GEN3001_Workshop 3

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S… Speaker 1 (GEN3001_Workshop 3)

Thank you.

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S… Speaker 1 (GEN3001_Workshop 3)

Thank you.

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S… Speaker 2 (GEN3001_Workshop 3)

okay um hi morning um

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S… Speaker 2 (GEN3001_Workshop 3)

You all know where to get your materials,

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S… Speaker 1 (GEN3001_Workshop 3)

right?

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 3 (GEN3001_Workshop 3)

very good.

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S… Speaker 1 (GEN3001_Workshop 3)

We

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S… Speaker 2 (GEN3001_Workshop 3)

have two sessions today,

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S… Speaker 2 (GEN3001_Workshop 3)

correct?

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S… Speaker 2 (GEN3001_Workshop 3)

Later on,

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S… Speaker 2 (GEN3001_Workshop 3)

I will upload the workshop number four.

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S… Speaker 2 (GEN3001_Workshop 3)

But maybe we might not...

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, we'll see how it goes.

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S… Speaker 2 (GEN3001_Workshop 3)

We might not have enough time to do the activity,

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S… Speaker 4 (GEN3001_Workshop 3)

but okay.

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S… Speaker 2 (GEN3001_Workshop 3)

Starting from this

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S… Speaker 2 (GEN3001_Workshop 3)

topic, we are going to work on...

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S… Speaker 2 (GEN3001_Workshop 3)

organisms or rather rather than a study on organisms.

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S… Speaker 2 (GEN3001_Workshop 3)

So the first one that you see here today is zebrafish.

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S… Speaker 2 (GEN3001_Workshop 3)

The one in the afternoon will be on

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S… Speaker 2 (GEN3001_Workshop 3)

drosophila.

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S… Speaker 3 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so you will be introduced to the models and how some genetic approaches

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S… Speaker 2 (GEN3001_Workshop 3)

are applied to the models.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so for this topic,

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S… Speaker 2 (GEN3001_Workshop 3)

our first part,

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S… Speaker 2 (GEN3001_Workshop 3)

we are going to focus on some of the more

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S… Speaker 2 (GEN3001_Workshop 3)

straightforward approaches.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so I will introduce you to two of those.

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S… Speaker 2 (GEN3001_Workshop 3)

We have forward,

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S… Speaker 2 (GEN3001_Workshop 3)

we have reverse,

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S… Speaker 2 (GEN3001_Workshop 3)

genetics.

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S… Speaker 2 (GEN3001_Workshop 3)

And then we are going to look at examples of forward genetic

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S… Speaker 2 (GEN3001_Workshop 3)

approaches.

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S… Speaker 2 (GEN3001_Workshop 3)

So we have GWAS,

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S… Speaker 2 (GEN3001_Workshop 3)

we have whole exome sequencing.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so these are two examples of forward genetics.

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S… Speaker 2 (GEN3001_Workshop 3)

And then later we will see how we can apply it to our

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S… Speaker 2 (GEN3001_Workshop 3)

animal models.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so to start,

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S… Speaker 2 (GEN3001_Workshop 3)

Right, I have a very cute image here.

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S… Speaker 2 (GEN3001_Workshop 3)

So this one,

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S… Speaker 2 (GEN3001_Workshop 3)

okay, gives you an idea about what is forward,

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S… Speaker 2 (GEN3001_Workshop 3)

what is reverse.

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S… Speaker 2 (GEN3001_Workshop 3)

So in a forward genetic,

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S… Speaker 2 (GEN3001_Workshop 3)

if you follow the arrow,

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S… Speaker 2 (GEN3001_Workshop 3)

we already know what the phenotype is.

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S… Speaker 2 (GEN3001_Workshop 3)

But we don't know the underlying genes that causes this phenotype.

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S… Speaker 2 (GEN3001_Workshop 3)

So we are going to apply things like GWAS,

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S… Speaker 2 (GEN3001_Workshop 3)

right, because we know about the phenotype of people.

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S… Speaker 2 (GEN3001_Workshop 3)

and then fish out what are the genes that may have been associated

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S… Speaker 2 (GEN3001_Workshop 3)

to the phenotype.

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S… Speaker 2 (GEN3001_Workshop 3)

So that's forward genetics.

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S… Speaker 2 (GEN3001_Workshop 3)

Similarly,

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S… Speaker 2 (GEN3001_Workshop 3)

you can also use your whole exome sequencing to

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S… Speaker 2 (GEN3001_Workshop 3)

determine what is the sequences of the genes that causes

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S… Speaker 2 (GEN3001_Workshop 3)

that particular phenotype.

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S… Speaker 2 (GEN3001_Workshop 3)

Then you will be able to fish out that causal gene.

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S… Speaker 2 (GEN3001_Workshop 3)

And then the reverse is also true,

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S… Speaker 2 (GEN3001_Workshop 3)

it's just the opposite.

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S… Speaker 2 (GEN3001_Workshop 3)

Here,

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S… Speaker 2 (GEN3001_Workshop 3)

you know about the genes,

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S… Speaker 2 (GEN3001_Workshop 3)

information about the genes.

