Animal Models

Question 1

What is contractarianism

Answer 1

Employs the idea of contracts between individuals to govern their interactions

Each of us have our own interests that we are entitled to pursue, however we can benefit from the help of others

E.g. we should care because consumers demand it

E.g. we should care for their welfare, as it will improve quality of research e.g. by not stressing them, and altering hormones

Question 2

What is ultilitarianism

Answer 2

Cost V benefit - Actions are right if they are useful or beneficial to the majority

Animals deserve moral considerations, in our dealing with animals we must consider the welfare consequences as well as the potential benefits to humans/animals

E.g. mass animal production is problematic, but animal research may be justified

Question 3

What is the relationship view

Answer 3

Highlights the importance of our relationships to animals and its based upon considering animals in a sort of hierarchical order

Have special duties to domestic animals because they are in our care

Considers how our treatment of animals might reflect on us and our treatment of humans

E.g. a dog is a man’s best friend, so its treated better than other animals, mice/rats do not matter as much

Question 4

What is the animal rights point of view

Answer 4

Directly opposes the concept of animal use, putting clear and definitive limits on our treatment of animals

Animals should have the same considerations as human beings, the right not to suffer or be killed for human benefit

E.g. experiments are unacceptable regardless of the benefits - animals are no our slaves

Question 5

What is the respect nature point of view

Answer 5

We have a duty to protect not just individual animals, but the species to which they belong - and in particular the integrity of each species

The preservation of species is morally good, should resect nature and its rich genetic structures

Not genetically modify species/ selectively breed- disrespectful inference

E.g. endangered species should be protected from extinction - and leave animals the way they naturally are

Question 6

Why is animal research needed

Answer 6

Cells behave differently in vitro to in vivo

Most medicines come from animal research of some form - and also contributed to 70% of Novel prizes

Helps to also understand animal ill health

Scientists seek to alleviate pain and suffering
UK law - animals shouldn’t be used if there’s an alternative, but no new medicines may be trialled in humans unless it has been thoroughly tested in other ways

Question 7

Describe In vitro methods

Answer 7

Many different forms - different cell lines

Cheaper and quicker

iPSC/organoids are very promising form of investigation

There is not many ethical concerns - other than patient consent
BUT it cannot fully replicate a living system

Question 8

What is computer modelling

Answer 8

Basic understanding needed before programming is possible

Limited processing power

Question 9

Why are animals used

Answer 9

Drug toxicity without prior testing on animals is risky…

Systems are similar to humans

Most human diseases exist in other species - and can be used in other ways e.g. veterinary science

Side effects and efficacy of drugs only show up in in vivo tests

Whole system complexity of interactions only replicable in vivo

Common = Mice, fish, rats, birds, 1% = horses, dogs, primates, cats (specially protected)

Question 10

Describe the ‘Animals - Scientific procedure - act 1986’

Answer 10

Act of UK parliament that permits the use of animals in scientific producers

Regulates the use of protected animals in any experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to animal

Protected animals - any living vertebrae animals (other than man) and any living cephalopod - octopus/cuttlefish/squid but not invertebrates

Animals cared for with the best standards of modern animal husbandry

Home office inspection system under place to ensure rules are not violated - incur fines/imprisonment
Widely consiidered as the most stringent animal welfare act in the world

Question 11

What is the licensing procedure for animal work

Answer 11

3 tier licensing system authorised by HO

Establishment license - certificate of designation

Project licence - specific research/testing programme

Personal licence - specific individual/competency

Only approved if
Benefits outweigh cost, there is no non-animal alternative
Minimum number of possible animals used, with lowest sensitivity to pain possible and pain minimised
Research premises have necessary facilities to care for animals

Question 12

What is a local ethics review

Answer 12

Every research/testing activity requires a committee of scientists and people to assess justification of use of animals

Cannot do work without licences AND approval from LEC

Question 13

What are the 3 R’s

Answer 13

Replacement - alternative techniques

Reduction - minimum number, fewer animals, more information

Refinement - better housing, improve procedures, welfare

Question 14

Why are zebrafish used as an animal mdoel

Answer 14

Tropical freshwater fish found in the river Ganges

Cheap, easy to manipulate and lays lots of eggs (which are also transparent, and develop ex-utero)

