Research Skills in Life Science
LECTURE 1
Autism Spectrum Disorder (ASD) difficult to study:
Defined based on behavioral symptoms, broad clinical definition.
Lack of known causal factors.
Poor access to brain tissue.
Understanding biological mechanisms as a first step towards more effective intervention
strategies.
Some advantages of using animal (= in vivo) models ?
1. Causality can be tested by experimental design.
2. Control over environmental and genetic background.
3. Relevant tissue (e.g. brain) can be obtained.
4. Developmental aspects can be studied (life-span), because a mice has a shorter life.
Correlation / causation?
Observational studies
Case-control studies
Genetic association studies (e.g. GWAS)
Cohort studies (e.g. LifeLines, group of people)
Experiments
Experimental manipulations:
Cellular models: “in vitro”
Animal models: “in vivo”
Clinical trials (randomized)
A correlation between variables, however, doesn’t
automatically mean that the change in one variable
is the cause of the change in the values of the other variable.
Causation indicates that one event is the result of the occurrence of the other event; i.e. there is a
causal relationship between the two events.
A correlation is the relationship between two sets of variables used to describe or predict
information.
Causation, also known as cause and effect, is when an observed event or action appears to
have caused a second event or action.
So sometimes when there is a correlation, you may think that you have found a causation.
Like when it is raining people use an umbrella, but when people use an umbrella is doesn’t
going to rain immediately.
Getrouwde mannen leven langer
Sommige mensen vertellen mij dat het in ieder geval langer lijkt, maar dat terzijde. Er is hier sprake van een omkering van de feiten.
Gezonde, aantrekkelijke en hoog opgeleide mannen hebben een hoge levensverwachting. Dat veroorzaakt een natuurlijke selectie en trekt
automatisch vrouwen aan. Laag opgeleid en niet succesvol verkleint gewoonweg de kans om te trouwen. Dit is dus geen causaliteit.
Kinderen worden door de ooievaar gebracht
De stad Hamburg in Duitsland heeft een van de grootste populaties ooievaars in en rond de stad. Daarnaast worden er gemiddeld meer
kinderen geboren per 1000 inwoners dan in de rest van Duitsland. De voor de hand liggende, maar zo foute, conclusie is dat baby’s worden
gebracht door ooievaars. Maar dit is dus geen causaliteit, want kinderen worden niet door ooievaars gebracht.
1
,Differences between observational and experimental studies:
An observational study is where nothing changes and just record what you see, but an
experimental study is where you have a control group and a testable group.
An observational study is where researchers simply collect data based on what is seen and
heard and infer based on the data collected. The researcher has no control over the variables
in an observational study. An experiment is a method of applying treatments to a group and
recording the effects
LECTURE 2
Genetic association study (“forward genetics”): phenotype -> genotype.
Genetic manipulation (“reverse genetics”): phenotype <- genotype.
Familiality is not heritability
Heritable due to genes?
Familial due to environment?
Twin studies can be used dissect genetic and environmental influences
Twin designs allow to assesses the genetic and environmental influence on a trait using twin
pairs.
Monozygotic twins (MZ) share ~100% of their genes, while dizygotic twins (DZ) share only
~50%.
Estimate heritability by assessing whether MZ twins are more similar (“concordant”) than DZ
twins.
Twin studies indicate genetic contribution to psychiatric conditions
Estimate heritability by assessing whether MZ twins are more similar (“concordant”) than DZ twins.
Heritability = 2 x (rMZ – rDZ)
Note: Heritability always depends on the environmental context in which it was measured
(e.g. see heritability IQ and the Flynn-effect (that IQ can be influenced by the environment))!!
Linkage study (1990s): association of genetic locus with behavioral disorder running in family e.g.
Mendelian trait: Aggressive/violent behaviour in a Dutch family was linked to defect in MAOA gene:
Mendelian traits are traits that are passed down by dominant and
recessive alleles of one gene.
Only present in males suggests X-chromosome, because females have
an extra copy of this chromosome and males don’t have. So it would
be a mutation on this chromosome.
Investigation of common genetic markers and “haplotypes” (i.e.
combination of markers) unique for affected family members.
Mutation likely inherited via combination of markers “2-a-3-7-0-1”
and pointing at marker for MAOA gene.
Using “Sanger sequencing” technique (1977) to read the full implicated DNA sequence:
PCR is presence of fluorescent, chain-
terminating nucleotides.
Fragments rung through gel electrophoresis.