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S… Speaker 2 (GEN3001_Workshop 3)

But you don't know what kind of

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S… Speaker 2 (GEN3001_Workshop 3)

phenotype you get.

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S… Speaker 2 (GEN3001_Workshop 3)

So you're going to apply reverse genetics approach for example.

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S… Speaker 2 (GEN3001_Workshop 3)

You can apply CRISPR.

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S… Speaker 2 (GEN3001_Workshop 3)

You can apply zinc finger nucleases.

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S… Speaker 2 (GEN3001_Workshop 3)

You can apply morpholinos,

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S… Speaker 2 (GEN3001_Workshop 3)

which we will look at later on.

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S… Speaker 2 (GEN3001_Workshop 3)

that will help you determine what kind of phenotype you get from applying those

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S… Speaker 2 (GEN3001_Workshop 3)

approaches on the genes.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so if you can remember this very cute picture,

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S… Speaker 2 (GEN3001_Workshop 3)

then you will have no problem understanding what is forward,

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S… Speaker 2 (GEN3001_Workshop 3)

what is reverse.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so I'm going to start off with forward genetics,

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S… Speaker 2 (GEN3001_Workshop 3)

looking at GWAS as an example.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so in a GWAS study,

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S… Speaker 2 (GEN3001_Workshop 3)

you guys know that in GWAS,

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S… Speaker 2 (GEN3001_Workshop 3)

we are looking at population level studies.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, so what does population mean to you?

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S… Speaker 2 (GEN3001_Workshop 3)

It means that it is a large -scale study.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so to deploy this large -scale study,

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S… Speaker 2 (GEN3001_Workshop 3)

there must be certain study design that is involved,

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S… Speaker 1 (GEN3001_Workshop 3)

right?

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S… Speaker 2 (GEN3001_Workshop 3)

So for the study design,

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S… Speaker 2 (GEN3001_Workshop 3)

we will need to recruit participants.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so it's very important we get participants to do this study.

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S… Speaker 2 (GEN3001_Workshop 3)

So one of our group of participants will be what we

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S… Speaker 2 (GEN3001_Workshop 3)

call controls,

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S… Speaker 2 (GEN3001_Workshop 3)

those that we determine them to be healthy.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, and then those that are also known on the other group here

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S… Speaker 2 (GEN3001_Workshop 3)

called the cases.

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S… Speaker 2 (GEN3001_Workshop 3)

So these are the people with the disease.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so I put inverted comma healthy because there

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S… Speaker 2 (GEN3001_Workshop 3)

is no such thing as 100 % healthy.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

but in comparison with the cases,

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S… Speaker 2 (GEN3001_Workshop 3)

these are people who are so -called healthy individuals.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now you will also need to bear in mind that

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S… Speaker 2 (GEN3001_Workshop 3)

when you conduct a GWAS study,

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S… Speaker 2 (GEN3001_Workshop 3)

you have to take note of the following factors.

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S… Speaker 2 (GEN3001_Workshop 3)

For example,

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S… Speaker 2 (GEN3001_Workshop 3)

things that need to be controlled are age.

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S… Speaker 3 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so you need to select your cases,

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S… Speaker 2 (GEN3001_Workshop 3)

your controls of around the same age group and you need to vary

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S… Speaker 2 (GEN3001_Workshop 3)

the age group.

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S… Speaker 2 (GEN3001_Workshop 3)

Otherwise,

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S… Speaker 2 (GEN3001_Workshop 3)

it will be a very biased study.

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S… Speaker 2 (GEN3001_Workshop 3)

You also need to factor in gender.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, because you cannot only select for one gender and ignore the other.

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S… Speaker 5 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

and then the last one,

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S… Speaker 2 (GEN3001_Workshop 3)

which is also quite important,

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S… Speaker 2 (GEN3001_Workshop 3)

will be ethnicity.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

this one also has to be factored in.

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S… Speaker 1 (GEN3001_Workshop 3)

Right,

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S… Speaker 2 (GEN3001_Workshop 3)

so if we talk about population,

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S… Speaker 2 (GEN3001_Workshop 3)

let's say I name a population here,

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S… Speaker 2 (GEN3001_Workshop 3)

I call it an Asian population,

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S… Speaker 2 (GEN3001_Workshop 3)

and we are trying to study maybe type 2 diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

For example,

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S… Speaker 2 (GEN3001_Workshop 3)

our cases are people with type 2 diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

Our controls are people without type 2 diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

But it doesn't mean that these people who

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S… Speaker 2 (GEN3001_Workshop 3)

are controlled won't have predispositions.

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S… Speaker 2 (GEN3001_Workshop 3)

For example,

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S… Speaker 2 (GEN3001_Workshop 3)

they could be people who are obese that may drive type 2

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diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

But because at that point when you recruit them,

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S… Speaker 2 (GEN3001_Workshop 3)

they don't have type 2 diabetes,

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S… Speaker 2 (GEN3001_Workshop 3)

we will still consider them as controlled.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, so in my Asian population,

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S… Speaker 2 (GEN3001_Workshop 3)

when I talk about ethnicity,

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S… Speaker 2 (GEN3001_Workshop 3)

that means that I will need to recruit,

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S… Speaker 2 (GEN3001_Workshop 3)

let's say,

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S… Speaker 2 (GEN3001_Workshop 3)

my Chinese Asians,

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S… Speaker 5 (GEN3001_Workshop 3)

right?

