Viral vectors, RNAi interference, Antisense Flashcards
Name the two approached to studying gene expression:
Gene overexpression
Gene knockdown
Describe the chemical method of altering gene expression and its major limitation:
Chemical –> Cell cultures/animal models –> Endogenous gene increases/decreases
Other genes/proteins may also change in expression
Describe the genetic method of altering gene expression:
Genetic (transgenic, viral vector) –> Model, cell culture, animals –> Endogenous/exogenous gene expression increases/decreases (cDNA, RNAi antisense)
Describe the difference between endogenous and exogenous gene expression methods:
Endo - put in TF, binds to promotor, observe change in gene expression, with no change from basal level with controls
Exo - isolate gene interested in studying, express in cell type (e.g. human form in mice)
Describe a limitation of genetic methods for altering gene expression (think animals vs. humans):
Transgenes are often not from the same species as the recipient, thus need to be transfected, electroporated, viral vectored in etc.
Describe transfection using DNA plasmids:
Plasmids act as a carrier for the GoI (transgene)
Transgene under prmotor control
DNA (plasmid) mixed with transfection reagent
DNA/reagent mix is applied to cells
Visualised by GFP tagging, immunocytochemistry, western blotting etc.
Describe viral vectors:
Genetically modified viruses
Exploits natural ability of viruses to infect mammalian cells, viral genomes genetically modified to carry and deposit GoI inside cell
Viral genes involved in replication are removed
Name some viral vectors:
Oncorectrovirus, lentivirus, adenovirus, adeno-associated virus (small, non-pathogenic, vector of choice for human gene therapy)
Describe the THREE unique properties of each viral system:
- Differences in DNA insert size/capacity
- Tropism (selectivity) for a given cell types (dependent on the ligand present on virsu surface and whether the target cell expresses the receptor for that ligand)
- Differing ability to transduce dividing and non-dividing cells
Describe the three-step process of generating a viral vector:
- Molecular cloning (using bacterial plasmid systems to genetically engineer viral genome)
- Vector packaging (using immortalised cell lines to assemble viral vector particles - cell factories)
- Viral vector purification (from cell lysate to give pure viral vector stock)
Describe the ways of developing an adeno-associated viral (AAV) vector:
A. AAV expression plasmid
Removed rep and cap genes, replace with therapeutic gene cassette (only retains AAV ITRs- packaging signal)
B. AAV packaging plasmid(s)
Incorporate rep and cap into separate plasmids together with adenoviral genes essential for packaging
Describe AAV vector packaging in the HEK293 line:
Transfect cells with AAV expression plasmid and helper plasmid(s)
Culture cells
Harvest, wash and lyse cells
Treat with DNase and RNase
Vector is purified from cell lysate using affinity column chromatography or ultracentrifugation using density gradients
Purified vector is concentrated and dialysed
Names the three steps of vector characterisation:
- Estimation of titre
- Estimation of purity
- Assess functionality
Describe how to estimate vector tire:
Genomic tire- use quantitative RT-PCR based methods to measure the number of vector genomes per volume of vector stock
Describe how to estimate vector purity:
SDS-PAGE of vector stock and coomassie blue staining
Western blots - use an antibody to detect viral capsid proteins
Describe how to assess vector functionality:
Apply vector to cultured cells
Immunocytochemical western blotting or ELISA techniques can be used to detect and quantify transgene protein
Name 4 uses for viral vectors:
Gene function studies Generating new animal models of disease Gene therapy (therapeutic application) Validating/identifying gene targets (e.g. viral vector based rat model of AD)
Why would we want to knockdown a gene?
To definitively prove that gene is involved in a particular function (reverse phenotype)
Name three ways of knocking down a gene:
- Mutagenic screens (drosophila, zebrafish, c.elegans) - creats a mutagenised population, look for phenotype and try to match to specific gene
- Antisense techniques
- RNA interference
Describe antisense technology:
Antisense oligonucleotides (13-25 bases) single stranded fragments of DNA that hybridise to an mRNA of interest.