Easier to trace cells as the organisms are smaller

Easy to destroy cell by lasers, move them around, and knock down genes with morpholinos/RNA injection/CRISPR
Pharmacological mediation by adding drugs to the water

Question 15

How is zebrafish eggs collected

Answer 15

Automated systems regulate the fish

Breeding - 28.5°C, pH 7 and salinity of freshwater with some salt as 500 microsiemens
10% water change per day to prevent organic waste build up
Development can be speed up by increasing temperature to 32°C, or slowing it down by decreasing it to 22°C

Egg collection - 14 hr day/light cycle, trays with marbles/slats are put overnight and then the lights on which induce mating, eggs are laid in the trays which are taken out and the eggs are sieved out

Slightly more females than females to prevent reproductive stress

Females - round whitish belly and paler in colour | Male - pinky orange brown colour

Question 16

How do zebrafish develop

Answer 16

Incubate the eggs at 28.5 degrees for correct staging and to standardise the development as above

After the first cleavage you can the yolk with the cells on the top

The cells start dividing at roughly 15 minute intervals getting a bundle of cells on top of the yolk forming a blastula (hollow sphere of cells)

This forms yolk layer, required for cellular movement and is important for cell specification

The process of epiboly begins - they migrate over the yolk and allows convergence, involution and extension (gastrulation)

The three primary germ layers are formed - endoderm, mesoderm and ectoderm

Endoderm - respiratory system, digestive system, organs associated with digestion, liver and pancreas
Mesoderm - somite’s; muscles, cartilage, dermis, notochord, blood vessels, connective tissue
Ectoderm - epidermis, neural crest cells, nervous system

Timeline
24 hours - muscles are twitching, the heart is beating and partitioning of the brain
48 hours - the fish hatch out of the chorion after 48 hours, pigment forms
5 days - the yolk has gone, you can see the eye and cartilage forming in the head

*Zebrafish have 2 chamber heart

Question 17

How do we study genes and disorders using zebrafish

Answer 17

Investigating gene function - are there any clues from the DNA sequence/structural motifs

Where and when its expressed

Where does it act - function in cell and where, or is it secreted

What occurs when you KO or suppress function and its effects on surrounding cells/tissue

What are the morphological/structural consequences

What occurs during overexpression - phenotype changes, cell death etc.

What pathways are affected by loss and gain of function

Is there any indication of a way to rescue gene from KO

Question 18

Describe in situ hybridisation for gene expression analysis in zebrafish

Answer 18

Staining endogenous mRNA by hybridising via labelled complementary strand of RNA (probe)

Detected via colorimetric reaction

Antisense RNA probe is synthesised from a plasmid containing the gene, and assembled using a transcription mix with uridine conjugated with a plant steroid called digoxigenin

Embryo is washed by detergent which punches holes into cell
Probe enters cell, hybridising at 65 degrees binding mRNA of GOI
Washing via salt concentrations to remove non-specific binding of probe
Add blocking reagent to prevent non-specific binding of secondary antibody
Anti-digoxigenin antibody conjugated to alkaline phosphatase is added and binds to dig labelled uridine in probe
Substrate for alkaline phosphatase is added, which changes colour in presence of alkaline phosphatase

Question 19

Describe protein localisation via antibody detection

Answer 19

This is carried out in zebrafish as they are transparent

Antibodies are developed via injection of protein of interest resulting in an immune reaction

These antibodies are purified, fluorescently labelled and thus is able to bind the protein within the zebrafish

Question 20

What are transgenic fish and how are they created

Answer 20

This is when a gene is inserted e.g. via plasmid

Plasmid contains promoter and a GFP/fluorescent protein sitting between TOLL2 recognition sites

This is injected into the zebrafish alongside transposase

Transposase recognises and cuts the TOLL2 sites, allowing plasmid integration into the genome

