L17 Flashcards
What is the scientific name for mouse
Mus musculus
What is the scientific name for clawed frog
Xenopus sp.
What is the scientific name for zebrafish
Danio rerio
What is the scientific name for fruit fly
Drosophila melanogaster
What is the scientific name for nematode worm
Caenorhabditis elegans
How much of our genes do we share with mice
95%
Why is the fact that mice have a short lifecycle useful?
rapid breeding of new strains
What’re the cons re: mice?
relative expensive to keep
When is animal research carried out in the UK
if there is no feasible alternative
What are the three licenses required for animal experiments?
‒ Personal license for the researcher
‒ Project license for the study
‒ Establishment licence for the place where the study is carried out
What percent of animal research carried out in the UK uses mice, rats, fish and birds
97%
What is the issue with in vitro models?
Informative studies but not representative of a protein’s role in a biological system like a body - interactions btwn diff. cell types are impossible to model outside of animals
What are the three mouse models used to study human development and disease
transgenic, knock out and knock in mice
What do all of the approaches (transgenic, knock out and knock in mice) require
vector construct
What differs between the different technique (transgenic, knock out and knock in mice)
level of complexity differs between each technique And whether it is targeted to a specific part of the mouse genome or not
What are the steps in standard transgenic approach
DNA (gene of interest) is microinjected into
the pro-nucleus of a fertilized mouse oocyte
Injected oocytes are transferred to a pseudo-
pregnant recipient mouse
ALL offspring are screened for expression of
the transgene by DNA analysis
What are the steps in gene-targeted transgenic approach
A isogenic transgene with a drug selection gene is
introduced into embryonic stem (ES) cells
Drug selection is used and surviving cells are
screened for the correct integration of transgene
Correctly targeted cells are micro-injected in
mouse blastocysts
Blastocysts are transferred to pseudo-pregnant
recipient mouse
Chimaeric offspring are identified and mated
to test for germline transmission of transgene
What is the difference between standard transgenic approach vs gene targeted transgenic approach
In transgenic approach =
DNA micorinjected into the pro-nucleaus
IN gene-targeted transgenic approach
A isogenic transgene with a drug selection gene is
introduced into embryonic stem (ES) cells
What does transgenic approach require?
Requires simple vector construct
What does the simple vector construct contain
gene of interest
relevant promoter
3’ protein tag for detection
poly A tail
What are the strengths of using transgenic mouse models?
Cheap & relatively easy to make
Multiple founders are generated
What are the weaknesses of using transgenic mouse models?
Need to characterise numerous mouse lines
Can not control site of integration into genome
(number of copies & variable expression levels
off target effects)
Wild type gene product is still present
(may interfere & influence phenotype
can express (tagged) human genes in mice
Is gene targeting approach for knock out mice more precise?
Yes, requires a complex vector and relies on
homologous recombination between vector and host genome
What is gene trapping
Gene trapping is a high-throughput approach that is used to introduce insertion
mutations across the genome in mouse embryonic stem cells
How is a gene trap vector inserted?
disrupts gene function
reports gene expression
provides a convenient tag for the identification of the insertion site
What does the IGTC do?
IGTC administers all publicly available gene trap cell lines
Researchers can search and browse the IGTC database for cell lines of interest
Typical ‘Genetrap’ from International
Mouse Phenotyping Consortium
To provide targeted inactivation through recombination using ___ and ___
FRT and loxP
How do FRT and
loxP sites mediate site-specific
recombination
DNA recognition sites
What is Cre recombinase?
tyrosine recombinase enzyme derived from the
P1 Bacteriophage and catalyses site-specific recombination between
two LoxP sites
What is flippase?
tyrosine recombinase enzyme derived from the the
baker’s yeast - Saccharomyces cerevisiae - and catalyses site-specific
recombination between two FRT sites
What do loxP allow ?
Strait forward knock outs
What do FRT and loxP allow ?
conditional knock outs
Describe the cre-lox system for conditional alleles
- Following manipulation the
mouse has a targeted but “floxed
allele”. - A second mouse is transgenic
for cre recombinase expressed
under the control of a tissue
specific promoter. - The two mice a crossed
together to generate a mouse line
that carries both alleles: “floxed”
and “cre”. - Result is the tissue specific
deletion of the “floxed allele”
ONLY in tissues where cre
recombinase is expressed. - Can also have inducible cre
expression.
What provides genetraps for all genes?
èInternational Mouse Phenotyping Consortium
How can we make conditional KO?
Using cre recombinase
What is the con of using a mouse?
May not accurately model a known human disease
What is knock-in technology used for
introduce
a human mutation into the mouse genome
What has changed the approached use for knock-in models?
crispr/cas9
Why would the remaining loxp side by a problem?
might interfere with mRNA stability and/or
expression
List an example of disease modelling using a variety of mouse transgenics
genetic skeletal diseases - the cartilage growth plate of a long bone
What bone diseases have mouse models for human genetic using ‘knock-in’ mouse
Targeted mouse models of genetic skeletal
diseases
Mouse model of a genetic skeletal disease
And ER stress
give examples of Generation of transgenic ‘phenocpies’ to
provide proof of concept for
understanding disease mechanisms
Transgenic ‘phenocopy’ using
Col2-TgRdw
Col2-TgRdw targeting, expression and retention in cartilage
Col2-Tg Rdw mice have severe short-limb dwarfism. . . .
