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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