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S… Speaker 2 (GEN3001_Workshop 3)

Chinese Asians can range from a very big group.

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S… Speaker 2 (GEN3001_Workshop 3)

I can have my Taiwanese,

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S… Speaker 2 (GEN3001_Workshop 3)

I can have my Singaporeans,

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S… Speaker 2 (GEN3001_Workshop 3)

I can have my Malaysians,

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S… Speaker 1 (GEN3001_Workshop 3)

right?

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S… Speaker 2 (GEN3001_Workshop 3)

So I need to factor in ethnic groups as well when

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S… Speaker 2 (GEN3001_Workshop 3)

I consider Asian.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now one of the important things here is that because you're going to

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S… Speaker 2 (GEN3001_Workshop 3)

group many, many people together to do this study,

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S… Speaker 2 (GEN3001_Workshop 3)

you will get lots of data.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

now from the data,

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S… Speaker 2 (GEN3001_Workshop 3)

what can you find out?

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S… Speaker 2 (GEN3001_Workshop 3)

You can find out about individual down to the basis.

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S… Speaker 2 (GEN3001_Workshop 3)

So we can use SNPs to inform us about any slight changes

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S… Speaker 2 (GEN3001_Workshop 3)

between the disease versus non -disease.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now we will be able to identify

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S… Speaker 2 (GEN3001_Workshop 3)

variants in the

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genes.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so what do I mean by variants?

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S… Speaker 2 (GEN3001_Workshop 3)

For example,

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S… Speaker 2 (GEN3001_Workshop 3)

if I have a gene here,

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S… Speaker 1 (GEN3001_Workshop 3)

okay,

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S… Speaker 2 (GEN3001_Workshop 3)

let's say this is the wild type.

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S… Speaker 2 (GEN3001_Workshop 3)

alleles, a TT,

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for somebody who is controlled without the disease.

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S… Speaker 2 (GEN3001_Workshop 3)

Now,

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S… Speaker 2 (GEN3001_Workshop 3)

in comparison to somebody who is a case,

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S… Speaker 2 (GEN3001_Workshop 3)

I will probably have a variant,

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S… Speaker 2 (GEN3001_Workshop 3)

let's say an AA.

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S… Speaker 2 (GEN3001_Workshop 3)

So it's a variant because it is no

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S… Speaker 2 (GEN3001_Workshop 3)

longer like the wild -type alleles.

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S… Speaker 2 (GEN3001_Workshop 3)

So this is what your GWAS will be able to pick down,

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S… Speaker 2 (GEN3001_Workshop 3)

down to the individual nucleotide sequence.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now it sounds like it's very good,

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S… Speaker 2 (GEN3001_Workshop 3)

right? You can recruit many people,

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S… Speaker 2 (GEN3001_Workshop 3)

you have a lot of data,

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S… Speaker 2 (GEN3001_Workshop 3)

you can pin through those layers of the data.

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S… Speaker 2 (GEN3001_Workshop 3)

However,

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S… Speaker 2 (GEN3001_Workshop 3)

one of the more important thing here to consider is that it

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S… Speaker 2 (GEN3001_Workshop 3)

can be very costly because you're getting so many people,

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S… Speaker 2 (GEN3001_Workshop 3)

you have to pay for them or at least provide an incentive to recruit these people.

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S… Speaker 2 (GEN3001_Workshop 3)

You have to hire very expensive bioinformaticians to help

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S… Speaker 2 (GEN3001_Workshop 3)

you layer down all these different data sets.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now the other important thing is to consider also

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S… Speaker 2 (GEN3001_Workshop 3)

the fact that you are just looking at associations,

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S… Speaker 2 (GEN3001_Workshop 3)

right,

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S… Speaker 2 (GEN3001_Workshop 3)

between your variants.

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S… Speaker 2 (GEN3001_Workshop 3)

and your disease state,

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S… Speaker 2 (GEN3001_Workshop 3)

example like the one I told you about type 2 diabetes,

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S… Speaker 2 (GEN3001_Workshop 3)

you may have come across many different types of genes that may be associated

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S… Speaker 2 (GEN3001_Workshop 3)

with type 2 diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

For example,

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S… Speaker 2 (GEN3001_Workshop 3)

genes that are related to obesity,

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S… Speaker 2 (GEN3001_Workshop 3)

genes that may cause transcription factor increase in

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the level of let's say cholesterol and all that.

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S… Speaker 2 (GEN3001_Workshop 3)

This is only just purely by association,

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S… Speaker 2 (GEN3001_Workshop 3)

but it is not the same as co -relation.