Question 21

Describe zebrafish gene KO

Answer 21

This is done using antisense morpholinos

They can inhibit gene specific translation by binding propoter regions/initiation sites to prevent ribosomal translation

They can target splice donor sites leading to intronic inclusion/in-frame premature stops/exon skipping leading to RNA mediated decay

Question 22

Describe problems with morpholinos

Answer 22

Difficult to measure the efficacy of MO’s without a good antibody

Difficult to rule out the possibility that the MO inhibits the function of an irrelevant gene

It can be difficult to inject precise and reproducible volumes of MO’s

MO’s only last to 5dpf since they’re metabolised

Question 23

Describe controls used in gene KO in zebrafish

Answer 23

Compare with existing mutants to reveal off-target MO effects - not always possible

Loss of protein should be verified using antibody staining or other assays

Incorrectly spliced pre-mRNA should be verified by RT-PCR and altered splice product sequenced

At least 2 MOs per target gene should be used to ensure they give similar phenotypes , testing for synergism is also good here

RNA rescue - co-inject MO and target gene RNA that is not recognised by MOs

Control MO’s, use of a standard control that affects a gene not expressed in the cells of interest e.g. human bet globin/ 5 base mismatch / p53MO

Question 24

What is ENU, and describe its use in screening in forward and reverse genetics

Answer 24

ENU = potent mutagen targeting spermatogonial stem cells

Forward genetics - ENU screening (phenotype based) = find genetic cause of a phenotype

Reverse genetics - ENU screen (genotype based) = find phenotypic consequence of genotype change
WES to find mutations then look for relevant phenotype

Question 25

Describe CRISPR

Answer 25

CRISPR - origins in bacterial adaptive immune system

Associated with Cas9, an endonuclease that causes ds-breaks, which binds an RNA enabling specificity, targeting sequences ending in NGG (PAM sequence)

Cas9 causes ds-breaks 3 bases upstream of the PAM sequence

There is 2 ways of repairing breaks = non-homological end joining, or homology directed repair

NHEJ = error prone = purposefully creating mutations = KO

HDR = used to add in DNA sequences from donor DNA but less efficient = knock-in

Question 26

Why is zebrafish good for use in drug discovery

Answer 26

Ease and low cost at raising large numbers

Highly conserved genetic and biochemical pathways between ZF and mammals

Wildtype/transgenic screens - target organs/systems

Morpholino or mutant rescue screens

Drug reprofiling and FDA approved libraries

Automated screening is now available

Question 27

Describe case study 1 -zebrafish as a chemical screen for anti-cancer drugs

Answer 27

RAS encodes GTPases most commonly activated in cancer

Injection of human RAS causes early lethality

HRAS was put inserted into ZF under a heat shock promoter
Heat shock leads to more HRAS which showed via microarray that it caused upregulation of dusp6

This upregulated version was used to screen for drugs that can inhibit the upregulation

Question 28

Describe case study 2 - zebrafish in behaviour profiling

Answer 28

Screened 5648 compounds for changes in multiple parameters: rest bouts, rest latency, waking activity

547 drugs significantly affected behaviour
Assigned behaviour fingerprints to each compound (reveals relationship between drugs and their targets

Question 29

Describe case study 3 - zebrafish in identification of a primary target of thalidomide teratogenicity

Answer 29

Prior to public availability it was originally a sedative used for many symptoms including nausea

Research found It was almost impossible to give a lethal dose

Testing:
Thalidomide bound to ferrite beads, incubated with HeLa cell lysate
Beads with bound protein washed, elution of bound proteins with free thalidomide

SDS gel electrophoresis and silver staining, mass spectroscopy to identify proteins that bind thalidomide

interacting proteins identified - cereblon and damaged DNA binding protein 1 (DDB1)

Cereblon KD causes thalidomide-like defects on ZF
Injection of cereblon that doesn’t bind thalidomide rescues effects of thalidomide

Question 30

Describe the pitfalls of genetic models as disease mdoels

Answer 30

Many genetic models are also good disease models, but:

Replicating a human genetic defect doesn’t necessarily create a disease model

Generating a functional defect does not necessarily replicate the human genetic disease

