Transcript Transgenic.142.ppsx

Dr. Azhar Chishti
Department of Medical Biochemistry
Dr. Azhar Chishti
LECTURE OUTLINES
•Transgenic animals
•Overview of transgenic mice
•How to create transgenic animals
•Transfer of DNA into eukaryotic cells
•Confirming mutation in germ cells
•Overview of knockout mice
•How to make gene targeted knockout mice
•Making ES cell containing knockout mutants
•Forming chimeras
•generating homozygous knockout mice
•Use of transgenic & knockout mice to study human genetic diseases
•Use of transgenics in gene therapy e.g. SCID
Dr. Azhar Chishti
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Transgenic animals can be produced by injecting a cloned gene
into the fertilized egg.
A giant mouse called "Supermouse“
Transgenic goats and cows can produce human hormones in
their milk.
Sometimes, rather than introducing a functional gene into a
transgenic mouse, a mutant gene is used to replace the normal
copies of that gene in the cells of the mouse.
This can be used to produce a colony of "knockout mice" that
are deficient in a particular enzyme.
Transgenic animals can serve as models for the study of a
corresponding human disease. For example, transgenic mice of
dystrophin gene serve as animal models for the study of
muscular dystrophy.
Dr. Azhar Chishti
1. Transgenic mice
2. Knockin and knockout mice
3. Scid mice
Dr. Azhar Chishti
Transgenic mice
 Developments in molecular biology and stem cell biology over the last 20
years have allowed researchers:
 To create custom-made mice through gene targeting in mouse embryonic
stem (ES) cells.
 Site-directed mutagenesis in embryonic stem cells and the phenotypic
characterization of the corresponding knockout and/or knockin mouse
 allows researchers to study gene function as it relates to the entire organism
 Now, certain diseases that normally do not present in mice, such as cystic
fibrosis and Alzheimer's disease can be induced by manipulating the
mouse genome and environment.
Dr. Azhar Chishti
Transgenic mice
 Transgenesis is the introduction of DNA from one species
into the genome of another species.
 Transgenic mouse is generally refers to any mouse whose
genome contains an inserted piece of DNA, originating
from the mouse genome or from the genome of another
species.
 To study gene function in a mouse is exogenous expression of a
protein in some or all tissues.
Dr. Azhar Chishti
Transgenic mice (cont.)
 The piece of DNA includes:
a. the structural gene of interest
b. a strong mouse gene promoter
c. Enhancer to allow the gene to be expressed
d. Vector DNA to enable the transgene to be
inserted into the mouse genome.
Dr. Azhar Chishti
Transgenic mice (cont.)
 Successful insertion of DNA results in the expression of the
transgene in addition to the wild type, basal (endogenous)
protein levels in the mouse.
 Depending on the goal of the experiment, the transgenic
mouse will exhibit over-expression:
a. non-mutated protein,
b. expression of a dominant-negative form of a protein,
c. expression of a fluorescent-tagged protein.
Dr. Azhar Chishti
Transgenic mice (cont.)
 To generate a standard transgenic mice include bacterial or
viral vector containing the transgene and any desired markers
are injected into a fertilized mouse egg.
 The DNA usually integrates into one or more loci during the
first few cell divisions of preimplantation development.
 The number of copies of the transgenic fragment can vary from
one to several hundred and the transgenic founder mice are
mosaic for the presence of the transgene.
 Founders are very likely to have germ cells with the integrated
transgene, and therefore will be able to vertically transmit the
integrated gene, and all cells of the progeny transgenic mouse
contain the transgene.
Dr. Azhar Chishti
Microinjection equipment
Dr. Azhar Chishti
Transgenic mice (cont.)
 This method is relatively quick, but includes the risk:
a. DNA may insert itself into a critical locus, causing an
unexpected, detrimental genetic mutation.
b. Transgene may insert into a locus that is subject to gene
silencing.
c. If the protein being expressed from the transgene causes
toxicity, excessive overexpression from multiple insertions can
be lethal to some tissues or even to the entire mouse .
 several independent lines mice containing the same transgene
must be created and studied to ensure that any resulting
phenotype is not due to toxic gene-dosing or to the mutations
created at the site of transgene insertion.
Dr. Azhar Chishti
Why mice and why
transgenics?
 Mice are a common
“platform” shared with other
biomedical researchers
 Mice can recapitulate key
pathologies observed in
human disease
 Trangenes allow access to all
forms of a protein - better
than toxicological phenocopy
of disease.
 ENU no good - need
humanized genes
The retina as a target for spontaneous
prion formation
 simple architecture
 photoreceptors replicate
Brain
prions.
 prions can transit
between the retina and
the CNS.
 prions formed in the
retina can be amplified
in the CNS, travel to the
periphery.