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S… Speaker 1 (GEN3001_Workshop 3)

Sorry,

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S… Speaker 2 (GEN3001_Workshop 3)

not the same as causal relationship.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so we cannot drill down to those variants that shows up a difference in

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S… Speaker 2 (GEN3001_Workshop 3)

our control to our cases.

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S… Speaker 2 (GEN3001_Workshop 3)

And so then,

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S… Speaker 3 (GEN3001_Workshop 3)

okay,

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S… Speaker 2 (GEN3001_Workshop 3)

I have variants 1,

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S… Speaker 2 (GEN3001_Workshop 3)

2,

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3.

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S… Speaker 2 (GEN3001_Workshop 3)

All these are the differences I get.

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S… Speaker 2 (GEN3001_Workshop 3)

between my case and my control.

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S… Speaker 2 (GEN3001_Workshop 3)

And I say that these differences are what drives

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S… Speaker 2 (GEN3001_Workshop 3)

type 2 diabetes.

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S… Speaker 2 (GEN3001_Workshop 3)

You cannot say this.

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S… Speaker 2 (GEN3001_Workshop 3)

What you can say is that they are probably related

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S… Speaker 2 (GEN3001_Workshop 3)

to the disease.

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S… Speaker 2 (GEN3001_Workshop 3)

So you cannot nail down the

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S… Speaker 2 (GEN3001_Workshop 3)

actual disease -causing gene in a GWAS because it's

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S… Speaker 2 (GEN3001_Workshop 3)

a lot of people.

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S… Speaker 2 (GEN3001_Workshop 3)

And all these people,

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S… Speaker 2 (GEN3001_Workshop 3)

they are not related.

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S… Speaker 2 (GEN3001_Workshop 3)

The only thing that they have in common is either they have the disease or they don't

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S… Speaker 2 (GEN3001_Workshop 3)

have the disease.

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S… Speaker 2 (GEN3001_Workshop 3)

That's all you can say about them.

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S… Speaker 2 (GEN3001_Workshop 3)

And you've controlled them for the different varying ages.

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S… Speaker 2 (GEN3001_Workshop 3)

You've controlled for different gender groups.

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S… Speaker 2 (GEN3001_Workshop 3)

You have also controlled them based on their ethnic groups.

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S… Speaker 2 (GEN3001_Workshop 3)

So they are not related by blood.

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S… Speaker 2 (GEN3001_Workshop 3)

So the best you can do is to say that the differences you see

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S… Speaker 2 (GEN3001_Workshop 3)

that you have obtained from the results is purely just because they are

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S… Speaker 2 (GEN3001_Workshop 3)

associated.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, now if you want to know the actual cause of the disease,

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S… Speaker 3 (GEN3001_Workshop 3)

right,

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S… Speaker 2 (GEN3001_Workshop 3)

what do you do?

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S… Speaker 2 (GEN3001_Workshop 3)

You take those causal variants like your 1,

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S… Speaker 2 (GEN3001_Workshop 3)

2, 3 over here,

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S… Speaker 2 (GEN3001_Workshop 3)

then you need to do individual pedigree studies.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so what is pedigree studies?

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S… Speaker 2 (GEN3001_Workshop 3)

You study people who are related by blood,

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S… Speaker 1 (GEN3001_Workshop 3)

right,

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S… Speaker 2 (GEN3001_Workshop 3)

within a family.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

and then you can nail down to which one is the one that caused the disease

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S… Speaker 2 (GEN3001_Workshop 3)

in that family.

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S… Speaker 2 (GEN3001_Workshop 3)

Okay, so this is GWAS.

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S… Speaker 2 (GEN3001_Workshop 3)

And then if we look at the other example of our forward

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S… Speaker 2 (GEN3001_Workshop 3)

genetics, here we have whole exome sequencing or

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S… Speaker 2 (GEN3001_Workshop 3)

WES for short.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

now the name here would have already given you

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S… Speaker 2 (GEN3001_Workshop 3)

a clue what you are trying to analyze.

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S… Speaker 2 (GEN3001_Workshop 3)

So exome,

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meaning the compilation of all the exomes from a single

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person.

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S… Speaker 2 (GEN3001_Workshop 3)

So we are looking at only exonic sequences.

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S… Speaker 1 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

we will ignore the introns.

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S… Speaker 5 (GEN3001_Workshop 3)

Okay,

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S… Speaker 2 (GEN3001_Workshop 3)

so from the axons,

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S… Speaker 2 (GEN3001_Workshop 3)

these are your information that will be made into your

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messenger that will be made eventually into the proteins.

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S… Speaker 2 (GEN3001_Workshop 3)

But at the level,

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S… Speaker 2 (GEN3001_Workshop 3)

this is what we want,

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S… Speaker 2 (GEN3001_Workshop 3)

the exonic sequences.

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S… Speaker 2 (GEN3001_Workshop 3)

So if you compare GWAS and WES,

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S… Speaker 2 (GEN3001_Workshop 3)

the difference here is the scale of what

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S… Speaker 2 (GEN3001_Workshop 3)

you're comparing.

