Week 3 Flashcards
What is the aim of forward genetics?
To identify mutations that produce a certain phenotype ie mutate a gene then isolate and characterise it
What is the process of forward genetics?
Use common mutagenic agents such as EMS and radiation
A random process
What is the aim of revsere genetics?
A targeted approach to investigating gene function, determines the phenotype that results from mutating a specific gene
How can a targetted modification of a particular gene in yeast, bacteria and mice?
Generate ‘knockout’ or knock in (if in case of a dominant mutation)
What are alternative methods for reverse genetics?
Random induction of DNA deletions and subsequent selection for deletions in a gene of interest, as well as the aplication of RNA interference
What are the three proteins in the Escherichia coli mismatch repair system?
mutS, mutL and mutH
What happens if there is a mutation in either mutS, mutL or mutH Escherichia coli gene?
Mutations in any of those genes will result in defective MMR
How can you use revserse genetics to determine bacterial gene function?
Use recombinant DNA technology to construct mutated version of your target gene
Transform into E.Coli
Screen for antibiotic resistance
Analyse phenotype
What would you expect if a bacteria had a non-functional MMR (mismatch repair)?
Higher levels of mutation
How does Rifampicin work against bacteria?
Rif binds to and inhibits the beta subunit of RNA polymerase encoded by rpoB
What can make a bacteria resistant to Rifampicin?
As this involves amino acids in the rpoB protein with aromatic side chains. If the residues are altered due to mutation, the protein can become insensitive to rifampicin. Mutated rpoB gene allows bacteria to grow on rifampicin
What would you expect if a bacteria had a non-functional MMR (mismatch repair) when grown on rifampicin?
They should be highly mutable and in the Luria-Delbruck fluctuation assay they will show a high frequency of spotaneous Rif resistance
How can you assay for defective MMR in E.coli?
Each strain is grown under a peirod of non-selective growth (Luria-Delbruk Fluctuation Assay)
In WT stains any mutations introduced are corrected by MMR system
Some mutations in the rpoB gene, that results from defective MMR, make the rpoB insensitive to rifampicin
These mutants are detected by plating onto LB+Rifampicin after the period of non-selective growth
What are the results of assay for detecting defective MMR in E.Coli?
LOW numbers of rifampicin-resistant colonies are observed in WT strains but larger numbers will be observed in mutants defective in MMR
What would you expect the results of an assay be if the plasmid pRB318 contains the WT copy of mutS?
The mutS will also have a low mutation frequency so little to no colonies when plated into LB+Rifampicin plate
How can you tell if a sequence is critical to a neccesary function?
Amino acid sequence alignment of the gene region will show conserved homologous sections for a gene function
What are transcriptional fusions?
Cells/tissues in which the gene is transcrobed are investigated using transgenics where the promoter is fused to a reporter gene e.g. GFP
What are translational fusions?
Cells/tissues or the subsullular location where the protein is expressed/active are investigated using transgenics expressing chimeric proteins
What is the origin of GFP?
It is a Escherichia coli beta-glucuonidase gene (a staining technique used in plants and most bacteria). The technique based on Beta-glucuronidase, when incubated with some specific colorless or non-fluorescent substrates, transforms them into coloured or fluorescent protein product
What are transgenic mice?
Used in research for investigating gene function as seen in the XX mouse expressing the SRY gene
What is RNAi?
RNA interface is a biological process which RNA molecules are involved in sequence-specfic supression of gene expression by double stranded RNA, through translational or transcriptional repression
How are microRNAs produced in eukaryotic cells?
Primary miRNAs are processed in the nucleus by the enzyme ‘Drosha’
In the cytoplasm ‘Dicer’ reduces the miRNA to a short-lived miRNA:miRNA duplex
The duplex is recognised by the RNA-Induced Silencing Complex
miRNA is eliminated and a functional miRISC is formed
How is miRNA processed by ‘Drosha’?
miRNA is epressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA. This is processed by the enzyme Drosha, to a 70-nucleotide stem-loop structure called pre-miRNA
What does the enzyme Dicer do to the pre-miRNA?
The pre-miRNA is cleaved by the RNase III enzyme Dicer. Thie endoribonuclease interacts with 5’ and 3’ ends of the hairpin and cuts away the loop joing the 3’ and 5’ arms, yielding an imperfect miRNA:miRNA* duplex about 22 nucleotides in length
What happens to the duplex to create the the functional miRNA?
Only one strand is usually incorporated into the RNA-induced scilencing complex (RISC) where the miRNA and its mRNA target interact
How can microRNAs control mRNA expression?
Through two modes of RNA interference - mRNA cleavage and translation inhibition
How does microRNAs cleave mRNA?
miRNA is complementary to a part of one or more mRNAs. Perfect or near perfect base pairing with the target RNA promotes cleavage of the RNA
Where are miRNAs complementary to mRNA?
In animal miRNAs they are usually complemetary to a site in the 3’ UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. In plants matchups are perfect or near perfect but in animals they more likely to be imperfect rather than perfect
How does mRNAs inhibit translation?