Fluorescent fragments detected by laser and
represented on a chromatogram.
2
,Association of rare mutations with neurodevelopmental disorders based on Whole Exome
Sequencing (2010-now)
Next generation sequencing (NGS) allows to read all the exomes of a genome at once:
180,000 exons
30 million base pairs (1% of the human genome)
NGS is used to identify genetic mutations that are likely detrimental to the gene:
rare inherited mutation (i.e. also present in parent).
de novo mutation only found in affected child..
Note: Because so many exomes are tested, it can be difficult to know which mutation is
really causing the disorder (all individuals have some ‘faulty genes’) !!
Polygenic traits basically mean the traits that are a result of multiple intertwined traits like
for example skin color.
Mendelian inheritance are dominant traits, no blending whichever dominant or recessive so
like albinism or blood type. So it determine one characteristic or one phenotype.
Mendelian disorders are often rare disorders and you can mostly identify them in families,
but their effect size is very big. So like aggression these people are very aggressive.
Polygenic disorders: are more common disorders that are driven by many genetic variance
and they have a much smaller effect size. So autism is a polygenic disorder.
Genome-wide association study to identify common genetic variation linked with schizophrenia
Testing associations between Schizophrenia and genetic makers across the entire genome (in
a large population).
GWAS requires extremely large sample sizes as the effect sizes for these markers (i.e. SNPs)
are often small.
Note: Significant markers (i.e. SNPs) are correlated with the actual causal variant, but not
necessarily causal!
Genetic association studies can also be used across species
There is a study between two kind of mice: the P. pollonotus that live in wet environments
and the P. maniculatus that live in the forest. The first makes much larger burrow with an
escape route, while the second mouse makes a small burrow.
They did an experiment if the mice did also make this burrow in a sand environment:
Burrowing behaviors in Peromyscus are heritable
3
, Genetic associations for burrowing behaviors
Multiple loci explaining difference in tunnel length - 3 cm each
Locus explaining probability to make escape tunnel
From genetic disorders to animal models
Animal models focus on genes disrupted by rare mutations
Study in fixed genetic and environmental background
The Fmr1 KO mice -> Fmr1 gene encodes for the protein FMRP that is involved in many tissues
No differences in general brain morphology in Fmr1 KO mice.
Increased testicular weight and abnormal exploratory behaviors in Fmr1 KO mice.
Impaired Morris Water Maze Performance in Fmr1 KO mice, they have to find a platform.
Lack of preference for a conspecific mouse in Fmr1 KO mice.
To create a knockout animal homologous
recombination of a targeting vector is used. You see
the Fmr1 gene with exons that code for the protein.
We want to disrupt the function of the protein. So
we insert, by using the long homologous
recombination, a piece of DNA. Now there is no
longer transcription of the protein, so you get a
non-functional protein. And with PCR you can look
with animals this KO gene has. First make a targeting
conduct, than you add that to embryonic stem cells and then you
provide electroporation. Now you can select the positive cells that are
taken up for the targeting constructor and inject the targeted ES into
blastocytes. Than you put this into a normal mouse and keep dividing.
Faster and more precise generation of genetically modified animals using Crispr/Cas9 (since 2013)
CRISPR RNA with variable targeting sequence (crRNA) allows binding to target DNA.
Single-stranded donor oligonucleotides (ssODN) contains alternative sequence.
Cocktail with CRISPR/Cas9 can be injected directly into zygotes: very fast!!
This technique allows to: slide 33 and 35!
Create efficient gene knocking or knockout
Create point mutations
Modify sequence segments
Crispr/Cas9 has also enabled genetic manipulation of ‘exotic’ species,
such as monogamous prairie voles:
When prairie voles choose a mate, there’s no turning back—the “love chemical” oxytocin
increases in their brains and they devote themselves to only each other.
Reduced preference for social novelty in male prairie voles with Oxytocin receptor knockout.
Genes are often expressed in multiple cell-types and brain regions: how to deal with this?
A conditional KO mouse is an animal in which the gene of interest is:
Inactivated only in specific cell types in a certain tissue; other cell types and tissues exhibit an
unmodified, functional gene expression (= Tissuespecific KO model).
Temporally inactivated at a given time-point in embryonic, post-natal or adult animals (=
tamoxifen induced KO model), slide 42/43. This can be done with LoxP system.
Flex switches enable endless possibilities to switch genes on/off at the desired timepoint
(=temporal) or tissue (=spatial). https://www.youtube.com/watch?v=I21NmFq4F8A
4
LECTURE 1
Autism Spectrum Disorder (ASD) difficult to study:
Defined based on behavioral symptoms, broad clinical definition.