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S… Speaker 2 (GEN3001_Workshop 3)

In GWAS,

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S… Speaker 2 (GEN3001_Workshop 3)

you're comparing all the entire genome,

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S… Speaker 2 (GEN3001_Workshop 3)

introns,

13:47

S… Speaker 2 (GEN3001_Workshop 3)

exons.

13:48

S… Speaker 2 (GEN3001_Workshop 3)

But in WES,

13:50

S… Speaker 2 (GEN3001_Workshop 3)

purely just the exome.

13:52

S… Speaker 2 (GEN3001_Workshop 3)

Okay, then the second level of difference here is usually

13:56

S… Speaker 2 (GEN3001_Workshop 3)

for this kind of WES,

13:58

S… Speaker 2 (GEN3001_Workshop 3)

because you're nailing down to the correct gene

14:03

S… Speaker 2 (GEN3001_Workshop 3)

that causes the disease,

14:04

S… Speaker 2 (GEN3001_Workshop 3)

we will study them in pedigrees.

14:07

S… Speaker 2 (GEN3001_Workshop 3)

So these are for people who are related.

14:10

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

14:12

S… Speaker 2 (GEN3001_Workshop 3)

not the same as GWAS where they are not related.

14:14

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

14:17

S… Speaker 2 (GEN3001_Workshop 3)

so when they are related by blood,

14:20

S… Speaker 2 (GEN3001_Workshop 3)

you can then put them into a chart,

14:22

S… Speaker 2 (GEN3001_Workshop 3)

a family chart like that.

14:24

S… Speaker 2 (GEN3001_Workshop 3)

that shows their relationship between one another.

14:26

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

14:27

S… Speaker 2 (GEN3001_Workshop 3)

so this is something that you've already learned in Gen 2,

14:30

S… Speaker 2 (GEN3001_Workshop 3)

right? You know how to read the pedigree chart.

14:32

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

14:33

S… Speaker 2 (GEN3001_Workshop 3)

the square is male,

14:35

S… Speaker 2 (GEN3001_Workshop 3)

circle is female,

14:36

S… Speaker 2 (GEN3001_Workshop 3)

colored means affected by the disease,

14:41

S… Speaker 2 (GEN3001_Workshop 3)

uncolored means unaffected by the disease.

14:45

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so using this pedigree chart,

14:47

S… Speaker 2 (GEN3001_Workshop 3)

you can then tell whether there is a form of inheritance

14:51

S… Speaker 2 (GEN3001_Workshop 3)

or not from that disease genes.

14:53

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

14:54

S… Speaker 2 (GEN3001_Workshop 3)

and you can also tell whether it has arisen very recently

14:58

S… Speaker 2 (GEN3001_Workshop 3)

or not.

15:00

S… Speaker 2 (GEN3001_Workshop 3)

generation.

15:01

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

15:10

S… Speaker 2 (GEN3001_Workshop 3)

then similarly like what we discussed in GWAS,

15:12

S… Speaker 2 (GEN3001_Workshop 3)

right, they are also good and bad about the technique itself.

15:16

S… Speaker 2 (GEN3001_Workshop 3)

WES compared to GWAS,

15:19

S… Speaker 2 (GEN3001_Workshop 3)

the scale is very,

15:20

S… Speaker 2 (GEN3001_Workshop 3)

very different because one is large population study.

15:23

S… Speaker 2 (GEN3001_Workshop 3)

This is just within the family.

15:25

S… Speaker 2 (GEN3001_Workshop 3)

So cost -wise,

15:27

S… Speaker 2 (GEN3001_Workshop 3)

it's cheaper because one is looking at bulk.

15:30

S… Speaker 2 (GEN3001_Workshop 3)

Here is looking at individuals.

15:32

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

15:33

S… Speaker 2 (GEN3001_Workshop 3)

so it's much more cheaper.

15:35

S… Speaker 2 (GEN3001_Workshop 3)

and the data that you get is much lesser as you can imagine

15:39

S… Speaker 2 (GEN3001_Workshop 3)

because of the scale and the size and the number of people involved okay

15:43

S… Speaker 2 (GEN3001_Workshop 3)

so less data but doesn't mean data

15:48

S… Speaker 2 (GEN3001_Workshop 3)

is not quality okay so we are talking about quantity over

15:52

S… Speaker 2 (GEN3001_Workshop 3)

quality here so quantity is much less but quality

15:56

S… Speaker 2 (GEN3001_Workshop 3)

still the same okay and perhaps even more because now

16:00

S… Speaker 2 (GEN3001_Workshop 3)

you are looking at family

16:02

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

16:03

S… Speaker 2 (GEN3001_Workshop 3)

now the other thing that is a plus point for

16:07

S… Speaker 2 (GEN3001_Workshop 3)

WES is because you are looking at the axonic sequences,

16:11

S… Speaker 2 (GEN3001_Workshop 3)

it will give you an idea about protein

16:15

S… Speaker 2 (GEN3001_Workshop 3)

function.

16:16

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

16:17

S… Speaker 2 (GEN3001_Workshop 3)

and the protein function can be directly related to the disease itself.

