Reverse Genetics Flashcards
What does reverse genetics seek to find?
phenotypes liked to specific sequences of DNA
deleting, disrupting or producing mutations in specific gene may reveal phenotypes that give clues to functions
What are the three steps in reverse genetics?
- Alter gene in vitro
- Introduce into cell
- Determine phenotypic effects
What are the different ways to knockout a gene? (5)
- Gene knockout
- replace with expressed in wrong tissue
- certain genes deleted in certain tissues
- slight changes in protein structure
- observing the effects of not having the genes
How do you alter a gene in vitro?
string nucleotides together in lab
create DNA sequence
use recombinant DNA techniques
- design a primer
- extension step happens
- included mutation
go through multiple cycles, plasmid created as PCR product
How do you introduce DNA into cell?
Take in plasmid through pores
transfection not transformation in animals
direct uptake of DNA:
incubate DNA with component cells
- bacterial/yeast transformation
- animal cell transfection
methods to induce DNA uptake:
- electroporation
- microinjection
- virus-mediated
- ballistic (gene gun)
fate of the transgene:
- transient expression
- replaces on a plasmid
- chromosomal integration
How does transient transfection work?
promotor sequence will be expressed
eventually plasmid sequence will break down unlike Bacteria
How do you determine phenotypic effects?
not unusual for no effect
can see the wild type then compare with symptoms on mutant e.g. premature ageing
What is gene targeting?
transgenic organisms = genetically engineered organisms
transgenes = modified genes
DNA carrying mutated gene in cell, inserts into genome by homologous recombination
specific genes can be targeted by gene crossing over
create unique barcode sequences
then undergo homologous recombination and create a knockout
Targeting deletion of diploid yeast requires two marker genes
knock out both copies
introduce disruption cassette can undergo homologous recombination
targeted the gene and introduce to different genes on different strands
identify to show what copies have been knocked out
What is the marker recycling scheme?
using Credit/Lox recombination system
knocked out target gene but also removed the markers
can then go and knock out another gene
knock out multiple different strains
Targeted gene deletion or replacement of diploid animal
more complex
lines of mice with functional gene altered/removed called knockout mice
used to determine function of many mice genes
put targeted gene in embryonic stem cells
inject & will get different mice from hybridised embryos
What is CRISPR?
first coined in 2002
Clustered Regularly Interspaced Short Palindromic Repeats
high likelihood for mutation in the gene, usually non functional
What is step 1 in CRISPR?
Short viral DNA sequence is integrated into CRISPR locus
What is step 2 in CRISPR?
RNA is transcribed from CRISPR locus, processed and bound to Cas protein
What is step 3 in CRISPR?
Small crRNA in complex with Cas seeks out and destroys viral sequences
when reinfected, the incoming viral DNA is destroyed by the crDNA
What can CRISPR Cas9 be used for?
- Disrupt or knockout a gene or sequence of choice
- Add an altered version of the gene by using homologous recombination
if overloads cell with recombinant DNA containing a mutation
- Targeted genome engineering
- combined with gene insertion enhances homologous recombination - Off the shelf CRISPR systems
- commercially available systems for use in mammals
Ethical concerns with regard to humans
What are the ethical concerns with CRISPR?
use in humans
concerns for long terms wellbeing
What is targeted regulated expression of native genes?
Replace native promoter for YFG with promotor
Determine phenotype when over and under repressed
What are reporters?
clone reporter after promotor sequence
replace native open reading frame
level detected reflects reporter activity
can identify when an gene is on or off
What is RNA interference?
specific knockdown of gene expression
RNAi
translational repression
RNAi inactivation of specific gene prevents migration and coming together of egg & sperm in fertilised egg
Why do we need high throughput technologies?
because organisms are complex
none of the proteins will work in isolation
do all of the techniques in an efficient way
screen phenotypes together on a plate
using robotics
screen phenotypes together by competition
particular mutations associated with a barcode
more complexity
