Transcript Recitation 5

Mouse as a Model Organism
Tuesday, February 7, 2012
Overview
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Reproduction
Grafting
Non-Homologous Recombination
Homologous Recombination
Cre / loxP Recombination
Tissue Growth
Histology
Models of Human Disease
Reproduction
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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
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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
TUNEL
Nile Blue
Acridine Orange
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 Models
Mouse
Chick
Zebrafish
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 mm
Gestation time
19-21 days ~20 days
~1-2 days
4 days to
swimming
tadpole
Development
Environment
In utero
External
External
Genome
Sequenced Sequenced
Sequenced
Sequenced
Genomic
Manipulation
Common
Surgical
Manipulation
Cannot Do It Common
In ovo
Cannot Do It Common
Xenopus
Difficult
Cannot Do It Common