16:21

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

16:25

S… Speaker 2 (GEN3001_Workshop 3)

now what is the negative side of this is that because you are

16:29

S… Speaker 2 (GEN3001_Workshop 3)

going to be studying and tracing down the generation within the family,

16:34

S… Speaker 2 (GEN3001_Workshop 3)

You can only focus on one gene at one

16:38

S… Speaker 2 (GEN3001_Workshop 3)

time perhaps,

16:39

S… Speaker 5 (GEN3001_Workshop 3)

right?

16:39

S… Speaker 2 (GEN3001_Workshop 3)

So we are looking at mostly monogenic diseases.

16:43

S… Speaker 2 (GEN3001_Workshop 3)

But a little different for GWAS because you are looking at a group

16:47

S… Speaker 2 (GEN3001_Workshop 3)

of people,

16:47

S… Speaker 1 (GEN3001_Workshop 3)

right?

16:48

S… Speaker 2 (GEN3001_Workshop 3)

And you are looking at variants.

16:51

S… Speaker 2 (GEN3001_Workshop 3)

The variants could highlight different chromosomes,

16:53

S… Speaker 2 (GEN3001_Workshop 3)

what are the problems,

16:54

S… Speaker 2 (GEN3001_Workshop 3)

the faults in those genes over there,

16:57

S… Speaker 3 (GEN3001_Workshop 3)

right?

16:58

S… Speaker 2 (GEN3001_Workshop 3)

And then that will give you an indication about diabetes risk profile.

17:02

S… Speaker 2 (GEN3001_Workshop 3)

But you can also pick up profile risk for other type of diseases

17:06

S… Speaker 2 (GEN3001_Workshop 3)

that are associated with type 2 diabetes,

17:09

S… Speaker 2 (GEN3001_Workshop 3)

for example.

17:10

S… Speaker 2 (GEN3001_Workshop 3)

So maybe things like cardiovascular risk,

17:13

S… Speaker 2 (GEN3001_Workshop 3)

you can also pick up.

17:14

S… Speaker 2 (GEN3001_Workshop 3)

Obesity risk,

17:15

S… Speaker 2 (GEN3001_Workshop 3)

you can also pick up.

17:16

S… Speaker 1 (GEN3001_Workshop 3)

Now,

17:19

S… Speaker 2 (GEN3001_Workshop 3)

the other minus point about here is that it would be better

17:23

S… Speaker 2 (GEN3001_Workshop 3)

off if you were to study things in newly...

17:29

S… Speaker 2 (GEN3001_Workshop 3)

formed offspring so children for example would

17:33

S… Speaker 2 (GEN3001_Workshop 3)

be much more effective because you're tracing down the family history rather than

17:37

S… Speaker 2 (GEN3001_Workshop 3)

to study adults because the adults perhaps would have already gotten the disease

17:42

S… Speaker 2 (GEN3001_Workshop 3)

themselves okay so this is more informative for you within

17:46

S… Speaker 2 (GEN3001_Workshop 3)

a family if you are trying to find out about the risk of

17:51

S… Speaker 2 (GEN3001_Workshop 3)

maybe having an offspring with that amount of risk or that high

17:55

S… Speaker 2 (GEN3001_Workshop 3)

amount of chance

17:56

S… Speaker 2 (GEN3001_Workshop 3)

Okay, the other important thing over here is that because

18:00

S… Speaker 2 (GEN3001_Workshop 3)

you're only studying the axonic sequences,

18:03

S… Speaker 2 (GEN3001_Workshop 3)

there is a chance that you're going to miss out on all the intronic information.

18:08

S… Speaker 2 (GEN3001_Workshop 3)

Because the introns,

18:10

S… Speaker 2 (GEN3001_Workshop 3)

even though we say that they don't make up into the protein,

18:12

S… Speaker 2 (GEN3001_Workshop 3)

but they are regulators.

18:18

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so they regulate what?

18:20

S… Speaker 2 (GEN3001_Workshop 3)

They regulate things like transcription levels,

18:23

S… Speaker 2 (GEN3001_Workshop 3)

transcription rate,

18:24

S… Speaker 2 (GEN3001_Workshop 3)

which will also affect the final protein functional

18:28

S… Speaker 2 (GEN3001_Workshop 3)

products and also the activity levels.

18:45

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so if we apply this to animal models,

18:47

S… Speaker 2 (GEN3001_Workshop 3)

earlier on what you saw were just applied to humans,

18:50

S… Speaker 2 (GEN3001_Workshop 3)

okay?

18:51

S… Speaker 2 (GEN3001_Workshop 3)

If we were to do it in animal models,

18:54

S… Speaker 2 (GEN3001_Workshop 3)

how do we do this?

18:55

S… Speaker 2 (GEN3001_Workshop 3)

So just to recap,

18:57

S… Speaker 2 (GEN3001_Workshop 3)

when we talk about forward genetics,

18:59

S… Speaker 2 (GEN3001_Workshop 3)

we are saying phenotype to

19:04

S… Speaker 2 (GEN3001_Workshop 3)

genotype.