Binds to the mRNA which makes the translation into proteins less efficient
How important of miRNAs?
In mammalian cells they target about 60% of the genes. Many miRNAs are evolutionary conserved, which they have important biological functions
What is the most wll-studied outcome for miRNAs?
Post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute 2 (Ago2), the catalytic component of the RISC.
Why is RNAi important reseach tool?
It is important for both cell culture and in living organisms because synthetic dsRNA introduced into cells can selectively and robustly induce supression of specific gene of interest. This can be used for large-scale screens that systematically shut down each gene in the cell, helping identify components necessay for particular cellular process or an event such as cell division.
How are mice embryotic stem cells are used for a disrution gene can be used to produce homozygous ‘knock outs’?
Inject ES cells into blastocoel cavity of early embryos
Surgically transfer embryos into pseudopregnant female
Chimeras are easily identified according to their coat colour
Select for chimeric mice for crosses to wild-type black mice
If any of the agouti cells have formed part of the germ-line in the chimera then they will go on to produce either fully agouti mice or pure black mice
How can the chimeric cells produce heterozygous offspring?
If transgenic mice ES cells contribute to the germ line, crossing chimeras to wild type mice may result in heterozygous offspring
What does the fact that agouti is dominant and the gene knockout is likely to be recessive for knockout proportion in the population?
It means only half of the agouti mice will carry it. Identify by PCR an inbreed to see expression
How can reverse genetics be used to model for human genetic disorders?
Mouse can be used as a model for human genetic disorders eg achondroplastic dwarfism, cystic fibrosis, achondroplasia
What causes achondroplastic dwarfism?
Most common form of short limb dwarfism (a dominant mutation)
A single amino acids substituation in the mouse FGFR3 otholog induces a similar phenotype in mouse
This is due to auto-activation of the FGFR3 protein kinase domain
What are the symptoms of achondroplastic dwarfism?
Skeletal abnormalities
Shorter face
Overgrown incisor teeth
Imporper head-to-spine connection
How can you generate null alleles?
Targeted gene disruption is used to generate null alleles in transgenic mice
What are the advantages of generating null alleles?
Some null alleles are not lethal ans can provide clues to the function og the gene product
What are the disadvantages of generating null alleles?
Many null mutants are embryonic lethal and therefore not informative with regards to overall gene function
What are conditional lethal mutants?
These can be generated where the target gene is deleted in a defined subset of cells/tissues
What is the process of LoxP/Cre-recombinase mediated gene deletion in target cells of mice?
A mouse carrying a Cre recombinase gene controlled by a tissue-specific promoter x is bred with a mouse carrying conditional (floxed (surrounded by LoxP)) alleles of gene Y. This produces an offspring where gene Y is inactivated by Cre in tissue X
How does the LoxP/Cre recombinase mediated gene deletion work?
It exploits the properties of a prokaryotic recombination mechanism- Cre/Lox
What is Fgfr2b and why is it important?
The epithelial isoform of fibroblast growth factor receptor 2 (Fhfr2b) is essential for embryogenesis (tyrosine kinase receptor)
What happens if a mice is Fgfr2b-null?
It dies at birth
What happens if you use Cre-Lox transgenics to delete Fgfr2b in cells expressing keratin 5?
This shows that mice lacking epidermal Fgfr2b survive into adulthood but display abmormalities in hair and sebaceous gland development
What are the impacts on mice when using Cre-Lox transgenics to delete Fgfr2b in cells expressing keratin 5?
Hair cells go through terminal differentiation but fail to orient properly. Hairs are very thin- with disoriented zig zag pattern structure. After day 6. the rate of sebacous gland growth in k5-R2b barely increasing whereas control glands coninued to grow
What happens to the mouse phenotype over time?
The mice got older the hair phenotype become more pronounced
How did they show that Fgfr2b in post-natal skin development and in adult skin homeostasis?
Epidermal hyperthickening develops with age, and 10% of mutant mice develop spontaneous papillomas, demonstrating the role of Fgfr2b in post-natal skin development and in adult skin homeostasis
What happens to mice with the Fgfr2b mutation when exposed to chemical carcinogenic insult?
They show greater sensitivity, displaying several oncogenic ha-ras mutations with dramatic developement of papillomas and squamous cell carcinomas. They have an increased imflammation in the skin, with an increased number of macrophages and γδT (gamma delta T) cells with abnormal morphology
What novel role of Fgfr2b does removing the gene in mice describe?
The first evidence of a tyrosine kinase receptor playing a tumor suppressive role in the skin
What does CRISPR stand for?
clustered, regularly interspaced, short palindromic repat systems
What is the function of CRISPR in nature?
Provides prokaryotics with adaptive immunity to viruses and plasmids
Who were the co-discoverers of the application of CRISPR?
Emmanuelle Charpentier and Jennifer Doudna (Nobel prize in Chemistry 2020)
How does the bacteria start the CRISPR process?