Lack of known causal factors.
Poor access to brain tissue.
Understanding biological mechanisms as a first step towards more effective intervention
strategies.
Some advantages of using animal (= in vivo) models ?
1. Causality can be tested by experimental design.
2. Control over environmental and genetic background.
3. Relevant tissue (e.g. brain) can be obtained.
4. Developmental aspects can be studied (life-span), because a mice has a shorter life.
Correlation / causation?
Observational studies
Case-control studies
Genetic association studies (e.g. GWAS)
Cohort studies (e.g. LifeLines, group of people)
Experiments
Experimental manipulations:
Cellular models: “in vitro”
Animal models: “in vivo”
Clinical trials (randomized)
A correlation between variables, however, doesn’t
automatically mean that the change in one variable
is the cause of the change in the values of the other variable.
Causation indicates that one event is the result of the occurrence of the other event; i.e. there is a
causal relationship between the two events.
A correlation is the relationship between two sets of variables used to describe or predict
information.
Causation, also known as cause and effect, is when an observed event or action appears to
have caused a second event or action.
So sometimes when there is a correlation, you may think that you have found a causation.
Like when it is raining people use an umbrella, but when people use an umbrella is doesn’t
going to rain immediately.
Getrouwde mannen leven langer
Sommige mensen vertellen mij dat het in ieder geval langer lijkt, maar dat terzijde. Er is hier sprake van een omkering van de feiten.
Gezonde, aantrekkelijke en hoog opgeleide mannen hebben een hoge levensverwachting. Dat veroorzaakt een natuurlijke selectie en trekt
automatisch vrouwen aan. Laag opgeleid en niet succesvol verkleint gewoonweg de kans om te trouwen. Dit is dus geen causaliteit.
Kinderen worden door de ooievaar gebracht
De stad Hamburg in Duitsland heeft een van de grootste populaties ooievaars in en rond de stad. Daarnaast worden er gemiddeld meer
kinderen geboren per 1000 inwoners dan in de rest van Duitsland. De voor de hand liggende, maar zo foute, conclusie is dat baby’s worden
gebracht door ooievaars. Maar dit is dus geen causaliteit, want kinderen worden niet door ooievaars gebracht.
1
,Differences between observational and experimental studies:
An observational study is where nothing changes and just record what you see, but an
experimental study is where you have a control group and a testable group.
An observational study is where researchers simply collect data based on what is seen and
heard and infer based on the data collected. The researcher has no control over the variables
in an observational study. An experiment is a method of applying treatments to a group and
recording the effects
LECTURE 2
Genetic association study (“forward genetics”): phenotype -> genotype.
Genetic manipulation (“reverse genetics”): phenotype <- genotype.
Familiality is not heritability
Heritable due to genes?
Familial due to environment?
Twin studies can be used dissect genetic and environmental influences
Twin designs allow to assesses the genetic and environmental influence on a trait using twin
pairs.
Monozygotic twins (MZ) share ~100% of their genes, while dizygotic twins (DZ) share only
~50%.
Estimate heritability by assessing whether MZ twins are more similar (“concordant”) than DZ
twins.
Twin studies indicate genetic contribution to psychiatric conditions
Estimate heritability by assessing whether MZ twins are more similar (“concordant”) than DZ twins.
Heritability = 2 x (rMZ – rDZ)
Note: Heritability always depends on the environmental context in which it was measured
(e.g. see heritability IQ and the Flynn-effect (that IQ can be influenced by the environment))!!
Linkage study (1990s): association of genetic locus with behavioral disorder running in family e.g.
Mendelian trait: Aggressive/violent behaviour in a Dutch family was linked to defect in MAOA gene:
Mendelian traits are traits that are passed down by dominant and
recessive alleles of one gene.
Only present in males suggests X-chromosome, because females have
an extra copy of this chromosome and males don’t have. So it would
be a mutation on this chromosome.
Investigation of common genetic markers and “haplotypes” (i.e.
combination of markers) unique for affected family members.
Mutation likely inherited via combination of markers “2-a-3-7-0-1”
and pointing at marker for MAOA gene.
Using “Sanger sequencing” technique (1977) to read the full implicated DNA sequence:
PCR is presence of fluorescent, chain-
terminating nucleotides.
Fragments rung through gel electrophoresis.
Fluorescent fragments detected by laser and
represented on a chromatogram.