19:07

S… Speaker 2 (GEN3001_Workshop 3)

Okay, knowing how the characteristic of the animal is,

19:10

S… Speaker 2 (GEN3001_Workshop 3)

the characteristic appearance of the animal.

19:14

S… Speaker 2 (GEN3001_Workshop 3)

And then now we are trying to find out what is the gene that causes that characteristic.

19:18

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

19:19

S… Speaker 2 (GEN3001_Workshop 3)

so you start off with parental organism.

19:25

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

19:26

S… Speaker 2 (GEN3001_Workshop 3)

so here we are focusing on zebrafish.

19:29

S… Speaker 2 (GEN3001_Workshop 3)

So assuming here they are all zebrafish.

19:31

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

19:32

S… Speaker 2 (GEN3001_Workshop 3)

so we have a breeding pair of the parental fishes.

19:36

S… Speaker 2 (GEN3001_Workshop 3)

okay so plus plus this is the genotype right so this is complete

19:40

S… Speaker 2 (GEN3001_Workshop 3)

wild type female fish and then this is bred with a male fish

19:45

S… Speaker 2 (GEN3001_Workshop 3)

that is chemically induced with a mutation okay

19:49

S… Speaker 1 (GEN3001_Workshop 3)

so

19:56

S… Speaker 2 (GEN3001_Workshop 3)

we add this enu treated male so it's a chemically

20:00

S… Speaker 2 (GEN3001_Workshop 3)

mutation in the male fish okay so

20:04

S… Speaker 2 (GEN3001_Workshop 3)

when we breed them with the wild type female this is the

20:08

S… Speaker 2 (GEN3001_Workshop 3)

very first offspring we get so a set of fish f1 we

20:12

S… Speaker 2 (GEN3001_Workshop 3)

call okay so if you do

20:17

S… Speaker 2 (GEN3001_Workshop 3)

the genotyping process okay so one of your fish

20:21

S… Speaker 2 (GEN3001_Workshop 3)

will have carried over the mutation from

20:25

S… Speaker 2 (GEN3001_Workshop 3)

the male

20:26

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so we have a plus slash M.

20:29

S… Speaker 2 (GEN3001_Workshop 3)

So M here would be the mutant or the mutation.

20:33

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

20:35

S… Speaker 2 (GEN3001_Workshop 3)

one of the mutant allele.

20:37

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

20:38

S… Speaker 2 (GEN3001_Workshop 3)

but this is still considered heterozygous mutant,

20:41

S… Speaker 2 (GEN3001_Workshop 3)

right?

20:41

S… Speaker 2 (GEN3001_Workshop 3)

So not what we want because if we want to find

20:46

S… Speaker 2 (GEN3001_Workshop 3)

out about the genotype,

20:47

S… Speaker 2 (GEN3001_Workshop 3)

we will have to pick a fish that is completely homozygous mutant.

20:51

S… Speaker 3 (GEN3001_Workshop 3)

Okay,

20:52

S… Speaker 2 (GEN3001_Workshop 3)

so then we continue breeding it,

20:54

S… Speaker 1 (GEN3001_Workshop 3)

right?

20:55

S… Speaker 2 (GEN3001_Workshop 3)

Our next level of the offspring we get will be the F2 generation.

20:59

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

20:59

S… Speaker 2 (GEN3001_Workshop 3)

so similarly,

21:00

S… Speaker 2 (GEN3001_Workshop 3)

F2 generation,

21:01

S… Speaker 2 (GEN3001_Workshop 3)

when you do the crossing over of the genotype,

21:03

S… Speaker 2 (GEN3001_Workshop 3)

you get a half -half genotype here that

21:08

S… Speaker 2 (GEN3001_Workshop 3)

is heterozygous.

21:11

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so with the heterozygous,

21:12

S… Speaker 2 (GEN3001_Workshop 3)

again, I can't study them because there will be mixed signals coming

21:17

S… Speaker 2 (GEN3001_Workshop 3)

from the wild -type allele.

21:18

S… Speaker 2 (GEN3001_Workshop 3)

So I will need to cross them again to final generation

21:22

S… Speaker 2 (GEN3001_Workshop 3)

here, F3.

21:23

S… Speaker 2 (GEN3001_Workshop 3)

So F3 over here,

21:25

S… Speaker 2 (GEN3001_Workshop 3)

what you will see is that there will be 75 %

21:29

S… Speaker 2 (GEN3001_Workshop 3)

of them that have no phenotype,

21:32

S… Speaker 1 (GEN3001_Workshop 3)

okay,

21:35

S… Speaker 2 (GEN3001_Workshop 3)

which is equivalent to three quarters.

21:36

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

21:38

S… Speaker 2 (GEN3001_Workshop 3)

and the last one quarter is the one I want.

21:42

S… Speaker 2 (GEN3001_Workshop 3)

Okay, that shows up the phenotype.

