Mouse

1. Why are mouse such a good model to study human disease?

Mice are mammals that are similar to humans in anatomy, physiology and genetics, sharing
approximately 95% of genes common with humans.
They allow us to save space, time and money, because they are small (saving space) and docile
(saving time), have a short gestation period (saving time and money), with large litters (saving
money and time). Pure, inbred lines are easily available. Any new lines that we need to engineer
are easily done. They are accepted as the primary model for analyzing and understanding
inherited human disorders.
They have a lifespan of approximately 2 years.
They are ideal for modelling complex human diseases and for drug efficacy testing, as they share
similar immune, endocrine, nervous, cardiovascular, skeletal and other complex physiological
systems that mammals share.
Their genome is susceptible to modification, allowing us to model diseases that may not
naturally occur in mice.

2. Explain what DBA mice are and why they are an important genetic resource.
DBA mice were the frirst inbred mice strain. DBA stands for dilute brown non-agouti. As it is
inbred and pure, this allows for replication of results. If results are assimilar, this may indicate a
lack of internal validity.
3. List 5 strains of pure inbred mice
C57BL
DBA
4. Compare and contrast the differences between the mice and human genome
Mice have telocentric chromosomes, whilst humans have metacentric chromosomes.
Telocentric means that the sister chromatids are joined at the telomeres, whilst metacentric
means that they are joined in the middle of the chromosome.
There is also a difference in the number of chromosomes. Mice have 19 autosomes, and 2 sex
chromosomes. Whilst humans also have 2 sex chromosomes, we have 22 autosomes (3 more
than in mice).
5. Outline the revolution in genetic map creation over the past few decades
6. Explain how GWAS studies are conducted
GWAS studies are conduycted by looking at several SNPs (single nucleotide polymorphisms), and
testing for the association between the SNP with the disease/phenotype of interest.
GWAS are common approach for mapping disease loci and QTL in many mammals including
humans.
7. Explain the concept of synteny
Synteny refers to the physical colocalization of genetic loci on the same chromosome within an
organism. There is some synteny present between the mouse and human chromosomes, as
seen in the phtoo below
KEY METHOD AIM: To find out where new mouse mutation may lie, you can use linkage mapping

8. You have just recently found an interesting phenotype within a mouse, known as redeye. You
would like to map out where this new and novel mouse mutation would be. Name the method
you would use to find out the loci of the gene/genes involved in the redeye phenotype, and
explain how you would discover the loci of the genes involved.
I would use linkage mapping.

9. Outline a case study where linkage mapping has led to the discovery of novel genes implicated
in a specific phenotype. Make sure to state the gene involved, along with the phenotype in
question
 10. Outline how gene therapy works, with reference to a specific gene
Retinitis pigmentosa comes from retinal defeneration. There is a 10kb insertion in peripherin 2.
This insertion can lead to retinitis pigmentosa or macular dystrophy.
Gene therapy can be used to treat for this.
Nanoparticles can be used to deliver functional peripherin2 (without te 10kb insert) into the
mutant mice (which have a mutated peripherin 2 gene). When injecting into the retina at
postnatal day 5, there were two types of injections:
- CBA-NMP – a beta actin propmoter that is expressed everywhere
- IRBP-NMP – a photoreceptor specific promoter, which is eye specific.

The injection resulted in long term expression of functional Rds.

When comparing the nanoparticle injection to the controls, there was a significant difference,
which no injection, saline and naked DNA.,

KEY RESEARCH: Transgenic mice can be used to research dominant mutations, and see when and
where genes are expressd (during which developmental timepoint and in which cells/tissues).

