W2L2 Thu Reverse genetics - Model for human genetic Flashcards
Way to investigate humans genetic
-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-inheritance pattern (no longer use due to being outdated)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
Limitations of humans as genetic models
Functional in vivo gene manipulations impossible
Classical inheritance studies slow
“Controls” fairly limited
Invasive phenotypic analysis difficult
What is used for genetic disease modeling
-reverse genetic to understand the final phenotype
Reverse Genetics: Animal models and technical approaches
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
Key consideration non human model
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
Evolutionary conservation - Vertebrates (phylogenetic and phylogenetic tree)
Phylogenetics - Evolutionary relationship between species
Morphological, molecular (nucleotide, amino acids)
Phylogenetic tree reveals accurate evolutionary distances (time) and branching points of common ancestors.
Genetic model organism
-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
Why use genetic model organism
Ease and low cost of husbandry
Short generation cycle
Genetic manipulation amenability
High number of offspring
What is relevance and suitability
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)
Mouse: Mammalian model Genomic vs. proteomic conservation
-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
Zebrafish
-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
Reverse Genetics - Gene knockdown Transient and variable efficiancy (MO)
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
EXP: Way of using genetic model
-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
Morpholino gene knockdown efficiency:what does it depend on?
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.
Translational MO and efficientcy detection
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.
Splice MO and detection
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
Morpholino controls method
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
Gene knockout vs knockdown
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.
Reverse Genetics - Gene knockout
Stable mutagenesis CRISPR
-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)
Conditional and Inducible Gene Expression
Conditional: gene are express if condition are met
Inducible: a condition that allow us to activate or shutdown gene
Reverse Genetics - Genetic compensation
-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
Reverse Genetics - Genetic compensation in human disease
- 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