21:44

S… Speaker 2 (GEN3001_Workshop 3)

So bear in mind,

21:48

S… Speaker 2 (GEN3001_Workshop 3)

even though what we really wanted is

21:52

S… Speaker 2 (GEN3001_Workshop 3)

to observe the phenotype,

21:54

S… Speaker 2 (GEN3001_Workshop 3)

you can't really pick any fishes that you want throughout the entire

21:58

S… Speaker 2 (GEN3001_Workshop 3)

generation of offspring.

22:00

S… Speaker 2 (GEN3001_Workshop 3)

You have to really look at the genotype itself because it

22:04

S… Speaker 2 (GEN3001_Workshop 3)

is the genotype that helps to manifest the phenotype.

22:06

S… Speaker 1 (GEN3001_Workshop 3)

Okay,

22:18

S… Speaker 2 (GEN3001_Workshop 3)

so then that will sum up 3 .1.

22:21

S… Speaker 2 (GEN3001_Workshop 3)

Okay, in 3 .2 here,

22:23

S… Speaker 2 (GEN3001_Workshop 3)

we are going to have a little bit of a

22:28

S… Speaker 2 (GEN3001_Workshop 3)

mouse model,

22:29

S… Speaker 2 (GEN3001_Workshop 3)

but there will be more emphasis in the later topic.

22:32

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so just take this as a little bit of that

22:36

S… Speaker 2 (GEN3001_Workshop 3)

reverse genetic technique.

22:39

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

22:39

S… Speaker 2 (GEN3001_Workshop 3)

so knocking out mice.

22:42

S… Speaker 2 (GEN3001_Workshop 3)

Then we also have a bit about how we can utilize the

22:46

S… Speaker 2 (GEN3001_Workshop 3)

mice models to help us model diseases.

22:49

S… Speaker 2 (GEN3001_Workshop 3)

And then finally,

22:51

S… Speaker 2 (GEN3001_Workshop 3)

that's the one that we want to focus on using zebrafish as a model

22:55

S… Speaker 2 (GEN3001_Workshop 3)

of study.

22:56

S… Speaker 2 (GEN3001_Workshop 3)

So we will use zebrafish as the ideal choice

23:00

S… Speaker 2 (GEN3001_Workshop 3)

of model to look at human diseases for this topic.

23:09

S… Speaker 2 (GEN3001_Workshop 3)

Slide 11,

23:10

S… Speaker 2 (GEN3001_Workshop 3)

how do you get a complete knockout mice?

23:12

S… Speaker 2 (GEN3001_Workshop 3)

So you will have to do this at the level of the cells.

23:16

S… Speaker 2 (GEN3001_Workshop 3)

So in the previous topic,

23:18

S… Speaker 2 (GEN3001_Workshop 3)

you've learned about stem cells,

23:20

S… Speaker 2 (GEN3001_Workshop 3)

right? Your embryonic stem cells.

23:23

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so what you do here with the embryonic stem cells is that

23:27

S… Speaker 2 (GEN3001_Workshop 3)

you take those embryonic stem cells from animal origin,

23:31

S… Speaker 2 (GEN3001_Workshop 3)

you culture them on a plate,

23:33

S… Speaker 2 (GEN3001_Workshop 3)

okay, and then what you do is that you probably have to switch

23:37

S… Speaker 2 (GEN3001_Workshop 3)

on certain genes or switch off certain genes depending on what your goal is,

23:41

S… Speaker 2 (GEN3001_Workshop 3)

and then take those that have already completely grown on a

23:45

S… Speaker 2 (GEN3001_Workshop 3)

full dish,

23:46

S… Speaker 1 (GEN3001_Workshop 3)

right,

23:47

S… Speaker 2 (GEN3001_Workshop 3)

and then you electroporate it into a blastocyst.

23:53

S… Speaker 2 (GEN3001_Workshop 3)

Okay, so in the first topic,

23:55

S… Speaker 2 (GEN3001_Workshop 3)

you've learned what blastosis is,

23:56

S… Speaker 2 (GEN3001_Workshop 3)

right? A growing ball of cells with lots of inner cell mass,

24:01

S… Speaker 1 (GEN3001_Workshop 3)

right?

24:02

S… Speaker 2 (GEN3001_Workshop 3)

So you electroporate this blastosis,

24:05

S… Speaker 2 (GEN3001_Workshop 3)

meaning you open up the pores using an electric current so

24:09

S… Speaker 2 (GEN3001_Workshop 3)

that you can deliver your ESCs that you have modified.

24:12

S… Speaker 2 (GEN3001_Workshop 3)

Okay,

24:13

S… Speaker 2 (GEN3001_Workshop 3)

once you have created this modified blastosis,

24:20

S… Speaker 2 (GEN3001_Workshop 3)

Can you see that here they sort of color -coded it for you?

24:23

S… Speaker 2 (GEN3001_Workshop 3)

These are your ESCs that you have grown in the culture.

24:26

S… Speaker 2 (GEN3001_Workshop 3)

Then you will implant this to a surrogate mother.

24:31

S… Speaker 2 (GEN3001_Workshop 3)

So the surrogate mother will

24:35

S… Speaker 2 (GEN3001_Workshop 3)

now carry the modified blastosis,

GEN3001_Workshop 3

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