1. What are the benefits and limitations of creating transgenic mice? When is it appropriate to
use transgenic mice?
Benefits are that it is faster to perform than complete knockouts.
Limitations is that it is slower than CRISPR, and it integrates at random places in the
genome. Furthermore, the addition of foreign DNA can induce copy number increases,
resulting in gain of function mutations, as more genes are being transcribed and translated
to proteins.
 It is only appropriate to use transgenic mice if we are modelling a dominantly acting
mutation.
2. Explain the procedure behind creating transgenic embryo
a) a zygote is isolated after fertilization, but before cell division
b) foreign DNA is injected into the zygote – this sep has tp be done before any cell
divisions, th ensure that all cells have a copy of the foreign DNA
c) the zygote is implanted into a foster mother.
d) Offspring are screened to find the mutation of interest
3. Explain the procedure behind creating a recombinant DNA vcector
Key concept- transgene is the foreign DNA that is inserted into the transgenic embryo. A
recombinant DNA vector is used, containing the structural gene of interest. In mice
experiments, this is sually a human gene carrying a dominant disease.
the construct also has to have promoter seqauences, which allow the gene to be expressed
by the host cells
reporter genes are also necessary, to identify which cells are expressing the transgene

4. Explain nthe benefits of using reporter genes

Reporter genes allow us to visualize whether a gene has been expressed in a cell. This
consequently allows us to study cell migration and development, isolate specific cells via
flow cytometry, target cells for electrophysiology and evaluate drug treatments.
5. Explain what knockout mice are
Knockout mice are genetically engineered mice where a gene is inactivated, by replacing it
or disrupting it with an artificial piece of DNA. Inactivation of a gene can cause changes in
mouse phenotype.
6. Explain how knockout mouse are created

Knockout mouse are created from the embryonic stem cells. These can differentiate into all
cell types, and are used for more precise modifications of the mouse genome. These are
crucial to create, not only knockoutmouse, but also knock ins and conditional mutants.

The ingredients for KO mouse creation are:

- DNA from a 129 mouse

- At least 4kb of unterrupted sequence on one arm ,with 2kb of uninterrupted
sequence on another arm – this is to increase the frequency at which homologoys
recombination occurs.
- Some form of double ES selection – for positive and negative selection
o Negative selection – use thymidine kinase. Thymidine kinase should be
included at the distal end of the genomic DNA construct.
The presence of thymidine kinase means that the homologous
recombination event hasn’t occurred properly (there needs to be two
 homologous recombination events). Therefore, the addition of ganciclovir
kills TK-positive cells.
The absence of TK is what we WANT (as operationalized by using
ganciclovir as well)
o Positive selection – use neomycin. Noemycin should be at the middle of the
construct. If homologous recombination has occurred, then it confers
resistance to the drug neomycin (allowing it to survive).

o
7. Describe the symptoms of Duschenne Muscular Dystrophy and how mice have been used to
find treatments or DMD
DMD is characterised by progressive muscle wasting, heart and respiratory failure as well as
early death (mid 20s – due to the cardiac muscle wasting away). It is an X-linked recessive
disorder, and caused by mutations in the DMD gene, which is very large. The mutations are
often deletions, and these deletions lead to the production of non functioning proteins. The
normal dystrophin gene is functional, and is a structural protein found in muscle fibres. It
bridges the inner cytoskeleton and the extracellular matrix, acting as a crosslinker.
Mice models have been very helpful for finding treatments for DMD, because of the various
mice models that can be created, such as mdx52, which is a mouse model for the DMD
mutation in exon52. The different models are useful, because they allow us to study various
aspects of the gene function.
Two differences between WT and mdx52 mice are the muscle fibre organisation and
dystrophin presence. In WT, dystrophin is present and the muscle fibres are organised, but
in mdx52 mice, there are disorganised muscle finbres and no dystrophin present.
To fix the mdx52 phenotype, exon51 can be deleted, restoring the reading frame. This can
be done via the systemic delivery of antisense oligonucleotides to mdx52 mice. Injection
facilitated the restoraton of dystrophin in the muscle within 2 weeks of a single coinjection,
of either 80, 160 and 320 mg/kg/dose of the antisense oligonucleotides. This was meaured
by RNA, protein, uscle pathology and function.
Based off these successful results in mice, there are considerations for human studies now.
The mice model established that thetreatment is:
- Non toxic
- Efficient DMD exon deletion and restorastion of reading frame
- Expression of RNA and protein
- Improved muscle pathology
- Imrpoved motor function
1. Why would you choose to do a knock-in experiment instead of a knockout experiment?
Knockout experiments are only reasonable if the mouse has the capability to have the mutation
naturally. However, many mice would not have the same mutation as humans do – hence we
 need to imitate the human mutation, by knocking in the specific mutation. The benefit of this is
that we get a more realisitc model of the human disease.
The strategy for a knock in is the same as for knock out – you use a positive and negative
selection agent.
2. Outline what conditional knockouts are and why you would conduct a conditional knockout
experiment
Conditional knockouts are used to knockout genes in a specific tissue and a specific timepoints.
These are useful when the tradtiional knockout would be lethal. By doing conditional knockouts,
we can see what cell types would be involved in a disease (which we otherwise wouldn’t be able
to do because the knockout would be lethal).
3. Outline the process of creating a conditional mutant
Creating a conditional mutant involves crossing two strains of mice together. One mouse strain
has to have the genes for the enzyme Cre ( a tissue specific recombinase), which is only
expressed under the control of a specific gene promoter, and the other mouse strain has to
have the gene of interest, flanked by LoxP (recombinase recognition sites).
Instead of Cre/LoxP, we could also use FLP recombinase/Frt sites. However this combination is
only mostly used to remove neo from yeast.
4. Discuss a case study where conditional mutants have been used to investigate a particular
disease
Conditional mutants have been used to investigate spinal muscular atrophy (SMA). SMA is a
neuromuscular disorder.

