Animal Models Flashcards
What is contractarianism
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
What is ultilitarianism
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
What is the relationship view
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
What is the animal rights point of view
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
What is the respect nature point of view
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
Why is animal research needed
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
Describe In vitro methods
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
What is computer modelling
Basic understanding needed before programming is possible
Limited processing power
Why are animals used
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)
Describe the ‘Animals - Scientific procedure - act 1986’
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
What is the licensing procedure for animal work
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
What is a local ethics review
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
What are the 3 R’s
Replacement - alternative techniques
Reduction - minimum number, fewer animals, more information
Refinement - better housing, improve procedures, welfare
Why are zebrafish used as an animal mdoel
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
How is zebrafish eggs collected
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
How do zebrafish develop
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
How do we study genes and disorders using zebrafish
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
Describe in situ hybridisation for gene expression analysis in zebrafish
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
Describe protein localisation via antibody detection
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
What are transgenic fish and how are they created
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
Describe zebrafish gene KO
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
Describe problems with morpholinos
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
Describe controls used in gene KO in zebrafish
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
What is ENU, and describe its use in screening in forward and reverse genetics
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
Describe CRISPR
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
Why is zebrafish good for use in drug discovery
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
Describe case study 1 -zebrafish as a chemical screen for anti-cancer drugs
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
Describe case study 2 - zebrafish in behaviour profiling
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
Describe case study 3 - zebrafish in identification of a primary target of thalidomide teratogenicity
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
Describe the pitfalls of genetic models as disease mdoels
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
Describe the constraints in using animal models as genetic models of disease
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
Describe the basis of transgenesis
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
Describe embryo manipulation
Embryo manipulation is important for transgenesis to generate a germ-line genetic modification so mutation can be passed on
Describe random transgenesis
DNA microinjection into zygotic pronucleus
Relatively quick & easy
Transient assays possible (we can look at embryos, for example)
Describe targeted transgenesis
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
Describe mouse pre-implantation development
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
Describe pronuclear injection transgenics
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)
Explain the uses of pronuclear injection transgenesis
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
Describe transgenes
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)
Describe promoter traps
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
Describe gene traps
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
Describe embryonic stem cells
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
Describe homologous recombination
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
Summarise selection strategies for ES cells
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
What are the Tet-On and Off systems
2 transgenes, one making a drug responsive repressor or activator protein
The other has a response element with the gene of interest
What is the Tet-On syste
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
What is the Tet-Off system
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
What is the Tet repressor
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
Describe Tet-Off
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
Describe Tet-On
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
Describe the estrogen receptor pathway
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
Describe knock-out mic
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)
Describe mouse models of cystic fibrosis
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
Describe conditional KO
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
Give some examples of recombinase enzymes
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
Describe cre-lox transgenics
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
How can you generate a conditional targeting construct
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
How do you make cre conditional
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
How do you perform targeted conditional gene inactivation via mouse gene KO
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
Compare transgenesis V genome editing
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
What are the 3 genome editing methods
Zinc finger
TALENs
CRISPR-Cas9
Describe genome editing using zinc finger
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
Describe genome editing using TALENs
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
Describe genome edition using CRISPR-Cas9
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
Describe the possible modifications to CRISPR
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
Describe how mouse mutants were used to study 22q11 deletion syndrome (DiGeorge)
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
Describe the role of TBX1 in 22q11/DiGeorge syndrome
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