Creating a disease model can require more than simple genetic modification – e.g. “humanization

Question 31

Describe the constraints in using animal models as genetic models of disease

Answer 31

Although many genes have highly conserved sequence and function, there are differences

Genetic background makes a difference

Gene dosage can have different effects between human and mouse

Complex phenotypes are difficult to replicate e.g. neurobehavioural defects

Question 32

Describe the basis of transgenesis

Answer 32

Transgenesis - stable insertion of exogenous DNA into host cell’s chromosomal DNA
- Doesn’t include transient transfection

Exogenous DNA with the same sequence as endogenous DNA can lead to exchange: known as homologous recombination

Genome engineering techniques use modified nucleases to cut, or nick genomic DNA

Endogenous DNA repair enzymes cause mutations or can be directed to insert novel DNA sequence

Question 33

Describe embryo manipulation

Answer 33

Embryo manipulation is important for transgenesis to generate a germ-line genetic modification so mutation can be passed on

Question 34

Describe random transgenesis

Answer 34

DNA microinjection into zygotic pronucleus

Relatively quick & easy

Transient assays possible (we can look at embryos, for example)

Question 35

Describe targeted transgenesis

Answer 35

Targeted - Homologous recombination in embryonic stem (ES) cells
Relatively slow and difficult
Very powerful and flexible

Targeted - Genome engineering techniques in ES cells or embryos

Ease and speed depend on the technique used
Possibility of off-target effects

Question 36

Describe mouse pre-implantation development

Answer 36

Fertilisation - large male pronucleus injected into egg, but doesn’t fuse with the female immediately

Zona pellucida constrains the growth of the early embryo

After 32 cells, there starts to be a distinction between two cells - trophectoderm , and the blastocyst containing the inner cell mass

Trophectoderm mainly create external embryonic tissue

Question 37

Describe pronuclear injection transgenics

Answer 37

4 week old females are superovulated and mated → fertilised eggs are collected and allowed to mature until the male pronuclei starts to move to the centre of the zygote

DNA is microinjected into the male pronucleus (200-400 eggs/day, 75% survival) → embryos are transferred into the oviduct of pseudo pregnant females (25-35 embryos per mouse)

18 days later pups are born (25% success rate) → screen DNA to identify transgene incorporation (10% success)

Question 38

Explain the uses of pronuclear injection transgenesis

Answer 38

For disease models, most commonly trans-speciation e.g. human gene in mouse

Repression of a gene - shRNAi (or morpholino antisense) inhibition of expression

(Over)-expression of a gene
“Normally” – its own promoter
“Ectopically” – another promoter

Express in different cell types, different times, and Cre

Reporter of gene expression - attach easily detectable protein/enzyme

Promoter or non-coding region analysis

Pronuclear injection is also used in genome editing

Question 39

Describe transgenes

Answer 39

The transgene contains promoter, enhancer, cDNA, some 5’ and 3’ UTR, 3’ intron and polyA signal

Important to distinguish the transgene from the endogenous gene

You can find the expression by

Promoter can be changed - ubiquitous/tissue specific, inducible and vary length of promoter/enhancer
Gene - what are its properties, gene from which species, attach tags (epitope tags or reporter)

Question 40

Describe promoter traps

Answer 40

Promoter traps - constructs containing a reporter gene and selectable marker but no promoter

Splice acceptor at 5’ end of gene biases for integration into ‘active’ regions of the genome

E.g. Gene = beta-galactosidase (lacz) and neomycin resistance

Thus the protein is only expressed when it is successfully integrated into DNA under a promoter, thus the protein expression pattern can be observed to learn about it

Question 41

Describe gene traps

Answer 41

Similar to promoter traps but instead you’re focusing on the gene - constructs containing a reporter gene and selectable marker but no promoter

Splice acceptor at 5’ end of gene biases for integration into ‘active’ regions of the genome

This is transfected into embryonic stem cells growing in culture

Reporter gene has a selectable marker so only transgenic cells are left
These cells are then implanted into a blastocyst which is implanted into a female mouse

