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
How are the ES cells containing the targeted gene knockout produced and isolated?
- This is usually done by creating a targeting vector that will encorporate into the exon of the gene you are knocking out that contains a neomycin resistance gene
- The cells that have encorporated the targeting vector will be positively selected for by adding neomycin to the plate
- In order to determine if the ES cells have actually encoporated the targeting vector into the genome by adding a thymidine kinase gene into the targeting vector that when the targeting vector incorporates will be lost
- The ES cells are exposed to ganciclovir to kill all cells that have not incorporated the vector (negative selection)
What is a conditional knockout mouse?
- Creating a system in a mouse that can conditionally express the knockout phenotype depending on
- The stage of development (you can have the gene be expressed during embryonic development)
- The tissue it is in
- This process involves adding a conditional allele that contains the neomycin resistance gene
- The enzyme cre recombinase will flip out the neomycin gene so only the conditional allele is left (and thus the conditional allele is no longer knocked out)
- This process requires genetically modified mice that will only express cre recombinase at certain times/tissues
What is a gene knock-in mouse?
- Made with a similar procedure as knock-out mice; relies on recombination in ES cells
- You create a targeting construct with the neomycin gene in middle of exon that has been mutated
- You select ES cells that have incorporated this targeting vector using neomycin
- You then use flip recombinase to remove the neomycin resistance gene
- This leaves the ES gene with the only change being a specifically mutated region
- Allows you to look at the phenotype when a section of a gene is swapped (mutated)
What are the 3 main systems for achieving gene editing?
- Zinc Finger
- TALEN
- CRISPR/Cas9
How does Zinc Finger Nuclease editing work?
- The ZF proteins specifically recognise DNA sequences (based on a whole series of ZF domains)
- The ZF protein is fused to a non-specific endonuclease Fok1
- If the ZF proteins bind to the DNA sequence the Fok1 will cause a double stranded break
- This results in:
1. Non-homolgous end joining: default pathway- causes gene disruption
2. Homology-directed repair: occurs when extra DNA with overlapping sequence i added- can be used to add in new genetic material (but very low success)
How does the TALEN editing system work?
- Uses: Transcription Activator-Like Effectors (TALE):
- These TALEs contain DNA binding tandem repeats (of 33-35 residues)
- Like in the ZF system, the TALEs are fused to Fok1
- When the TALE binds to a specific DNA sequence, Fok1 will cleave it (creating double stranded break)
- Also results in non-homologous end joining or homology directed repair
How does CRISPR/CAS editing work?
CRISPR = Clustered Regular Interspaced Short Palindromic Repeats
- Newer and more effective tehnique than ZF and TALEN
Process:
1. PAM sequences (nearly always NGG) are scattered through the genome- you must find a PAM next to/embedded in target gene
2. Design a guide RNA sequence (gRNA) complementary to the downstream (3’) sequence from the PAM (in the target gene)
3. Attach the gRNA to the enzyme Cas9 which is a nuclease
4. Cas9 will recognise the PAM and then check to see its the bound gRNA in complementary to the downstream region
5. If the gRNA is complementary, Cas9 will cause a double stranded break in the DNA at this point
6. The double stranded break causes:
- Non-homologous end joining (NHEJ)
- Homology directed repair (HDR)- if you add extra DNA
What are the pros and cons of the CRIPSR system?
- Simple
- Very high efficiency of mutation
- Micro-injected directly into zygote
- Multiple mutations can be introduced immediately
Cons:
- Off-site effects in genome (but these are countered by high-efficiency- just do many experiments)
- Can reuslt in mosaic insertion (when injected in zygote)
What gene editing system has the lowest off-target binding?
- TALEN
