W2L2 Thu Reverse genetics - Model for human genetic

Question 1

Way to investigate humans genetic

Answer 1

-GWAS- indentification gene associated with phenotype
-Monozygotic twin: epigenome reset in early gestation, but each twin accumulate their own Epigenetic changes
-inheritance pattern (no longer use due to being outdated)
-Human steam cell- genetic manipulation easy, individualized diseas studies and treatment screening

Question 2

Limitations of humans as genetic models

Answer 2

Functional in vivo gene manipulations impossible
Classical inheritance studies slow
“Controls” fairly limited
Invasive phenotypic analysis difficult

Question 3

What is used for genetic disease modeling

Answer 3

-reverse genetic to understand the final phenotype

Question 4

Reverse Genetics: Animal models and technical approaches

Answer 4

Animal models:
Evolutionary distance, genetic conservation
Relevance, suitability
Reverse Genetics:
Gene knockdown vs. knockout
Morpholino technology - controls
CRISPR mutagenesis
Genetic compensation
Conditional and inducible gene expression

Question 5

Key consideration non human model

Answer 5

Evolutionary and genetic conservation (relevance)
Relevance vs. technical suitability of animal models
Genomic vs. proteomic conservation
Cell vs. organ vs. integrated function vs. higher order behaviour

Question 6

Evolutionary conservation - Vertebrates (phylogenetic and phylogenetic tree)

Answer 6

Phylogenetics - Evolutionary relationship between species
Morphological, molecular (nucleotide, amino acids)
Phylogenetic tree reveals accurate evolutionary distances (time) and branching points of common ancestors.

Question 7

Genetic model organism

Answer 7

-Non-human species used to study fundamental, highly conserved processes
-Used to research human health and disease aspects
-Relevant conservation of cellular, tissue or organ function or process
-Relevant underlying genetic conservation

Question 8

Why use genetic model organism

Answer 8

Ease and low cost of husbandry
Short generation cycle
Genetic manipulation amenability
High number of offspring

Question 9

What is relevance and suitability

Answer 9

Relevance: Is the process conserved in humans?
1. Evolutionary / genetic distance - phylogeny & conservation
2. Complexity (cellular vs. organ or behaviour)
Suitability:
What is the most suitable model (e.g. with genetic tools)

Question 10

Mouse: Mammalian model Genomic vs. proteomic conservation

Answer 10

-Closest genetic model, i.e. highest relevance to human genetics
-40% nucleotide alignment
-May lead to no or functionally irrelevant AA changes
80% of mouse gene have single ORTHOLOGUE
one-to-one equivalent of gene with conserved expression (time and location) and function
-<1% of mouse genes have no detectable orthologue
-Conservation: Nucleotide < Amino acid < functional protein
-Same principle that explains why mutation size cannot predict phenotype (although there are trends).
Many similar cell, organ and body organ function

Question 11

Zebrafish

Answer 11

-Large set of genetic tools as a vertebrate, and whilst evolutionary distance is greated from human, genetic conservation remains high.
-Many basic vertebrate specific conserved cell types / organs
1. 70% of human genes have orthologues
2. Whole genome duplication:
* Potential for identifying multiple roles of genes temporal (one in development, one in adult) or spatial (in two different cell types or organs)
* Loss of gene often viable in contrast to mammals for example

Question 12

Reverse Genetics - Gene knockdown Transient and variable efficiancy (MO)

Answer 12

Morpholino (MO) technology / Antisense oligonucleotide
1. 25 bp synthetic oligomer complementary to bind mRNA of target gene.
2. Transcription process for other occurs unperturbed, MO acts at mRNA level.
3. Two types:
A. Translational MO
B. Splice MO

Question 13

EXP: Way of using genetic model

Answer 13

-find volunteer with rare genetic problem
-accept and sequence the genome
-informatics analysis of submitted gene (find orthologs of model organism) and prioritized some gene
-test on drosophila and zebra fish, potential discovery of new gene and genotyping expansion

Question 14

Morpholino gene knockdown efficiency:what does it depend on?

Answer 14

Efficiency depends on numbers of MOs vs number of mRNA molecules. With every cell division, MOs are roughly halved and knockdown often becomes inefficient by the end of the rapid developmental period in the first 3 / 4 days.

Question 15

Translational MO and efficientcy detection

Answer 15

Binds start site or just upstream and prevents translation by steric hindrance. Thus, mRNA formed, but no protein.
Protein loss can be tested using antibodies against the protein either in immunostaining or Western blots.

Question 16

Splice MO and detection

Answer 16

Binds to donor or acceptor splice site and prevents proper splicing (mRNA processing).
Spliceosome cannot recognise the correct , side and aberrant splicing leads to a defective protein
-Check by looking at protein length, Change in mRNA length can be detected by PCR with primers at either end of the relevant region followed by gel electrophoresis. But it can lead to fuction change that is unpredictable

Question 17

Morpholino controls method

Answer 17

MO controls to address off target / non-specific effects, i.e. binding of MO to the wrong sequence / gene or procedural phenotype
1. Controls for experimental procedure:
Water, control MO (binds nothing) or 5-pair mismatch (same nucleotides jumbled up).
2. Common sense for phenotype (spatial and temporal): Affected organ should be expressing the gene at the same time as the phenotype.
3. RNA rescue:
Phenotype should be completely reversed, if gene mRNA is provided that cannot be bound by the MO.
4. Using two different MO at concentrations that alone show no phenotype - additive effect

Question 18

Gene knockout vs knockdown

Answer 18

When targetting gene knockout for stable mutant, efficiency of gene loss does not need to be determined, no dilution.
However, for early developmental processes, maternal effect might occur:
Even if the embryo is homozygous for a recessive mutation, maternally deposited WT mRNA or protein in the egg may result in maternal rescue of a potential early phenotype prior to transcription of embryonic genes.

Question 19

Reverse Genetics - Gene knockout
Stable mutagenesis CRISPR

Answer 19

-CRISPR used by prokaryotes to destroy phage DNA
1. Site-directed by guide RNA
2. Requires PAM (protospacer adjacent motifs)
3. Cutting enzyme Cas9
4. Mutation either random (non-homologous end joining) or designed (homology directed recombination - repair template)

Question 20

Conditional and Inducible Gene Expression

Answer 20

Conditional: gene are express if condition are met
Inducible: a condition that allow us to activate or shutdown gene

Question 21

Reverse Genetics - Genetic compensation

Answer 21

-Altered gene expression would have a detrimental consequences but it is not seen. WT phenotype observers
-not dosage compensation or gene duplication but driven by molecular cue upstream of gene fuction
-Not all mutation induced genetic compensation

Question 22

Reverse Genetics - Genetic compensation in human disease

Answer 22

• Genetic compensatory responses have been identified in zebrafish, yeast and mice
• Large-scae sequencing identified loss-of-function mutations in human populations, expected to result in loss of genefunction, in genes that are critical for survival, and yet theseindividuals are, remarkably, healthy and non-symptomatic
• Suggests genetic compensation may be widespread in human populations and affect disease penetrance