Create chimeric mice as the edited stem cells will proliferate at different levels compared to others

Germline-transmission is then checked from its offspring

Question 42

Describe embryonic stem cells

Answer 42

Pluripotent cells isolated from the inner cell mass cells of a blastocyst

They proliferate indefinitely in culture (and remain pluripotent, under the right conditions)

Can be genetically manipulated in culture
Form the basis for the vast majority of targeted transgenics

Question 43

Describe homologous recombination

Answer 43

Homologous chromosomes pair up, create 2 ds-breaks forming a Holliday junction

Reminder - this can create CNV’s (non-allelic homologous recombination)

This processes causes swapping of a group of alleles
However, the repair enzymes only recognise the end regions
Thus the middle segments can be altered

DNA can be introduced between regions identical to host DNA
However, homologous recombination occurs at low frequency
Positive drug selection is needed to identify these rare events

DNA integration can also occur at random locations
These can also confer drug resistance/sensitivity, allowing negative drug selection

To select for this the gene would be at the END of the recognised sequence, so it would not undergo homologous recombination and only be integrated through this mechanism
You want to prevent this so you kill these cells of using a drug this gene causes sensitivity to

Question 44

Summarise selection strategies for ES cells

Answer 44

As homologous recombination is rare, we need to be able to select the ES cells in which it has taken place

Positive selection - include gene in the recombination region, conferring resistance to a toxic drug

Negative selection: include gene in the construct that confers sensitivity to a toxic drug
Outside of recombination region, to kill cells in which random integration has occurred (gene at end)

Positive selection: (PGKneo bpA, neo) - neomycin phosphotransferase, makes cells resistant to G418
Use phosphoglycerate kinase promoter and bovine growth hormone poly A tail
Can also use hprt and HAT medium

Negative selection: (MC1TKpA, tk) - Herpes simplex virus thymidine kinase, makes cells sensitive to ganciclovir or FIAU

Question 45

What are the Tet-On and Off systems

Answer 45

2 transgenes, one making a drug responsive repressor or activator protein
The other has a response element with the gene of interest

Question 46

What is the Tet-On syste

Answer 46

Four amino acid changes to TetR (repressor) alter its binding characteristics and create reverse TetR (rtTA) - thus becoming an activator

This binds the TRE (tet response element) in the presence of doxycycline and activates transcription

Question 47

What is the Tet-Off system

Answer 47

The tet-controlled transcriptional activator (tTA) is a fusion of the wild-type Tet repressor (TetR) to the VP16 activation domain (AD) of herpes simplex virus

tTA binds the Tet-responsive element (TRE) and prevents transcription in presence of tetracycline or doxycycline

Question 48

What is the Tet repressor

Answer 48

This is a starting point for inducible gene expression

Wild-type TetR is a repressor protein from bacteria - a basis for antibiotic response in bacteria

When present in the same cell TetR will bind the TRE and turn OFF gene expression

TET/DOX binds TetR, it can’t bind to the TRE = turns ON gene expression

Thus by default (no TET) gene expression is off

Binds TRE and turns off expression in absence of Tet/dox

Cannot bind TRE and activates transcription in presence of Tet/Dox

Question 49

Describe Tet-Off

Answer 49

TetR is modified to add the Trans-Activation domain from VP16 (Herpes Simplex Virus) = tTA

This tTA protein binds to the TRE and turns ON gene transcription in the absence of Tet

TET/DOX binds tTA, it can’t binding to the TRE = turns OFF gene expression
Thus by default (no TET) gene expression is on

Binds TRE and activates transcription in presence of Tet/Dox

Question 50

Describe Tet-On

Answer 50

TetR is modified to add the Trans-Activation domain from VP16
Thus by default, without binding it now deactivates promoter
AND 4 amino acids are altered to generate rtTA (reverse tTA)

Prevents TRE binding in the absence of antibiotic (Tet) keeping gene expression turned OFF

Tet permits rtTA binding to the TRE = turns ON gene expression
Thus by default (no TET) gene expression is off