The homozygous loss of the SMA gene is lethal in mice, because the gene is not duplicated
like it is in humans  Hence a conditional knockout is necessary.
Experiment Aim Method Results and
conclusion
1 Prove that SMA Mouse 1 : heterozygous loxP Offspring had cre-
mutation via mouse mediated deletion
deletion of exon 7 Mouse 2: Cre under the control of exon7
is lethal of general promoter Offspring2 had no
Cross to generate offspring live mice, because
Offspring intercrossed to they were
generate more offspring2 homozygous for
Cre-mediated
deletion of exon7.
2 Investigate the Mouse 1: heteroxygous loxP Offspring had NSE-
specificity of Cre- mouse Cre-mediated
 mediated deletion Mouse 2: Cre under the control deletio nof exon 7
of exon7, and how of specific neuron promoter conditional to
it impacts upon (NSE) neurons. Lower
the motor ability Offspring measured expression of SMN
of the offspring Also looked at motor ability, by in spinal cord
using a test for the mouse to compared to
climb back onto a rotating pole muscle.
However, Cre was
also found in
kidney – indicating
the ‘leakiness’ of
the Cre promoter.
Offspring found it
harder to get back
onto the rotating
pole.
3 Looking at the Looked at the AchR presence, as In mutants – we
skeletal muscle a marker for the neuromuscular can see AchR,
pathology to see if junction. howevrm they are
there is any not organised well
phenotypical like they would be
changes from the in the WT
deletion of exon7
4 Look at the spinal Same as exp 2, then staining SMA mutants have
cord pathology at conducted the hallmarks of
2 weeks to see the apoptosis.
differences At four weeks,
ebtween WT and there is
the SMA mutants fragmentation of
the neuron nucleus
5 Staining with an antobody In WT, the stain
showed coiled
bodies within the
cell, but in the
mutants, these
coiled bodies wrre
absent.
5. Explain the benefits of the inducible gene expression and how it is usually conducted.
Inducible gene expression is useful because it facilitates a temporal control over the conditional
knockout, not just a spatial control like the normal protocol allows.
The method by which this occurs is via fusing Cre to a mutant estrogen hormone which binds
Tamoxifen (instead of estrogen). The binding of the tamoxifen would then activate Cre, provided
that the Cre is under the control of a promoter.
This is a rapid and irreversible change in gene expresson.

