manipulating cells in culture; generation of in vivo gain-of-function and loss-of-function models Flashcards
what is loss of function?
reduced activity
- in heterozygous state to half normal levels of the protein product or complete loss of the gene product
what is gain of function?
increased levels of gene expression
- development of a new function of the gene product
how is gene silencing achieved?
- RNA interferance (RNAi)
- CRISPR/Cas9
how is gene overexpression achieved?
- expression vectors
- CRISPR/Cas9
siRNA (small interefering RNA)
- chemically synthesized
- consists of two RNA strands (sense and antisense)
- transfection to the cytosol
- siRNA integration to the RISC (RNA induced silencing complex)
- strand separation within RISC
- antisense strand hybridises to the complementary (target) mRNA
- cleavage of targeted mRNA within RISC
- further degradation by other endogenous nucleases
- transient silencing
short hairpin RNA (shRNA)
- synthesized within the cell, two RNA strands linked by a short loop
- DNA plasmid transfection or lentiviral transduction
- transcription, export to cytosol
- DICER removes the loop (siRNA production), siRNA loading to RISC, removal of one RNA strand
- target mRNA with complementary sequence, cleavage of mRNA + further degradation
- stable knock-down cell lines
three variations of CRISPR
- genome engineering with Cas9 nuclease
- genome engineering by double nicking with paired Cas9 Nickases (= HDR)
- localisation with defectie Cas9 nuclease
why do we carry out expression vectors to trigger gene overexpression?
- assess effect of having a protein/RNA of interes in excess
- overexpress mutant protein
- recover WT phenotype in mutant/defective background
- study function of heterologous proteins
- analysis of interacting molecules (co-immunoprecipitation)
what are the five types of vectors/plasmids?
- cloning plasmids
- expression plasmids
- gene-knock down plasmids
- reporter plasmids
- viral plasmids
what are cloning plasmids?
facilitate cloning of DNA fragments
- bacterial resistance gene, origin and MRS
what are expression plasmids?
used for gene expression
- promoter, transcription terminator sequence, inserted gene
what are gene knockdown palsmids?
reduce expression of endogenous gene
- shRNAm promoter for expression of short RNAs
what are reporter plasmids?
study the function of the genetic elements
- promoter, reporter gene (Luciferase, GFP)
what are viral plasmids?
deliver genetic material into target cells
where are epitope tags found?
carried by two commonly used mammalian expression vectors
- usually in 5’ region after MCS (multiple cloning site)
what are epitope tags?
method of expressing proteins whereby an epitope for a specific monoclonal antibody is fused to a target protein using recombinant DNA techniques.
- fusion gene cloned into an appropriate expression vector for the experimental cell type and host cells are transfected
GFP (green fluorescent protein)
- naturally produced in jellyfish; Aequorea victoria
- discovered in 1960s
- source of bioluminescence when exposed to UV light
why is using GFP useful?
- tags cells and helps us trace to a target protein
- acts a reporter gene, link it to another gene to show if it is expressed
what is site-directed mutagenesis?
introduces point mutations
- a method to create specific, targeted changes in double stranded plasmid DNA.
- used to study changes in protein activity that occur as a result of the DNA manipulation.
process of site directed mutagenesis
- mutant strand synthesis done by denaturing DNA template, annealing primers with desired mutation (1hour)
- Dpn I digestion of template , digestion of parental methylated and hemimethylates DNA with new Dpn I enzyme (5mins)
- transform mutated molecules into competent cells for nick repair (1.5hours)
methods for DNA and RNAi delivery; chemical transfection
- uses ; calcium phosphate, cationic polymers, lipofection, - transcient/stable transfection
- suitable for sensitive primary cells
methods for DNA and RNAi delivery; physical transfection
- microinjection
- electroporation (nucleofection-primary cells)
- transient/stable transfection
- not suitable for sensitive primary cells
what does transient transfection mean?
expression of foreign DNA for a limited time (24-96hours)
- NO DNA INTEGRATION
what is a stable transfection?
integration of foreign DNA in the genome of the cells
summary of manipulating genes in culture
- generating gain or loss of function in vitro models to study the functional impact of genetic variants
- different types of plasmids used for different purposes; from DNA cloning to protein expression
- expression vectors can contain tags e.g. GFP
- different cell transfection methods can be used to introduce genetic material inside cells in culture, they can be chemical or physical and trasnfection can be transient or stable
- site directed mutagenesis is used to introduce mutations in expression plasmid
- siRNA and shRNA are short RNA molecules that when introduced in host cell can downregulate gene expression by targeting mRNA in transient or more stable way.