The bacterium inserts a piece of DNA from a virus in the CRIPSR section of the genome. Between each viral DNA is a repeated sequence
What happens to the bacterial DNA which contains the viral DNA and the repeated sequences?
The CRISPR DNA is copied to make a long RNA molecule
What happens to the long RNA containing the viral sequences?
TracrRNA fits with the repeated section of CRISPR RNA like a piece of a puzzle. When tracrRNA attaches to to CRISPR RNA, the scissor protein Cas9 also links to the complex. The long moleculenis then cleaved into smaller pieces called RNase III
What happens to the CRISPR cas9 complex containing a single unique section of RNA corresponding to a virus?
The finished genetic scissors contain code from a single virus. If the bacterium is reinfected by the same virus, the genetic scissors will recognide and disarm the virus ny causing a double stranded break in the virus DNA
What are the 6 functional domains of the Cas9 protein?
Rec 1
Rec 2
Bridge Helix
PAM interacting
HNH
RuvC
Which domain of Cas9 binds to the gRNA?
Rec1 binds to the gRNA and the conformaitonal shape of the whole protein changes to allow it to bind to DNA. The Cas9 can only encircle DNA when bonded to gRNA
How does CRISPR know when to create a double stranded break?
It can bind to any NGG section and cause the DNA to unravel but will only cut when the sequence of the gRNA is complementary to the DNA sequence, due to being able to form a tight enough bond
What are the 3 main steps for using Cas9 in gene editing?
A Cas9:gRNA complex can bind to a target DNA sequence
Cas9 Nuclease produces dsDNA break
DNA repair, often by Non-homologous endjoining
What are the 4 different outcomes of Non-homologous gene editing?
Wildtype- Recovers the original DNA sequence
Insertion- During the cut back and reforming, a new DNA sequence is added
Deletion- During the cut back and reforming, a section of DNA is removed
Frameshift- During Insertion or deletion you could potentially cause a frameshift if it happens during a coding region of DNA
What is the key outcomes of NHEJ?
It is unpredictable but often results in a gene knockout
How can uncut DNA plasmids show to different band sizes on a Gel Electrophoresis?
The plasmid spends a lot of its time supercoiled tightly. When Gel electrphoresis the supercoiled plasmids travel further than the circular plasmids
How did Charpentier and Doudna show how and what was needed for CRISPR to cut RNA?
The showed that the plasmid, Cas9, the specific crRNA, tracrRNA and Mg2+ were need to cut the plasmid
What was the difference in gel electrophoresis in both circular and pre-linearized plasmid DNA?
Circular only has one band
Pre-linearized has two bands, with one being a lot smaller than the other
How did they show that you could replace the seperate crRNA and tracrRNA with a synthetic crRNA-tracrRNA chimera?
They joined the two together using a linker loop
What are the potential issues with using CRISPR in mammal cells?
Nuclear entry
Codon optimisation
Size of human genome
Dealing with chromatin
How did they use translational fusion to determine if CRISPR worked?
The expression of Cas9 also had a GFP attached to it as they both have the same promoter. Then everywhere you could see the GFP, the Cas9 protein would also be attatched
How did they find out whether the CRISPR had worked in mammalian cells?
The use of GFP and DAP (DNA staining), if you merged the images of both of them seperately and the coloured regions overlapped then the CRISPR worked
How did nuclear localised signals impact CRISPR results?
Only 5’= didnt work
Only 3’= does work
Both 5’ and 3’= Is the best, almost perfect nuclear translocation
What did initial experiments show with the difference between chimericRNA or the natural crRNA with tracrRNA in mammalian cells?
That overall the chimericRNA was less effective than the naturally occuring crRNA and tracrRNA, with variations depending on where you cut
What is the main outcome of HDR with respect to CRISPR?
You provide the DNA that has been broken with a template that you would want added in between
What was the Homologous recombinant efficiency of adding DNA with the use of HDR?
It was only 0.7%
How did they demostrate that they could add DNA to region that was cut?
They made the addition of the new gene create 2 new restriction sites meaning they could be seen on a gel electrophoresis
What are the pros and cons of TALENS?
They are slightly easier to use than Zinc Finger but still requires to be engineered for each new edit site
What are the advantages of CRISPR cas9?
Cheaper, quickier and easier to make
Cas9 multiplexing is possible
What are the limitations of Cas9 binding?
The requirement for the selection of Cas9 target sites is the prescence of a PAM sequence directly 3’ of the 20bp target sequence
Each cas9 ortholog has a unique PAM sequence, for example SpCas9 requires 5’-NGG PAM sequence - though it humans NGG occurs every 8-12bp on average
What are the limitation of using Cas9?
Never 100%
Never 100% accurate - off targets
Indels can be unpredictable
Knock-ins, difficult to engineer
Delivery of CRISPR/Cas9 as therapy to mature cells is difficult
What is InDelphi?
A machine learning that tries to predict the error percentage of using CRISPR on human cells
What are the applications of CRISPR?
Genome wide screening
Animal models
Cell therapy
Antimicrobials
Agriculture
Gene drives
