IA2.
Kontrola ztráty funkce:
Studium fenotypu:
• přežívání myší – význam genu pro základní životní funkce a vývoj;
embryonic lethal;
• projevy onemocnění;
• vývojové defekty;
The GMC offers the examination of mouse
mutants using a broad standardised phenotypic
check-up. The screens in the German Mouse Clinic
are designated to the areas of behaviour, bone
and cartilage development, neurology, clinical
chemistry, eye development, immunology,
allergy, steroid metabolism, energy metabolism,
lung function, vision and pain perception,
molecular phenotyping, cardiovascular and
pathology.
Př. patologie:
Ah-/- mice have smaller
hepatocytes than wild-type
mice. Livers of 1-year-old mice
were fixed in formalin, and 6mm
sections were examined
after staining with
hematoxylinyeosin.
Livers of Ah -/- mice exhibit
portosystemic shunting. With
perfusion of colloidal carbon,
livers of Ah +/+ mice become
black (Left), whereas livers of Ah
-/- mice remained pink (Right).
The livers of male miceare shown
above the livers of female mice.
ARNT-/- - embrya nepřežívají ED 10.5
Two loxP sites are inserted on each
side of an essential exon (2) of the
gene of interest (i.e., gene X) (blue)
by homologous recombination. These
sites do not disrupt gene function.
The loxP-containing mouse is
crossed to a transgenic mouse
carrying a cell-type-specific
promoter controlling expression of
the Cre recombinase, which induces
recombination between loxP sites.
This mouse is heterozygous for a
constitutive gene X knockout. In the
resulting loxP-Cre mouse, Cre
protein is produced only in those
cells in which the promoter is
active, and in those cells
recombination therefore occurs
between the loxP sites, leading to
deletion of exon 2. Since the other
allele is a constitutive gene X
knockout, deletion between the IoxP
sites results in complete loss of
function in all cells expressing Cre.
Cell-type-specific gene knockouts using
the loxP-Cre recombination system.
Targeted Modification of the Arnt Gene. The Arnt gene, targeting construct, targeted
allele, and schema of Cre-mediated deletion of the Arnt gene are shown. Hatched
triangles represent loxP sites, and the small arrow above the PGK-neo cassette shows
the direction of transcription that is opposite the direction of transcription of the ARNT
gene. The BamHI restriction fragments monitored and detected with the 200-bp probe
located in intron 4 are denoted by double end arrows. The restriction sites are
abbreviated as follows: A, AvrII; B,BamHI; E, EcoRI; S, SacI; X, XhoI. (Tomita et al.,
2000).
Conditional liver-specific ARNT
knockout:
Konstitutivně aktivní protein – CAAhR:
Neoplastic lesions and intestinal metaplasia in the stomach of CA-AhR mice. (A) Normal stomach
from a 12-month-old wild-type male. (B) At 3–4 months of age single small cysts. (C) In older CA-AhR
animals (6–12 months old) the number of cystic tumors increased and occupied a larger area of the
stomach. (D) In the most severe cases (9–12 months of age), the stomach was adherent to adjacent
organs such as spleen (sp), pancreas (panc), and liver (liv).
Fenotyp CA-AhR mouse – nádory žaludku:
Porovnání různých přístupů:
Huntington’s disease is a dominantly inherited disorder characterized by a
progressive neurodegeneration of the striatum that also involves other regions,
primarily the cerebral cortex. Single gene mutated – huntigtin (HD).
Three types of mouse models have been developed:
knockout, transgenic, and knock-in models.
1) Nullizygous animals die during embryonic development, they have demonstrated
that huntingtin plays a crucial role in embryogenesis.
2) The neuronal degeneration and behavioral phenotype detected in conditional
knockout, in which the huntingtin gene was inactivated in brain and testis at
early stages, indicate that huntingtin is required for neuronal function and
survival.
3) Transgenic models are those in which the human mutant HD gene,
or a fragment of it, is inserted randomly into the mouse genome. In this case,
the mouse will express a full-length or a fragment of the mutant gene in
addition to the two normal copies of the endogenous mouse huntingtin (Hdh)
gene.
4) Knock-in mouse models have the mutation inserted into the mouse huntingtin
gene and can be homozygous or heterozygous for the mutation. Because
knock-in mice carry the mutation in its appropriate genomic and protein context,
they are the most faithful genetic models of the human condition.