1. Describe what complex traits are and how they are investigated
 Complex traits are traits that are mediated by several genes, and the interaction between these
genes’ loci. To investgate them, you would use genome-wide methods, such as GWAS. Genome
wide methods allow for the mapping of the inheritance of complex traits in non-model
organisms. Exampls of compelx traits includes obesity, height, alcoholism, puberty. The goal
during compelx traits analysis would be to have many participants, to get a wide range
Complex traits can be influenced by many things, inclyuding genetics, environment. And hence
there is an equation we can use to model the complex traits
𝑝ℎ𝑒𝑛𝑜𝑡𝑦𝑝𝑒 = 𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 + 𝑔𝑒𝑛𝑒𝑠 + 𝑒𝑟𝑟𝑜𝑟
Measuring genes:
- We can use SNPs to build a relationship matrix. Traditionally we would use family,
pedigrees or twin studies. But currently its being powered by genomic tools. SNPS
are useful because they can be used to estimate the genetic similarity between
individuals and pinpoint any differences.
- GWAS – can be used to ifentify key SNPs (risk alelles with may predispose towards a
certain disease).

Hence we can modify the equation to be

𝑝ℎ𝑒𝑛𝑜𝑡𝑦𝑝𝑒 = 𝑒𝑛𝑣𝑖𝑟𝑜𝑛𝑚𝑒𝑛𝑡 + 𝑔𝑒𝑛𝑒𝑠 + 𝑆𝑁𝑃 𝑒𝑓𝑓𝑒𝑐𝑡 + 𝑒𝑟𝑟𝑜𝑟

2. How would you see the effect of SNPs?

SNP2 clearly has a relationship, whilst SNP1 doesn’t.

3. Using a case study of puberty, discuss how we can find key results for complex traits by using
genome wide analyses
Puberty is a complex trait, as the age of puberty varies. However, there are a few things that
play a role I nthe onset of puberty, such as leptin and BMI.
Coussminer et al. investigatred puberty in humans:

- Method = GWAS. They matched phenotype to the genotype. They measured the
pehnotype by using early puberty traits, such as via the Tanner scale (which rates it
off visual body changes), the age of menarche and pubertal growth (BMI, weight
and height). They measured genotypes by using SNPs from a high density chip
inputed to sequence data. They then conducted a linear regression model per
cohort. PCA for general genetic factors (to see for genetic similarity) and age when
the phenotype was obsertved.
- Results = different for male versus female
o male
 discovery of an SNP involved – SNP near the MKL2 gene
o female
 confirmed an SNP involved – an SNP near the LIN28B gene
 higher BMI linked to early puberty

fortes et al. investigated puberty within cattle

- method = GWAS
o phenotypes = weight, height, age at puberty (onset of puberty measured by
first ovulation in COWS and scrotal circumference in BULLS)
o genotypes – used 770,000 SNPs using illumina chip technology
 - results
o top SNP was located at the PLAG1 gene

1. Using the case study of alcohol preference, explain how you could find QTLs associated with
alcohol consumption using mice
2. Forward genetics – how could you use this in mice?
C. elegans
1. what features of C. elegans lends well to genetic analysis?
It is a transparent organism, allowing easy visualization of various systems.
Life cycle is short – this has benefits for time and money
Small – easy to store
Lends itself well to neuroscience because it has small neuron numbers but these neurons are
capable of so much.
Development is very stereotypic
Hermaphrodite, with low numbers of males in WT – this allows for preservation of genetic
background, instead of introduction of genetic variety. This is because the hermaphrodite
inbreeds.
2. Explain why the hermaphroditic nature of C. elegans is so important
The hermaphroditic nature of C. elegans is important because it allows for the preservation of
genes, as the hermaphrodites have to inbreed.
3. Outline how males are created
Males are created by a nondisjunction, and they consist of 1% of the C. elegans population
4. Outline a forward genetics approach to doing mutant screens in C elegans,
a) L4 stage worms are treated with EMS
b) Their offspring are hatched, and self-mated to produce homozygous offspring
c) The mutant phenotype of desire should be identified in F2
5. Describe the genome size
6. Microscopy can be used in C. elegans. Design an experiment investigating the role of the
movement gene, by using microscopy methods, along with other research techniques.
7. If you wanted investigate a gene’s function in C. elegans (i.e. using a reverse genetics approach),
what approach should you take? Note that there are many answers to this question.
Cell ablation
Mutant creation and million mutant project – using the million mutant project, you can order
different mutants that you are interested in
Transformation – you can inject the gene into the worm and see if the progeny takes up the
DNA. There is a high inheritance rate, which is a benefit of this method, however, this
 inheritance rate is not 100%. To make sure you know which progeny has taken up the gene, you
can inject a gene with a fluorescent marker to vusualise it.
Gene expression – lots of techniques are available here, such as:
- Microarray/RNA seq
- Quantitative rreal time PCR
- Distribution of GFP labelled tranbsgenes
- Metabolomics
- Insitu hybridization
RNAi – RNAi can be used to knockdown and strongly suppress genes. This can be done in C
elegans by injecting RNAi OR by feeding RNAi-filled E.coli to C. elegans worms.
Gene mapping – deep sequencing of outcrossed mutant lines. The process involves comparing
the outcrossed mutanrt lines to the non mutated reference sequences. It works well due to a
lack of heterozygosity.
Wormbase – wormbase is a digital repository of the knowledge and data that describes C.
elegans. It has good links to literature, and is a good place to find out more information if youre
interested in seeing if a gene is linked to C elegans already.
8. Explain the method involved in gene mapping
a) Divergent strains are crossed
b) The divergent strains are inbred for several generations
c) Interesting phenotypes are selected for
d) Deep sequencing reveals regions of the genome that contribute to the selected trait.