Binds TRE and activates transcription in absence of Tet/Dox

Question 51

Describe the estrogen receptor pathway

Answer 51

Normally the estrogen receptor (ER) is in the cytoplasm, complexing with HSP90 (heat shock protein)
When oestrogen diffuses into the cytoplasm oestrogen binds the ER
It then dissociates from HSP90

ER then travels into the nucleus to dimerise with other ER and drive transcription of downstream target genes

This system can be adopted by taking the ER ligand binding domain, fusing it to another protein (TF) so that the protein/TF will only turn on its target in presence of oestrogen

A reporter gene can be placed downstream of the ER-responsive element

Question 52

Describe knock-out mic

Answer 52

The creation of a null (completely non-functioning) mutation for a specific gene

Sometimes used to model a human loss of function mutation/disease

Usually done by removing all coding exons, or essential exons near the 5’ end of the gene

The gene is removed in all cells of an embryo, from the beginning of development

Many null mutants are lethal
Some null mutants have no effect (redundancy)

Question 53

Describe mouse models of cystic fibrosis

Answer 53

Inactivating mutations of the CFTR introduced by insertion of exogenous gene sequences

CFTR DF508 mutations introduced by homologous recombination

CFTR null mice have a more severe phenotype than DF508 mutations
Both mutants die shortly after birth

BUT from intestinal obstruction, not lung problems!
Specific strains of Tg mice show some lung pathology under specific conditions

Question 54

Describe conditional KO

Answer 54

Many gene knockouts are (prenatal) lethal, so there is the need to control gene deletion, using recombinases to create conditional knockouts

Cre is a site-specific recombinase with a 34bp recognition site (loxP)
2 inverted (palindromic) repeats and core GCATACAT

Cre recombinase induces recombination between two loxP sites, in cis (same Chr), or in trans (diff Chr)

Effect depends on the orientation of the loxP sites
Usually used to remove intervening sequence between the loxP sites

Image in notes shows 3 possible loxP deletion patterns

A = cis/homologous Chr, same strand - cut out, forms circle which is degraded (colour image)
B = cis/homologous Chr, different strand - translocation
C = trans/non-homologous Chr - translocation

Question 55

Give some examples of recombinase enzymes

Answer 55

Cre is not the only recombinase

Cre and Flp are the most widely used recombinases and others (eg Dre, PhiC31)
Uses frt sites instead of loxP for Flp

The principle is the same - a short recognition sequence, a separate, site-specific recombinase

Question 56

Describe cre-lox transgenics

Answer 56

The region of genomic DNA is cloned into bacteria and modified then used to generate targeted ES cells by homologous recombination to generate the loxP site

Insert loxP sites into the introns, as far away from exon-intron boundaries, to flank the exon to be deleted
This is to avoid splice site interference

Targeted ES cells are used to generate a mouse line with a “floxed” (flanking loxP) allele
These mice are normal

Cross the floxed mouse with a Cre mouse (this Cre mouse is a separate transgenic animal)
Offspring thus has that cre that removes the flanked allele = target exon deleted

Expression of Cre in the cre mouse can be driven by different methods

Knocked in to an existing gene, so controlled by that gene’s promoter

Driven by a promoter of its own – this could be expressed in all tissues, or some and expressed all the time, or at specific times e.g. combined with Tet

Can be turned on/off by specific drugs that are given to the mouse e.g. tamoxifen

Wherever Cre is expressed, it recognises the loxP sites and recombination removes the floxed exon
Once recombination has occurred, that DNA is totally deleted

Question 57

How can you generate a conditional targeting construct

Answer 57

Notes = Images

Insert flanking loxP
Insertion of neomycin (antibiotic resistance) flanked by frt
Use neomycin to select cells that underwent homologous recombination
You can then remove it with Flp the frt sites by crossing with Flp mouse

Mouse > add sequence that flanks the exon target and insert frt flanked neo > select successful via neomycin > cross with Flp mouse to remove neomycin gene > cross with Cre mouse to remove exon

Question 58

How do you make cre conditional

Answer 58

A fusion protein of Cre-ERT is used (expression driven in all tissues or in specific cell types)

