Techniques of Genomic Manipulations Flashcards
(54 cards)
Techniques of genetic manipulation of a random gene
Random mutagenesis screening using chemical mutagens
Random mutagenesis using transposons
Large scale RNAi screening
Techniques of genetic manipulation of a specific gene (ad hoc approach)
Exogenous expression of a gene via transgenesis
Targeted disruption of gene via HR (knock out)
Targeted modification of a gene via HR (knock in)
RNAi
Example to figure out what part of the enzyme is the active site
What can be modified/altered when interfering with a gene
Modifying expression level of a gene (under/over expression)
Modifying expression pattern of a gene (in time and space)
Modifying protein product of a gene (CDS mutations)
What does throughput mean?
How many experiments can physically be conducted
If phenotype is obvious from first look = high throughput screen
Time consuming experiment = low throughput
Eyeless gene in drosophila
In drosophila: wild type is red eyes
Mutant is eyeless
Eyeless is responsible for formation of the eye
If eyeless gene is expressed in other parts of the body, an eye will form there
How does EMS work?
EMS (Ethyl methanesulfonate) creates mutations in the genome
GC base pair –> EMS adds a group to O of G base –> creates O5-ethylguanine that is recognized as an A base –> Now is an AT base pair
Allows precise random modifications
EMS in drosophila
EMS given to male drosophila and modifies all cells (incl. Germline)
Male flies mated to female
Gives rise to mutant progenies
Mutant crossed with a balancer
Mutant mapping and identification of genes
Advantages of using EMS
Safe to use
Very efficient
Creates many mutations (on average one mutation every 5-10kb)
Creates random mutations but one mutation may be interesting to study
Drosophila life cycle
Drosophila are easy to work with due to quick life cycle
Life cycle lasts 12 days at 25 degrees
Male and female fly mate
Female flies are responsible for choosing their partner
Female fly lays embryos
Takes 24 hours for embryo to give rise to larvae
Larvae stage last for 5-6 days: eats continuously, grows in size
1st, 2nd, 3rd instar larvae
Metamorphosis: once larvae has grown to appropriate size grows pupil case around it
Get a new adult
Takes 24 hours for new adult to be sexually mature
Drosophila is good because have enough time to study larvae and adult flies
Genetic screenings in drosophila sorted by throughput
Random mutagenesis with EMS and then screening for phenotype development
Medium throughput = Olfactory (check if flies react to an odor)
Low throughput (time consuming)= Response to pain/body wall muscle (need to teach the fly, wait a few days, see if fly recalls information)
High throughput (efficient): Lethality, body size
Transposable elements for mutagenesis
Random mutagenesis
Transposable elements are fragments of DNA that can insert into new chromosomal locations
Often make duplicate copies of themselves in the process
Comprise 50% of human DNA
Responsible for spontaneous mutation, genetic rearrangements, horizontal gene transfer of genetic material
Speciation and genomic change
Cells must repress transposition for genetic stability
P-elements in drosophila
Drosophila has a particular type of TES called P-elements
P strains are found in the wild
M strains are found in the lab
Crossing P flies to M flies results in sex selected hybrid dysgenesis
Meaning that the effect of transposons on the genome will be different when crossing male transposon (P) with female (M) or female transposon (P) with male (M)
Sex-specific alternative splicing of P-elements in Drosophila
Gene for transposon can also encode for its own repressor via sex-specific alternative splicing
P-element contains 4 exons separated by 3 introns
In germline of female: intron 3 remains –> shorter protein (66kD) which is a repressor –> no transposable elements
In germline of male: complete splicing –> full length protein (87kD) –> transposase –> can jump around in the genome –> introduce random mutations –> sterile progeny
P male x P female cross
Repressor from P female prevents transposition of all P-elements
P male X M female cross
P element synthesizes transposase and M female does not produce repressor –> sterile progeny
M male x P female cross
Repressor from P female prevents transposition of all P elements
Transgenesis by transposons in drosophila to make transgenic animal
Use two plasmids to provide control
One is a vector and one is a helper
Vector: Recognition sequence + gene of interest (white+ that gives red eye colour)
Only injecting vector would give no red eyed progeny
Helper: contains transposase which recognises recognition sequence (inverted repeats) and integrates gene of interest into the genome randomly
Cross with white eye host strain
Select transformants
Don’t put transposase and gene in the same plasmid because we want to limit transposase activity
Injecting a developing drosophila embryo
Start with single cell fertilized egg (zygote)
First nuclear division (syncytial blastoderm) to create many nuclei in the same cytoplasm/cell
Nuclei migrate to periphery (cellular blastoderm) –> want to inject here to make germline changes
Cell division to create embryo
How can the P element be used for Transgenesis in Drosophila?
P-elements insert themselves in Drosophila genome
P-elements can be used to insert a gene of interest
Transposase must be present at moment of injection
But transposase must not be present after injection or for too long
Inject two independent plasmids in the embryo
One encoding for transposase and one with gene of interest flanked by recognition sequences
Gene of interest will insert itself in germline and progeny will inherit it
Stable transgenic is made
Transgenic individuals can be recognised by a collateral dominant marker
Usually mini white gene conferring red eyes
What would happen if a transposable element inserts itself in specific regions in the genome?
Intergenic region: Influences expression
Between enhancers: may interfere with ability of TFs to bind enhancers
Before/after 5’UTR: mutant gene/frameshift so protein is not produced. Most common place of P-element insertion
Junction between exons and introns: mutant gene/frameshift so protein is not produced
Within an intron: normally spliced away so doesn’t affect product but can affect splice site
Advantage of P-element insertion mutagenesis
Easier to find culprit
Random point mutations everywhere in the genome leads to specific phenotype
Need to identify gene causing the phenotype using gene mapping which takes very long
Using P-elements tells you that gene causing phenotype is the gene that contains the P-element
We know the sequence of the P-element
GAL4-UAS system
GAL4 is a transcription factor activator that binds to UAS cis-regulatory sequence
GAL-4 randomly inserted next to a to enhancer using P-elements
GAL4 can insert itself into 5’UTR of a gene so it is then regulated by the enhancer of that gene
GAL4 is now expressed in particular cells/tissues
Example if it is a gene expressed only in macrophages then all macrophages in transgenic animal will express GAL-4
Use P-element to insert UAS coupled to the gene X
So wherever GAL4 is produced (specific tissue) it will bind to UAS leading to expression of gene X
Cross male with GAL4 and female with UAS so part of the progeny will have both and express gene X
Transgenic mice
Microinject DNA into the nucleus in the egg as soon as it has fertilised
Implant embryo in foster mother
Embryo gives rise to transgenic progeny
This technique is quite inefficient
Only 5% or less of treated eggs become transgenic progeny
Need to check mouse pups for DNA (by PCR or southern blots), RNA (by northerns or RT-PCR) and protein (by western or by some specific assay)
This takes very long and is very expensive in mice vs in flies experiments are quicker
Gene targeting by Homologous recombination
Efficiency of transgenesis is low in every animal
Exploits mechanism of DNA repair that are endogenous to the cell
Works on many organisms from bacteria to mice
Plasmid containing homologous regions that pairs with homologous regions in the genome –> activates homologous repair machinery leading to integration of the gene in the targeted region
Requires access to a stem cell that will become an embryo
Knock out: Can disrupt/destroy the target
Knock in: Can change gene sequence (ex. Changing sequence of changing isoform)
Make it more efficient by adding antibiotic resistance gene
Inject DNA in blastocyst –> creates embryonic stem cells –> add antibiotic to petri dish –> select for cells that have incorporated the gene (successful knock-in or knock-out)