1. Explain what gasotransmitters are, with reference to edxamples and their function/
Gasotransmitters are small signaling molecules that can also act as toxic gases. There are three
main gasotransmitters – CO2, H2S and NO.
The functions of these gasotransmitters vary
NO H2S CO
Function Mediates vascular Mediates vascular
dilation dilation
Acts as a Acts as a
neuromodulator neuromodulator
PARTICIPATES in Mediates defence
defence against against pathogens
pathogens
Induces suspended Induces suspended
animation in animation in
mammals/invertebrates mammals/invertebrates
Protexts mammalian
organs for transplant
Activates defence
against severe hypoxia
2. Explain the cellular processes that occur during mild hypoxia, severe hypoxia and anoxia in C.
elegans adult worms.
Consider aerobic respiration, glycolysis, the presence of antioxidant enzymes and mobility levels
During mild hypoxia aerobic respiration decreases. This correlates with an increase in glycolysis
(which is expected, because of the lack of O2 present). The levels of antioxidant enzymes
increase, to anti-age, and the mobility levels are near norma.;
During severe hypoxia, the C. elegans dies
During anoxia, energy metabolism decreases, along with mobility levels
3. E xplain how development is impacted by varying O2 levels.
Mild hypoxia – full growth of the embryos
Severe hypoxia – the embryos are aborted. The stage of abortion varies, depending on how low
the O2 levels are, at various times
Anoxia – embryos are arrested and prevented from moving down to the vulva.
4. Explain how CO impacts upon development during O2 deprivation
During O2 deprivation, CO acts as a promoter for survival under lethal hypoxia.
5. Discuss how CO and hif1 interplay with each other to impact upon worm survival
CO serves as a primary modulator of worm survival, and hif1 serves as a back up modulator of
worm survival. If CO is present, hif1 is not necessary to be activated, because the CO will protect
the worm, by inducing diapause.
The O2 restriction level required to induce diapause varies between embryos and adults.
Embryos will enter diapause under severe hypzia (1000ppm), whilst adults enter diapause at
anoxia.
6. Discuss the factors involved in inducing embryonic diapause.
There appears to be two facfors impacting upon embryonic diapause – the environemnt the
embryo is in, the presence of san1. hif1.
The environment – embryos were more likely to be viable in the in-utero environment,
compared to when they were hatched outside
San1 – san1KO embryos were less likely to be viable than worms with san1 present.
Hif1 – hif1 KO embryos were not different in terms of emvryo viability. n
7. Say you wanted to investigate if a gene is implicated in the light sensitivity of the worm. How
would you co about investigating if the gene is implicated in this process?
Use a transgene
- Assay – score for fluorescence (because the transgene of interest will be cojoined
with a fluorescent marker). Then compare the number of cciable embryos beween
worms with the transgene and worms without the transgene.
- Ensure that you have good controls – by pairing it up with a control where the
transgene is under the control of say a msucle promoter, instead of a uterus specific
promoter.
- Results
o If muscle cells – hif1 not implicated
o If neural cells – hif1 involved in increasing embryo numbers in the uterus \
8. Compare and contrast hif1 mutants with the wild type, under mild hypoxia, severe hypoxia and
anoxia
WT Hif1 mutant
 Mild hypoxia – Increased glycolysis, Embryonic diapause
DIFFERENT EFFECT decreased aerobic and decreased
respiration, mobility mobility levels
levels near normqal,
increased antioxidant
enzymes
Severe hypoxia – Embryonic diapause Embryonic diapause
SAME EFFECT Decrease in mobility Decrease in mobikity
levels levels
Anoxia - SAME All energy mtabolism All energy
EFFECT decreases metabolism
Mobility levels decrease.
decrease Mobility levels
decrease
Lack of hif1 hence embulates more serious/deficient O2 level conditions

