L17

Question 1

What is the scientific name for mouse

Answer 1

Mus musculus

Question 2

What is the scientific name for clawed frog

Answer 2

Xenopus sp.

Question 3

What is the scientific name for zebrafish

Answer 3

Danio rerio

Question 4

What is the scientific name for fruit fly

Answer 4

Drosophila melanogaster

Question 5

What is the scientific name for nematode worm

Answer 5

Caenorhabditis elegans

Question 6

How much of our genes do we share with mice

Answer 6

95%

Question 7

Why is the fact that mice have a short lifecycle useful?

Answer 7

rapid breeding of new strains

Question 8

What’re the cons re: mice?

Answer 8

relative expensive to keep

Question 9

When is animal research carried out in the UK

Answer 9

if there is no feasible alternative

Question 10

What are the three licenses required for animal experiments?

Answer 10

‒ Personal license for the researcher
‒ Project license for the study
‒ Establishment licence for the place where the study is carried out

Question 11

What percent of animal research carried out in the UK uses mice, rats, fish and birds

Answer 11

97%

Question 12

What is the issue with in vitro models?

Answer 12

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

Question 13

What are the three mouse models used to study human development and disease

Answer 13

transgenic, knock out and knock in mice

Question 14

What do all of the approaches (transgenic, knock out and knock in mice) require

Answer 14

vector construct

Question 15

What differs between the different technique (transgenic, knock out and knock in mice)

Answer 15

level of complexity differs between each technique And whether it is targeted to a specific part of the mouse genome or not

Question 16

What are the steps in standard transgenic approach

Answer 16

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

Question 17

What are the steps in gene-targeted transgenic approach

Answer 17

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

Question 18

What is the difference between standard transgenic approach vs gene targeted transgenic approach

Answer 18

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

Question 19

What does transgenic approach require?

Answer 19

Requires simple vector construct

Question 20

What does the simple vector construct contain

Answer 20

gene of interest
relevant promoter
3’ protein tag for detection
poly A tail

Question 21

What are the strengths of using transgenic mouse models?

Answer 21

Cheap & relatively easy to make
Multiple founders are generated

Question 22

What are the weaknesses of using transgenic mouse models?

Answer 22

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

Question 23

Is gene targeting approach for knock out mice more precise?

Answer 23

Yes, requires a complex vector and relies on
homologous recombination between vector and host genome

Question 24

What is gene trapping

Answer 24

Gene trapping is a high-throughput approach that is used to introduce insertion
mutations across the genome in mouse embryonic stem cells

Question 25

How is a gene trap vector inserted?

Answer 25

disrupts gene function
reports gene expression
provides a convenient tag for the identification of the insertion site

Question 26

What does the IGTC do?

Answer 26

IGTC administers all publicly available gene trap cell lines
Researchers can search and browse the IGTC database for cell lines of interest

Question 27

Typical ‘Genetrap’ from International
Mouse Phenotyping Consortium
To provide targeted inactivation through recombination using ___ and ___

Answer 27

FRT and loxP

Question 28

How do FRT and
loxP sites mediate site-specific
recombination

Answer 28

DNA recognition sites

Question 29

What is Cre recombinase?

Answer 29

tyrosine recombinase enzyme derived from the
P1 Bacteriophage and catalyses site-specific recombination between
two LoxP sites

Question 30

What is flippase?

Answer 30

tyrosine recombinase enzyme derived from the the
baker’s yeast - Saccharomyces cerevisiae - and catalyses site-specific
recombination between two FRT sites

Question 31

What do loxP allow ?

Answer 31

Strait forward knock outs

Question 32

What do FRT and loxP allow ?

Answer 32

conditional knock outs

Question 33

Describe the cre-lox system for conditional alleles

Answer 33

1. Following manipulation the
   mouse has a targeted but “floxed
   allele”.
2. A second mouse is transgenic
   for cre recombinase expressed
   under the control of a tissue
   specific promoter.
3. The two mice a crossed
   together to generate a mouse line
   that carries both alleles: “floxed”
   and “cre”.
4. Result is the tissue specific
   deletion of the “floxed allele”
   ONLY in tissues where cre
   recombinase is expressed.
5. Can also have inducible cre
   expression.

Question 34

What provides genetraps for all genes?

Answer 34

èInternational Mouse Phenotyping Consortium

Question 35

How can we make conditional KO?

Answer 35

Using cre recombinase

Question 36

What is the con of using a mouse?

Answer 36

May not accurately model a known human disease

Question 37

What is knock-in technology used for

Answer 37

introduce
a human mutation into the mouse genome

Question 38

What has changed the approached use for knock-in models?

Answer 38

crispr/cas9

Question 39

Why would the remaining loxp side by a problem?

Answer 39

might interfere with mRNA stability and/or
expression

Question 40

List an example of disease modelling using a variety of mouse transgenics

Answer 40

genetic skeletal diseases - the cartilage growth plate of a long bone

Question 41

What bone diseases have mouse models for human genetic using ‘knock-in’ mouse

Answer 41

Targeted mouse models of genetic skeletal
diseases
Mouse model of a genetic skeletal disease

And ER stress

Question 42

give examples of Generation of transgenic ‘phenocpies’ to
provide proof of concept for
understanding disease mechanisms

Answer 42

Transgenic ‘phenocopy’ using
Col2-TgRdw

Col2-TgRdw targeting, expression and retention in cartilage

Col2-Tg Rdw mice have severe short-limb dwarfism. . . .