Mouse as a Model Organism

Tuesday, February 7, 2012

Overview

• Reproduction• Grafting• Non-Homologous Recombination• Homologous Recombination• Cre / loxP Recombination• Tissue Growth• Histology• Models of Human Disease

Reproduction

• 5-10 litters / year• 5-10 pups / litter• 19-21 day gestation• Sexually mature at

7 weeks• 4-5 generations per

year

Grafting

• Cannot do it!• Cells are too small

Techniques in Mouse

3 Types of Genetic Modifications

• Insertion – of a transgene or a modified allele, i.e., “knock-in” – can produce a gain of function mutation

• Knockout – of a particular gene or piece of DNA – to assess a gene’s function, i.e., is it necessary for a particular role in development

• Conditional Mutant – a spatially and temporally specific knockout!

Creating transgenic mice

Creating transgenic mice

I. Inserting DNA into Cells

• 1) Microinjection of cloned gene into nucleus of newly fertilized egg

• 2) Transfection incubate ES cells in solution that makes them take up the DNA, very inefficient need to identify cells that took up the DNA with reporter such as drug resistance

• 3) Electroporation – a high voltage pulse “pushes” DNA into cells

• 4) Retroviral vectors – a more natural way or getting genes into cells

Microinjection

Transfection

Electroporation

Highly efficient for the introduction of genes in mammalian tissue culture cells

Retroviral vector

II. Knocking out a gene

• Homologous recombination– Clone gene that is nonfunctional– Introduce DNA into cell by any method discussed above– Homologous recombination will occur replacing endogenous gene

with nonfunctional gene

Homologous recombination

BMP7

Conditional Mutant: Cre-LoxP

• Conditional mutants are needed when you want to study the effects of a gene in certain tissue late in development but the gene is also necessary early in development. A traditional knockout would result in a mutant that does not develop to stage needed.

• Cre is a recombinase that excises DNA located in between LoxP sites• You generate two transgenic lines one that expresses Cre in the tissue

you are interested and a second that contains gene of interest flanked by loxP sites. The gene will only be deleted where Cre is expressed. – Can also activate genes: In second line place stop signal flanked by loxP

between 5’ regulatory element and gene. When stop signal is removed gene will be activated.

Cre / loxP Recombination

Tissue Culture

• Possible to grow certain tissues in vitro

• Need to have isolated stem cell line

• Most tissue type has different protocols

• Does not form functioning organ

Embryo and Organ culture

• Can remove entire embryo or organ and maintain alive in culture for a short period of time– Add factors to embryo or organ: activators, inhibitors, drugs

• Afterwards do whole mount or sections in situ hybridization, RT-PCR, immunostaining ect. to analyze the embryo or organ.

• Can also do tissue transplantations• Can also remove at different stages to observe

development

Histology• Immunohistochemistry (Antibody staining)• In situ hybridization• Cell death staining• Bone and cartilage chemical stains

Cell Death Staining• TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), Nile

Blue, Acridine Orange• Used to detect apoptosis• Tunel: detects DNA fragmentation by labeling the ends of the DNA

Acridine OrangeNile Blue

TUNEL

How to detect Cell Proliferation

• BrdU is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues.

• BrdU labeling: (Bromodeoxyurdine) BrdU incorporated into cells that are undergoing DNA synthesis. Detected with antibody staining.

Model of Human Disease

• Many known gene mutations exist that reproduce human diseases in mice.

• Are these accurate models of human disease?– Not all mouse phenotypes correspond to human

phenotypes• Studies primarily done in C57BL/6 strain

– Is a study of a single strain sufficient to make conclusions about humans?

Comparison of Vertebrate ModelsMouse Chick Zebrafish Xenopus

Numbers of Eggs per ovulation

5-10 Development controlled by temp

Lots and lots Lots and lots

Initial Size ~100 µm 2-3mm 600 µm ~1 mmGestation time 19-21 days ~20 days ~1-2 days 4 days to

swimming tadpole

Development Environment

In utero In ovo External External

Genome Sequenced Sequenced Sequenced Sequenced

Genomic Manipulation

Common Cannot Do It Common Difficult

Surgical Manipulation

Cannot Do It Common Cannot Do It Common