Retinal photoreceptors as a target for de novo prion formation
*
Lens
To brain
Insult
Optic nerve
e globin MAR
lacZ
opsin promoter
non-Tg
Tg
Retinal transgenes
e globin MAR
lacZ
opsin promoter
PrP
E200K
PrP
F-CJD mutations
E200K V210I
PrP
Asymmetric photoreceptor cell degeneration in opsin/PrP mice
Superior Hemisphere (SH)
Inferior Hemisphere (IH)
ATG
TAG
APP695
KM670/671NL
V717F
42 kb Hamster PrP cosmid
• Cosmid injected into C57B6/C3H oocytes
• Resultant line is denoted “TgCRND8”
• Maintained on C57B6/C3H outbred or 129SvEv congenic
Protein expression from APP and PS1 transgenes
Tg CRND 8
60
APP
98
C99
and
Ab
120 180 240 300 Days
b-stubs
6
Ab
4
kDa
Ab 6E10
64
APP
50
36
30
1
3
4
5
36
PS1
16
12 kDa
APP CTF
kDa
2
Ab 6E10
PS1 NTF
30
kDa
Ab NT-1
Amyloid peptide in aging TgCRND8 mice
Age
(weeks)
n
Ab42 ng/g
Ab40 ng/g
Ab42/40
4
6
38 ± 3
54 ± 4
0.7 ± 0.02
6
7
54 ± 7
55 ± 7
1.1 ± 0.1
8
7
79 ± 30
56 ± 3
1.5 ± 0.5
10
5
409 ± 245
101 ± 76
5.2 ± 1.5
25
5
21780 ± 6600
10584 ±1495
2.0 ± 0.6
TgCRND8 Brain Pathology
Morris Water Maze Test
Hidden platform
“Swim path”
Learning acquisition in 11 week old TgCRND8 mice
40
Latency (seconds)
35
30
25
TgCRND8
20
non-Tg
15
TgCRND8
10
5
0
1
2
Day 1 Day2
3
Day 3
Session
4
5
Day 4
Day 5
Ab Immunization
Causes
50% Reduction Neuropathology
What effect
on ADa neuropathology?
of TgCRND8 Mice at Age of 25 Weeks
IAPP vaccine
Ab42 vaccine
Ab-immunization prevents/stops memory deficit in
the TgCRND8 AD mouse
Before
Training
Normal mouse
AD mouse, IAPP ctl
Normal mouse
AD mouse, IAPP ctl
AD mouse, Aß42
After
Training
AD mouse, Aß42
Creating lab models of AD
And the Oscar
goes to…
TgCRND8
Knockin mice
 A knockin mouse is generated by targeted insertion of the transgene at a
selected locus.
 Site-specific knockins result in a more consistent level of expression of the
transgene from generation to generation because it is known that the
overexpression cassette is present as a single copy.
 The targeted transgene is not interfering with a critical locus and the
resulting phenotype is due to the exogenous expression of the protein.
 The generation of a knockin mouse does avoid many of the problems of a
traditional transgenic mouse, this procedure requires more time to assemble
the vector and to identify ES cells that have undergone homologous
recombination.
Dr. Azhar Chishti
Knockout mice
 Homologous recombination allows to completely remove
one or more exons from a gene which results in the
production of a mutated or truncated protein or, more
often, no protein at all.
 knockout mice are generated to remove protein
information by elimination of a gene or the deletion of a
functional domain of the protein.
 This can be achieved through random mutation using
chemical mutagenesis or a gene trap approach, or through
gene targeting to generate a knockout mouse.
Dr. Azhar Chishti
Figure 1: Gene targeting for knockout mice
Knockout mice
Dr. Azhar Chishti
Conditional Knockout mice
 Many genes that participate in interesting genetic pathways are
essential for either mouse development, viability or fertility.
 Therefore, a traditional knockout of the gene can never lead to
the establishment of a knockout mouse strain for analysis.
 Conditional gene modification using Cre-lox technology allows
the gene of interest to be knocked-out in only a subset of
tissues or only at a particular time to avoid lethality.
 This genetic dissection allows researchers to define gene
function in development, physiology or behavior.
Dr. Azhar Chishti
Figure 1: Gene targeting for knockout mice
Conditional Knockout mice
Dr. Azhar Chishti
Conditional Knockout mice
 Cre recombinase is isolated from the P1 bacteriophage,
catalyzes recombination between two of its consensus DNA
recognition sites.
 These loxP sites are 34 base pairs in length, consisting of two
13bp palendromic sequences that flank a central sequence of
8bp which determines the directionality of the loxP site.
 Two loxP sites are most often placed on either side of an
essential, functional part of a gene so that recombination
removes that functionality and knocks-out the gene. (See
Figure 2)
 LoxP sites placed on different chromosomes can be used to
generate targeted translocations, though this recombination
event occurs at a relatively low frequency compared to the
highly-efficient intra-gene recombination
Dr. Azhar Chishti
Presenilins
Cytoplasm
N
C
ER
Superimposed
transgene
None
TgPS1(M146L)1
TgPS2(N141I)1032
TgPS2(M239V)1379
TgAPP6209
PS1 deficient mice
33 PS1
d.
genes
33 d. accelerate amyloid
deposition in TgCRND8 mice
33d
33d
minus
PS1(M146L+L286V)
62d
PS1(L286V)
A Transgenic Mouse Model of Human
Alzheimer’s Disease
HUMAN
Amyloid deposition in HUMAN
Alzheimer Disease Brain
(70 years of age).
MOUSE
Amyloid deposition in AD transgenic
MOUSE brain (92 days of age).
SCID mice
 These are immunodeficient mice
 Nude mice are T-cell deficient
 Scid mice are both T & B cell deficient
 They are use in Cancer studies
 They are use for gene therapy
Dr. Azhar Chishti
References
 Lippincott, Illustrated review of Biochemistry, 4th edition
 Transgenic and Knockout Mouse – Approaches (Website)
http://www.cellmigration.org/resource/komouse/komouse_approaches.s
html
 Chishti MA et al.,. Early-onset amyloid deposition and cognitive deficits
in transgenic mice expressing a double mutant form of amyloid
precursor protein 695. 2001 Journal of Biological Chemistry. 276:2156221570
Dr. Azhar Chishti
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