1. How do organisms recognise pathogens?

There are three different ways:
- PAMP - pathogen associated molecular patters
- DAMP - damage associated molecular patterns
- cSADD – cellular surveillance-activated detoxification and defence
surveillance pathways monitor core cellular activities, and senses whatever is
critical to the organisms’ function. If there is a disruption to the normal
surveillance, the organism takes this as a pathogen attack, and starts initiating
immune and detox responses, as well as aversion behaviour. This explains why
worms can remember the quality of the food.
2. Let’s say you want to investigate food aversion in C. elegans. How would you do this?
Via an aversion assay.
a. Use RNAi
i. CONTROL = empty RNAi vector
ii. EXPERIMENTAL = elt2 RNAi
b. Add the worms to a medium with bad quality food
c. Score the number of C elegans on the food medium, over the total number of C elegans.

Instead of food aversion, we can use the food aversion assay for toxins, therapeutic drugs
and pathogens.

We can observe in all cases, a xenobiotic response which is designed to contradict and
prevent further infection.

Another experiment

- Grew the C elegans

- Transfer to four test plates
o Bad quality + RNAi to KO the aversion gene  AVERSION
 o Bad quality + no RNAi – this is our control, because we expect the presence
of the aversion gene since there is no RNAi. - AVERSION
o OP50 – good qualitty food  NO AVERSION
o HB101- good quality food NO AVERSION
- As the E. coli learned that the HT115 was bad, regardless of if tge hsp60 gene was
knocked out, it shows that the C elegans can learn!

If you want to investigate therole of a gene in a particular location, you can suppress genes in
location-specific manner via RNAi under the control of specific promoters.

For example we can investigate rde-1, and see that it is specifically for the hypodermus. Genetically
engineeering this prevents us from having to abalte cells

3. Describe the evidence showing that serotonin is linked to food aversion response.
Inhibited serotonin using RNAi.
Investigated how serotonin inhibition would affect mutants of all different kinds,
Regardless of the type of mutatuo, impairment of 5HT lkeads to impaired food response.
THEREFORE serotonin is a neuromodulator of feeding
4. To increase confidence that your results are ‘real’, what are some possible things you could
implement in your experiiment?
Include controls
Investigate paralogs of the gene of interest. This tests a bit broadly, if we can see that the
paralog has no effect, it gives us greater confidence that the gene response is real, and not a
response based off a familial response.
In general just ttest more broadly to make sure that your results are specific for the area of
interest, and not based off whole-body response or the operationalisation method.

