Genetic Technologies Flashcards
What is forward genetics?
Phenotype –> Genotype
- Phenotype is observed and then the gene is determined
What is reverse genetics?
Genotype –> Phenotype
- A new gene is identified and then its function is determined via a model organism which when the gene is knocked out/in will show a phenotype
What are the 5 standard model organisms in order of least closely related to humans to most closely related to humans:
- Saccharomyces cerevisiae (Baker’s yeast)
- C. elegans (nematode)
- Drosophila (fruit fly)
- Danio rerio (Zebrafish)
- Mus Musculus (mouse)
What are the advantages and disadvantages of the mouse as a model organism?
Advantages:
- Highest amount of synteny with human genome
- 85% of mouse protein coding regions same as humans
- Readily undergoes homologous recombination
Disadvantage:
- Expensive
- Vertebrate = ethical issues
What are the advantages and disadvantages of the zebrafish as a model organism?
Advantages:
- Realtime mapping of early development due to translucent body of embryos and larvae
- Embryo manipulation easy (in vitro)
- Fast replication and many progeny
- Relatively closely related to humans
Disadvantages:
- Gene targeting not widely used
- Phenotypic tests limited in translation to humans
- Requires specific infrastructure
- Genomes of fish have massive duplication events
What are the advantages and disadvantages of the drosophila as a model organism?
Advantages:
- Many genetic tools available
- Good development model
- Good homology to human gene families (e.g. Hox genes)
- No ethics
- Fast generation time and many progeny
Disadvantages:
- Not vertebrate
- Not deuterostome
- Application to human diseases can be limited
What are the advantages and disadvantages of C. elegans as a model organism?
Advantages:
- Gene manipulation easy
- Transparent and small (all cells can be counted)
- Quick and cheap
- No ethics
Disadvantages:
- Not vertebrate
- Not deuterostome
- Gene targeting difficult
What are the advantages and disadvantages of Yeast as a model organism?
Advantages:
- Genome easy to manipulate- homologous recombination)
- Super cheap and super-fast
- Exist in haploid state
- Eukaryotes- Good for studying fundamental processes
Disadvantages:
- Evolutionarily distant
- Single celled organism- Phenome is limited
What is a genome?
The set of hereditary information encoded in DNA of an organism, including both the protein-coding and non-protein-coding sequences
What was the aim of the human genome project?
- Make a complete and accurate sequence of the human genome
- Identify all genes
- Understand the role of specific genes
- Determine the role of genes in diseases
- The role of the genome in personalised medicine
What are some basic facts found about the human genome?
- Only 1-2% of the human genome is protein coding
- There are 3 billlion base pairs in the human genome
- There are only 20,000 genes
What is first generation sequencing?
- The first strategies used in the HGP
- Includes: Sanger sequencing and capillary electrophoresis sequencing
- Very slow and quite expensive
What are the differences between clone-by-clone strategies and the whole shotgun method?
- In order to use first generation methods for the entire genome the genome must be broken down and replicated by:
- Clone-by-clone sequencing:
- Human chromosomes are broken apart and put in something smaller (yeast chromosomes), these are broken apart and put in plasmids, the DNA in plasmids is cloned and then sequenced using Sanger/Capillary
- Accurate but slow and expensive - Whole shotgun method:
- The genome is randomly broken apart
- These section are sequenced (e.g. Sanger or capillary)
- These short reads are then put back together using computation
- Quicker and cheaper
- Higher error rate- but overcome by redundancy (reading the same section multiple times)
What are next generation sequence techniques?
- Pyrosequencing (400bp reads)
- Illuminq Hiseq (50-150 bp reads)
- Illumina NexSeq (450 bp reads)
What is third generation sequencing?
- The newest methods of DNA sequencing
- Aims to be able to do long reads and use single stretches of DNA (or very small amounts)
- Includes PacBio and the Oxford Nanopore Technology
What is:
- Transcriptomics
- Proteomics
- Pharmacogenomics
- Transcriptomics:
- Sequencing DNA that has been transcribed (exons) from mRNA using cDNA
- Uses northern blots - Proteomics:
- Investigates proteins e.g. by mass spec - Pharmacogenomics:
- How an individuals genome affects their response to a drug
- Used to personalise treatment
What are the 3 basic methods for Reverse Genetics in a whole organism?
- Targeting the transcriptome:
- Transgene
- Target mRNA - Adding a gene to the germline by random insertion
- Pronuclear injection
- Virus delivered material - Targeted insertion/deletion of a gene in the genome:
- Knock-out
- Knock-in
- Gene editing
What are the two ways in which the transcriptome is transiently targeted?
- Adding more expression of a gene:
- The transient gene expression is introduced using a plasmid vector
- The endogenous gene is still in the background - Knocking down of gene expression:
- Diminishes translation of target gene mRNA by using RNA interference (RNAi)
How does RNAi work?
- RNAi is a process of knocking down a gene’s expression by targeting the endogenous mRNA
- RNAi molecules are complementary to the target mRNA
- The RNAi binds to the mRNA making a dsRNA molecule which is targeted by the RISC complex in cells and cleaved
- A transient technique developed quite well in Zebrafish and C. elegans
- Disadvantage: off-target effects
How do zebrafish morphilinos work?
- Similar to RNAi
- Uses synthetic nucleotides called morphilinos to make the oligonucleotide that bind the target mRNA
- These morphilino oligonucleotides are more resistant to nucleases (more stable) and there is less non-specific binding between them and endogenous mRNA (less off-target effects)
What is pronuclear injection?
- A method by which a gene can be randomly incorporated into the entire genome
- Only really possible in yeast and mice (where homologous recombination will encoroporate the transgene)
- Issue: the integration of the DNA is random and if it disrupts a gene the phenotype may be due to the gene disruption and not the incorporated gene
- An alternative to this is using viruses (but this is best for cell lines)
What does gene targeting involve?
- Targeted insertion/deletion of genes into the genome
- Includes gene knockouts, knockins and conditional knock outs
What are some issues with gene-knockout mice?
- A small amount of gene knock-out mice are phenotypically normal
- 15-40% of genes when knocked out are embryonic lethal
- The neomycin resistance genes can influence the phenotype
How do you produce a Knock-out mouse?
- 5 Basic steps
- In vitro mutation of target gene in embryonic stem cells (from mice that are white)
- ES cells containing the mutated gene being studied are introduced into blastocyst (of mouse that is brown)
- These blastocysts with ES cells incorporated are implanted into a pseduo pregnant mouse
- This creates chimeric pups (brown and white patches)
i. Chimeric mice contain tissues derived from either the transplanted ES cells (white) or the host cells (brown)- if one of the transplanted ES cells became a germline cell then we can achieve a knockout mouse
- These chimeric mice are mated with normal mice to assess whether mutation is incoporated into germline (white offspring appear) - Heterozygous white mice for the gene mutation are mated to produce white mice homozygous for the mutated gene