reverse genetics Flashcards
reverse genetics approaches to seek
the phenotype linked to specific sequences of DNA (including genes)
how may the gene responsible for a certain phenotype be revealed
producing mutations in a specific gene may reveal phenotypes that give a clue as to its function
reverse genetics method simple (3)
1.alter the gene in vitro 2. introduce into cell 3. determine phenotypic effect
alter gene in vitro
- first get it synthesised
- use recombinant DNA techniques e.g. site directed mutagenesis
e. g. using restriction endonucleases
site directed mutagenesis
in reverse geentics oligonucleotide mediated site directed mutagenesis (SDM) is used. -plasmid is dentured and hybridised to a mutant oligo. Then this transformed plasmid is frown up in E.coli. -then the desired clone is isolated
introduce DNA into ells
- direct uptake of DNA
- electroportation
- agrobacterium tumefaciens-mediated
direct uptake of DNA
incubate DNA with competent cells -bacterial/yeast transformation -animal cell transfection
transfection
animal cells
transformation
yeast and bacteria
electroporation
the action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to open the pores in the cell membranes briefly. -microinjection -virus mediated
ballistic
gene gun cells with walls e.g. plants
gene gun is for cells with
walls e.g. plants
agrobacterium tumefaciens mediated
plants and some fungi
Fate of the trangene
-transient expression -replicates on a plasmid -chromosomal integration random but can also be targeted to a particular locus
DNA can be introduced to four different types of cells
somatic germ haploid diploid
how are transgenic cells detected
using selectable marker genes or dominant or recessive nature
what can be utilised for gene targeting
homologues recombination -occurs in meiosis -breakage and rejoining of DNA -reciprocal -genetic rearrangement
targeted gene disruption by homologous recombination
Circular homologous DNA with SEL is introduced. Select for cells expressing marker. Single crossover occurs within the gene on the chromosome. Therefore it is disrupted and not expressed
targeted gene deletion by homologues recombination
linear homologous dna with SEL is introduced. Select for cells expressing marker. Gene is deleted due to a double crossover on each side of the the gene
targeted gene deletion of diploid yeast requires..
two marker genes or a marker recycling system
marker cycling scheme
.
deleted genes can be replaced with a..
mutant gene and selectable marker by homologous recombination
targeted gene deletion of a diploid animals..
more complex
CRISPR
Clustered regularly interspaced short palindromic repeats
CRISPR-mediated immunity bacteria
step 1: short viral DNA sequence is integrated into CRISPR locus step 2: RNA is transcribed from CRISPR locus, processed and bound to a Cas protein step 3: small crRNA in couple with Cas seeks out and destroys viral sequence
CRISPR/ Cas9 for gene activation/repression
-activation domain can attach to complex and switch gene on -repressor domain can attach and turn gene off
CRISPR is not
100%; off target effects are possible
there are ethical concerns with regard to humans when it comes to
CRISPR/Cas9 for gene editing
targeted regulated expression of native genes
replace native promoter for YOUR FAVOURITE GENE (YFG) with a very active one, or a regulatable promotor e.g. GAL 1p -determine phenotype when over or under expressed
targeted regulated expression of native genes
replace native promoter for YOUR FAVOURITE GENE (YFG) with a very active one, or a regulatable promotor e.g. GAL 1p -determine function of certain gene when phenotype is over or under expressed
promotor activity effects
how much of a specific protein is being produced due to the rate of transcription
what can be used to determine promotor activity
reporters
example of reporters used to determine promotor activity
B-Galactosidase, Lucifer’s, GFP
promotor activity- reporters : process
Clone reporters are inserted (homologous recombination) after the promotor sequence, replacing native ORF (open reading frame). –> the level of GFP protein produced will show the activity of that specific promotor. –> e.g. if lots of GFP is produced then the activity of the promotor is obviously high
ORF
open reading frame
ORF
open reading frame An ORF is a continuous stretch of codons that do not contain a stop codon (usually UAA, UAG or UGA).
protein localisation and movement
tag with GFP -microtubules can be seen this way as well as mitochondria as as well as neurones
GFP
Origin: Bioluminescent jellyfish (Aequorea victoria): Autocatalytic, fluorescent protein In vivo reporter, high signal to noise, no enzymatic activity Many spectral mutants available (RFP, YFP, BFP etc.)
how can we avoid genomic editing using RNAi
instead of editing DNA, RNAi targets RNA, that is perhaps being over translated and causes its degradation -specific ‘knockdown’ of gene expression
‘knockdown’ of gene expression
meaning that instead of DNA being altered the translation of RNA of a specific gene is reduced e.g. using RNAi or siRNA
‘knockdown’ of gene expression
meaning that instead of DNA being altered the translation of RNA of a specific gene is reduced e.g. using RNAi or siRNA
we need high throughput technologies because..
organisms are complex and processes that take a long time are expensive
examples of high throughput technologies
pic
high throughput reverse genetic study using C.elegans RNAi
- each well contains E.coli expressing a different dsRNA–> target diff RNA
- C.elegans (worm) is added to the 96 well plate
- worms injest E.coli.
- resulting phenotypes are recorded and analysed we can screen phenotypes separately
dsRNA
dsRNA forms the genetic material of some viruses (double-stranded RNA viruses).
during high throughput reverse genetic study using C.elegans RNAi, phenotypes can be
screened separately
Genome wide screens for fitness using a large pool of barcoded yeast deletion mutants
a pool of barcoded yeast mutants, each deleted for a diff gene is grown in a condition of choice.
Then purified.
The relative abundance of each barcode is then recorded . -screen phenotypes together by competition
during genome wide screens for fitness using a large pool of barcoded yeast deletion mutants phenotypes can be
screened together by competition
in conclusion reverse genetics
seeks to find the phenotype linked to specific sequences of DNA (including genes) whereas forward genetic seeks to find the DNA sequence responsible for certain phenotypes