2
,Association of rare mutations with neurodevelopmental disorders based on Whole Exome
Sequencing (2010-now)
Next generation sequencing (NGS) allows to read all the exomes of a genome at once:
180,000 exons
30 million base pairs (1% of the human genome)
NGS is used to identify genetic mutations that are likely detrimental to the gene:
rare inherited mutation (i.e. also present in parent).
de novo mutation only found in affected child..
Note: Because so many exomes are tested, it can be difficult to know which mutation is
really causing the disorder (all individuals have some ‘faulty genes’) !!
Polygenic traits basically mean the traits that are a result of multiple intertwined traits like
for example skin color.
Mendelian inheritance are dominant traits, no blending whichever dominant or recessive so
like albinism or blood type. So it determine one characteristic or one phenotype.
Mendelian disorders are often rare disorders and you can mostly identify them in families,
but their effect size is very big. So like aggression these people are very aggressive.
Polygenic disorders: are more common disorders that are driven by many genetic variance
and they have a much smaller effect size. So autism is a polygenic disorder.
Genome-wide association study to identify common genetic variation linked with schizophrenia
Testing associations between Schizophrenia and genetic makers across the entire genome (in
a large population).
GWAS requires extremely large sample sizes as the effect sizes for these markers (i.e. SNPs)
are often small.
Note: Significant markers (i.e. SNPs) are correlated with the actual causal variant, but not
necessarily causal!
Genetic association studies can also be used across species
There is a study between two kind of mice: the P. pollonotus that live in wet environments
and the P. maniculatus that live in the forest. The first makes much larger burrow with an
escape route, while the second mouse makes a small burrow.
They did an experiment if the mice did also make this burrow in a sand environment:
Burrowing behaviors in Peromyscus are heritable
3
, Genetic associations for burrowing behaviors
Multiple loci explaining difference in tunnel length - 3 cm each
Locus explaining probability to make escape tunnel
From genetic disorders to animal models
Animal models focus on genes disrupted by rare mutations
Study in fixed genetic and environmental background
The Fmr1 KO mice -> Fmr1 gene encodes for the protein FMRP that is involved in many tissues
No differences in general brain morphology in Fmr1 KO mice.
Increased testicular weight and abnormal exploratory behaviors in Fmr1 KO mice.
Impaired Morris Water Maze Performance in Fmr1 KO mice, they have to find a platform.
Lack of preference for a conspecific mouse in Fmr1 KO mice.
To create a knockout animal homologous
recombination of a targeting vector is used. You see
the Fmr1 gene with exons that code for the protein.
We want to disrupt the function of the protein. So
we insert, by using the long homologous
recombination, a piece of DNA. Now there is no
longer transcription of the protein, so you get a
non-functional protein. And with PCR you can look
with animals this KO gene has. First make a targeting
conduct, than you add that to embryonic stem cells and then you
provide electroporation. Now you can select the positive cells that are
taken up for the targeting constructor and inject the targeted ES into
blastocytes. Than you put this into a normal mouse and keep dividing.
Faster and more precise generation of genetically modified animals using Crispr/Cas9 (since 2013)
CRISPR RNA with variable targeting sequence (crRNA) allows binding to target DNA.
Single-stranded donor oligonucleotides (ssODN) contains alternative sequence.
Cocktail with CRISPR/Cas9 can be injected directly into zygotes: very fast!!
This technique allows to: slide 33 and 35!
Create efficient gene knocking or knockout
Create point mutations
Modify sequence segments
Crispr/Cas9 has also enabled genetic manipulation of ‘exotic’ species,
such as monogamous prairie voles:
When prairie voles choose a mate, there’s no turning back—the “love chemical” oxytocin
increases in their brains and they devote themselves to only each other.
Reduced preference for social novelty in male prairie voles with Oxytocin receptor knockout.
Genes are often expressed in multiple cell-types and brain regions: how to deal with this?
A conditional KO mouse is an animal in which the gene of interest is:
Inactivated only in specific cell types in a certain tissue; other cell types and tissues exhibit an
unmodified, functional gene expression (= Tissuespecific KO model).
Temporally inactivated at a given time-point in embryonic, post-natal or adult animals (=
tamoxifen induced KO model), slide 42/43. This can be done with LoxP system.
Flex switches enable endless possibilities to switch genes on/off at the desired timepoint
(=temporal) or tissue (=spatial). https://www.youtube.com/watch?v=I21NmFq4F8A
4