1. How could you monitor gene expression in a temporal and spatial manner?
You could use GFP, under the control of a specific promoter.when you activate the
promoter, this gives you temporal control over GFP expression. The specificity of the
promoter enables a spatial control.
2. How could you investigate if genes are coexpressed together?
Create a GFP reconstitution system.
GFP is split into two systems, with a zipper peptide attached to each GFP domain.
When both domains are expressed in the same cell, the zipper protein zips thw two GFP
inactive domains together and activates it, allowing or fluorescence.
Therefore wheneber you see GFP, you know that coexpression is occuring, between the
genes with the GFP domain are being expressed.
You would use this coexpression experiment to first show proof that there may be an
interaction between two genes
Limitations of this approach would be that the GFP tends to be unstable, and the
fluorescence tends to fade with time – so you cant do long term studies with this!
Create a reconstituted caspase system
This is the same protocol as the GFP, however, whenever coexpression occurs, cell death
occurs, because caspase is implicated in cell death. We can use the heatshock promoter,
because it gets turned on everywhere.
 3. When would you use tissue specific RNAi?
If you wanted to performe tissue specific gene suppression.
The rde1 gene is crucial for facilitating RNAi. If it is absent, the organism cannot do RNAi,
and gene suppression is impossible,
Therefore, to ensure that there is tissue specific gene suppression, we can get an organism
which is rde-1 mutant – meaning that it doesn’t have proper rde1 in its body and its whole
body cannot do RNAi. We grab this mutant, and we insert rde1 under the control of a tissue
specific promoter.
This means that rde1 WT will be in specific tissues that we control (with the use of a
promoter), and that we can do targeted gene suppression.
4. CRISPR
Targeted gene modification, based off bacterial immunity.
5. Optogenetics
Use light sensitive proteins, such as ion channels to sensitise or inhibit neurons when
exposed to light. Light specific proteins have beeb linkedtowards behavioural changes.
We can genetically engineer neurons with a light sensitive switch, and induce a transgene to
express a light sesntiive protein in a target neuron.muscle cell to activate it when exposed to
light,

1. Explain why conserved genes are sequestered at the middle of a chromosome, whilst variable
genes are present at the ends of a chromosome.
the genes at the end of a chromosome are more prone to being lost due to cell replication, or
mutated, as they are at the telomeric ends. The chromosome ends are perhaps the cutting ede
of adaptation and evolution. Furthermore, the existing configurations of gene sequences in the
middle of the chromosome are already optimal
2. Dexcribe the three gene classes

Genes can vbe expressed in different ways, with different regulatory mechanisms

a) Constitutive – these are highlky expressed. I would assume (due totheir high expression)
that their promoters have low mutation rates, as they are already at optimal capacity
b) Differential regulation between genotypes – low expressionm, and high promoter mutation
rate
c) Environemntall regulated – low expression, intermediate promoter mutation rate

1
-
C. elegans type of questions:

 Previously characterized mutation that you want to find if it is linked to a disease

Order the mutant from the C elegans genetic stock centre. Assay it for the disease of interest.
For example, if it s a metabolic disease, assay for the fat content, muscle content, etc.
 Uncharacterized mutation that you want to find if it is linked to a disease
If its based off a human model, you can inject in the DNA including the mutation of interest.
Confirm the validity of the worm model
If it is just a ranom model
 Uncharacterized mutation that you want to find if it plays a role in mediating a
disease/response, along with another mutation
Do a cross with your mutation of interest and the other characterized mutation. Observe if the
WT phenotype is restored – if restoration occurs, they are on different loci.
To determine if it plays a role in a disease/response, look at the F1 progeny, with the parental
strains, and see if the response to the disease is differential (i.e. not the sum of the parental
strains – either greater or lesser).
 Isolating where a previously characterized mutation is expressed in C. elegans
Can use reconstituted GFP
Or link GFP to a promoter for the mutation
 Youre given the results of a study and asked to interpret it
e.g. you have C ekegans with an uncharacterized mutation from the million mutant project.
When you exposed them to anaesthesia, 95% of the C .elegans were still mobile. Suggest
explanations to explain this phenomena, and ways to confirm if the phenomena is due to the
uncharacterized mutation
the million mutant project contains mutants with all sorts of mutations. So the results could be
due to a single mutation, or perhaps multiple mutations complementing each other.
To determine if the phenomena is solely due to the uncharacterized mutation, we could
generate a C. elegans worm with only the mutation of interest via transforming it with the DNA
for the mutation of interest. As transformation is not 100% effective, we would have to make
sure that the DNA construct also contains a fluorescent marker to identify the worms that have
taken up the construct.
Then, we could assay the C. elegans and observe if 95% of the C. elegans were still mobile. If
there are, then this suggests that the resistance to anaesthesia effect is due to this mutation.
To confirm your results, you can inject the cDNA back into the C. elegans and observe if the
numbers of mobile C. elegans significantly decrease after anaesthesia exposure.