transgenic animals
DESCRIPTION
Transgenic Animals. Dr. Azhar Chishti Department of Medical Biochemistry. 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 - PowerPoint PPT PresentationTRANSCRIPT
Transgenic Animals
Dr. Azhar ChishtiDepartment of Medical
Biochemistry
Dr. Azhar Chishti
LECTURE OUTLINES
Dr. Azhar Chishti
• 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
Transgenic Animals
Dr. Azhar Chishti
• 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.
Transgenic Animals
Dr. Azhar Chishti
1. Transgenic mice
2. Knockin and knockout mice
3. Scid mice
Transgenic miceDevelopments 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 interestb. a strong mouse gene promoter c. Enhancer to allow the gene to be expressedd. 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
Dr. Azhar Chishti
Microinjection equipment
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 architecturephotoreceptors replicate
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.
Brain
*
Insult
To brain
Optic nerve
Retinal photoreceptors as a target for de novo prion formation
Lens
opsin promotere globin MAR
lacZ
non-Tg Tg
Retinal transgenes
opsin promotere globin MAR
lacZ
PrP
PrP
PrP
E200K
E200K V210IF-CJD mutations
Asymmetric photoreceptor cell degeneration in opsin/PrP mice
Superior Hemisphere (SH) Inferior Hemisphere (IH)
KM670/671NL V717F
APP695
42 kb Hamster PrP cosmid
ATG TAG
• Cosmid injected into C57B6/C3H oocytes• Resultant line is denoted “TgCRND8”• Maintained on C57B6/C3H outbred or 129SvEv congenic
APP
Ab 6E10
Non-Tg
Tg CRND6
Tg CRND8
Tg APP 6209
98
64
50
36
30
16
kDa
12 kDaAPP CTF
C99 and Ab
Tg CRND 8
60 120 180 240 300 Days
Ab 6E10
6
4kDa
Ab
PS1
Ab NT-1kDa
36
30PS1 NTF
1 2 3 4 5
b-stubs
APP
Protein expression from APP and PS1 transgenes
Age(weeks)
n Ab40 ng/gAb42 ng/g
4
6
8
10
25
6
7
7
5
5
38 ± 3
54 ± 7
79 ± 30
409 ± 245
21780 ± 6600
54 ± 4
55 ± 7
56 ± 3
101 ± 76
10584 ±1495
0.7 ± 0.02
1.1 ± 0.1
1.5 ± 0.5
5.2 ± 1.5
2.0 ± 0.6
Ab42/40
Amyloid peptide in aging TgCRND8 mice
TgCRND8 Brain Pathology
Morris Water Maze Test
“Swim path”
Hidden platform
Learning acquisition in 11 week old TgCRND8 mice
1 2 3 4 50
5
10
15
20
25
30
35
40
non-Tg
TgCRND8
Late
ncy
(sec
onds
)
Session
Day 1 Day2 Day 3 Day 4 Day 5
TgCRND8
IAPP vaccine Ab42 vaccine
What effect on AD neuropathology?Ab Immunization Causes a 50% Reduction Neuropathology of TgCRND8 Mice at Age of 25 Weeks
Ab-immunization prevents/stops memory deficit in the TgCRND8 AD mouse
Normal mouse AD mouse, Aß42AD mouse, IAPP ctl
Normal mouse AD mouse, Aß42AD mouse, IAPP ctl
Before Training
AfterTraining
And the Oscar goes to…
TgCRND8
Creating lab models of AD
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
Knockout mice
Dr. Azhar Chishti
.
Figure 1: Gene targeting for knockout mice
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
Conditional Knockout mice
Dr. Azhar Chishti
.
Figure 1: Gene targeting for knockout mice
Figure 1: Gene targeting for knockout mice
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
N
C
ER
Cytoplasm
Superimposedtransgene
None
TgPS1(M146L)1
TgPS2(N141I)1032
TgPS2(M239V)1379
TgAPP6209
PS1 deficient mice
33 d. 33 d.
33d 33d 62d
PS1 genes accelerate amyloid deposition in TgCRND8 mice
minus PS1(M146L+L286V) PS1(L286V)
A Transgenic Mouse Model of Human Alzheimer’s Disease
Amyloid deposition in HUMAN Alzheimer Disease Brain
(70 years of age).
HUMAN
Amyloid deposition in AD transgenic MOUSE brain (92 days of age).
MOUSE
SCID mice
These are immunodeficient miceNude mice are T-cell deficientScid mice are both T & B cell deficientThey are use in Cancer studiesThey are use for gene therapy
Dr. Azhar Chishti
ReferencesLippincott, Illustrated review of Biochemistry, 4th edition
Transgenic and Knockout Mouse – Approaches (Website)
http://www.cellmigration.org/resource/komouse/komouse_approaches.shtml 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. 276:21562-21570
Dr. Azhar Chishti
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