Describe the two mechanisms by which antisense technology downregulates gene expression:
RNase-H dependent degradation of mRNA (up to 85-90% knockdown using sequences against any region in mRNA)
Steric blocker oligonucleotides physcially prevent translation or inhibit progression of splicing (efficient only when targeted to the 5’ or initiation codon region)
Name the types of antisense technologies:
Phosphodiester oligonucleotides
Phosphorothioates
Peptide nucleic acids (PNAs)
Morpholinos
Describe antisense phosphodiester oligonucleotides:
Use limited, rapidly degraded
Describe antisense phosphorothioates:
RNase-H dependent mechanism
Non-bridging O2 replaces by a sulfur at each phosphorus in the oligo
Nuclease stable and easy to synthesise and highly soluble
Weakness: Phosphorothioate backbone induces sequence-independent effects on expression of heparin growth factor family members
Describe antisense peptide nucleic acids (PNAs):
Not substrates for RNase-H
Contain an uncharged, flexible polyamide backbone of repeating N(2aminoethyl)glycine units to which bases are attached
Stable duplexes formed with nucleic acids - single or ds DNA or RNA
No negative charges leading to high affinity for nucleic acid binding
Describe antisense morpholinos:
Not substrates for RNase-H
Deoxyribose moiety replaced by morpholino ring
Stable, should have fewer non-specific effects compared to phosphothioate backbone derivatives
Potential weakness: Does not complex with cationic lipids - delivery could be an issue for mammalian cells
Describe RNA interference (RNAi):
A form of post-transcriptional gene silencing in which specific sequences of ds RNA can be used to knockdown the expression of a gene target
Commonly used as a tool to investigate links between genes and their function, also being developed for therapeutic applications
History of RNAi (ds more potent than antisense)
Describe the biochemical mechanism of RNAi:
- dsRNA introduced into cell
- DICER digests dsRNA into ~21bp dsRNA (short-interfering RNAs;siRNAs)
- siRNAs are integrated into RISC (RNA-induced silencing complex)
- siRNAs undergo strand separation - antisense strand binds to its complementary/target
- Endonucleases within the RISC degrade target mRNA
Describe how RNAi could be used as a tool for manipulating gene expression in mammalian cells:
Introducing long dsRNA initiates cellular interferon response that leads to apoptosis
For research/therapeutic applications RNAi can be delivered by siRNA, shRNA and miRNAs.
Describe synthetic short-interfering RNA (siRNA):
siRNA are typically 21-23bp of dsRNA with a dinucleotide overhang at the 3’ end
Algorithms are used to design RNAi sequences
Delivery:
Cell culture (transfection, electroporation)
In vivo models (injection, direct application)
RNAi effect is transient (3-7 days), partial to full knockdown of gene expression
Not much use in animals
Describe synthetic short-hairpin RNA (shRNA):
Like siRNA but form a hairpin structure chopped off via DICER allowing the cell to continue generating these molecules
Delivery via plasmids or viral vectors
Effects
In vitro - transient knockdown
In vivo - long-term (>1 week) effects using viral vectors
Describe microRNAs:
Endogenous RNAi pathway in animal cells
~21-mer small RNA molecules from non-coding RNA (introns, independent miRNA genes)
Regulate gene transcription by binding to the 3’-untranslated regions of specific mRNAs
Key regulators of early development, cell proliferation, cell death, apoptosis, cell differentiation, brain development
Describe miRNA processing:
Transcription of primiRNA Processing into premiRNA in the nucleus by Drosha Export of pre-miRNA to the cytoplasm Dicer complex processing miRNA strand selection by RISC
Describe miRNA reduction in steady state protein levels (3 types):
Translational repression: Imperfect duplex reduces protein expression without an effect on corresponding mRNA levels mRNA degradation: Perfect duplex, no mRNa=no protein Transcriptional regulation: Effects on chromatin methylation
Name some potential applications of RNAi:
Testing hypotheses of gene function
Validation of targets for drug development
New therapeutic approaches to treat diseases
Artifical miRNAs can be used as a tool for gene function studies.