ERT is a modified version of the ligand binding domain of the oestrogen receptor with high affinity for Tamoxifen (= lower affinity for endogenous oestrogen)

ER is normally held in the cytoplasm by Hsp90
Addition of Tamoxifen causes Hsp90 dissociation and the Cre-ERT protein translocate to the nucleus where Cre mediates loxP recombination

Question 59

How do you perform targeted conditional gene inactivation via mouse gene KO

Answer 59

Notes = image

Gene:
Exon 1 
Selection marker - neomycin resistance gene flanked by frt & loxP1/2
Reporter gene - lacz is flanked by frt
Exon 2 - flanked by loxP 2/3

Due to the large amount of exogenous DNA, the gene is inactivated = KO FIRST

Route 1:
Select for recombination
FLP = removes selection marker + reporter gene
Cre = removes exon 2

Route 2:
Select for recombination
Cre = removes resistance gene nd exon 2
Only reporter gene remains

Question 60

Compare transgenesis V genome editing

Answer 60

The vast majority of models are mouse models, and until recently, all made by transgenesis – that is the introduction into the mouse genome of “unnatural” DNA, artificial or from another species

Recently, genome editing technology has allowed changes to be made directly to genomic DNA sequences

Question 61

What are the 3 genome editing methods

Answer 61

Zinc finger
TALENs
CRISPR-Cas9

Question 62

Describe genome editing using zinc finger

Answer 62

Protein with a DNA cutting enzyme and a DNA-grabbing region

Can be programmed to recognise different genes

Off target cuts are a problem - not very precise

Question 63

Describe genome editing using TALENs

Answer 63

Protein with a DNA cutting enzyme and a DNA-grabbing region

Can be programmed to recognise different genes - easier to design than zinc finger

Off target cuts are a problem - not very precise

Question 64

Describe genome edition using CRISPR-Cas9

Answer 64

DNA-cutting protein (cas9) guided by an RNA molecule

Cas9 works on any sequence of guide RNA as long as it has a tracer RNA
Cas9 recognises the trcrRNA and binds RNA and DNA
nduces ds-DNA breaks at a location
Cellular machinery tries to repair the clean ds-break by NHEJ
Guide RNA can add a homologus DNA sequence as a template to repair the break, thus inserting the donor template (HDR)
More precise, but less efficient

Affordable, easy to use and useful in high-throughput multi-gene experiments

Can make off-target cuts - but more specific due to guide RNA

Question 65

Describe the possible modifications to CRISPR

Answer 65

Cas9 has off-target effects

Random nuclease activity can occur throughout the genome and generate non-specific effects/mutations

Modified versions of Cas9 have fewer off-target effects

Nuclease can be modified to have a nickase activity - so it cleaves only one strand at a time
By targeting the two separate strands, you can minimise the off-target effects as you need a ds-break

Cas9 can be modified into an activator, repressor or reporter

Also modifications with altered PAM specificity allow for more precise targeting

Question 66

Describe how mouse mutants were used to study 22q11 deletion syndrome (DiGeorge)

Answer 66

Affects face, heart, neural tube, thymus and parathyroids

Two common breakpoints - typically deleted regions and DiGeorge critical region

Involves intra-chromosomal recombination at low copy repeats - mediated by homologous regions

The Df1 Mouse Mutant
A multigene deletion of a homologous, syntenic region of 22q11
Df1/+ mice have 22q11DS-like aortic arch defects
22 genes are deleted including Tbx1
Complementation (rescue) and single gene KOs identify Tbx1 as key gene for pharyngeal phenotypes

Question 67

Describe the role of TBX1 in 22q11/DiGeorge syndrome

Answer 67

TBX1in 22q11 Deletion Syndrome

Tbx1 is expressed in the pharyngeal region

Tbx1 mutations in animal models phenocopy many physical defects of DGS

BUT, heterozygous Tbx1+/- mice have a transient, embryonic phenotype

Both copies of Tbx1 have to be inactivated to produce the full spectrum of human phenotypes