Test 3 Flashcards
Genomics
Chromosomes/DNA
DNA copy-number assessment
– Comparative genome hybridization to DNA microarray
Mutation screening
- DNA sequencing
- Mass-spectrometry-based genotyping
- Mutation-specific PCR
Transcriptomics
DNA/mRNA
Gene-expression profiling
- DNA microarray
- Multiples PCR
MicroRNA-expression profiling
- DNA microarrays
- Multiplex PCR
Proteomics
Protein
Proteomic profiling
– Mass spectrometry
Phosphoproteromic profiling
– Mass spectrometry after immunoprecipitation specific antibodies
Methods of determining the sequence of DNA
Sanger sequencing (chain termination/dideoxy method)
Shotgun sequence method
2nd gen (eg. pyrosequencing)
Sanger Sequencing: protocol
- Denaturation
- Primer attachment and extension of bases (like PCR)
- Termination w/dye-labeled ddNTPs
- Gel electrophoresis
- - Run four separate reactions each with different ddNTPs
- - Run on a gel in four separate lanes
- - Read the gel from the bottom up
(Can be automated)
Sanger Sequencing: disadvantages
Only good for 500-750bp rxn
Expensive
Takes a while
The human genome is ~3 bil bp
Why was the Human Genome Project started?
Began in 1990
To study:
Human evolution
Nature v. nurture
Causes of disease
Shotgun Sequencing: protocol
Used to sequencing whole genome
- DNA extraction
- DNA fragmentation: DNA is broken up randomly into smaller fragments
- - Clone into vectors
- - Transform bacteria, grow, isolate vector DNA - Sequence the library: dideoxy method produces reads
- Reconstruction: assemble contiguous fragments
- - Look for overlap of reads
Pyrosequencing
Visible light is generated and is proportional to the number of incorporated nucleotides.
– Double peak heights indicate incorporations of two nucleotides in a row
DNA + NTP + DNA pol = DNA-NP = PPi
PPi + APS + ATP sulfurylase = ATP + SO4^2-
ATP + Luciferin + O2 + Luciferase =
AMP + PPi + Oxyluciferin + CO2 + LIGHT!!!!
Pyrosequencing:
disadvantages
Smaller sequences
Nonlinear light response after more than 5-6 identical nucleotides
Clonal Single Molecule Microarray: protocol
Attach DNA to flow cell
1) Prepare genomic DNA sample: randomly fragment genome DNA and ligate adaptors to both ends of the fragments
2) Attach DNA to surface: bind singel stranded fragments randomly to the inside surface of the flow cell channels
Bridge Amplification
3) Bridge amplificaiton: add unlabled nucleotides and enzyme to initiate solid phase bridge amplification
4) Fragments become double-stranded
Cluster Generation
5) Denature the double-stranded molecules
- - repeat cycles of solid phase bridge amplification
6) Completion of amplification: on completion, several million dense clusters of double-stranded DNA are generated in each channel of the flow cell
~1000 molecules/ 1um cluster
~ 20-30,000 clusters/tile
~ 40 M cluster/flowcell
Sequencing by Synthesis (SBS): protocol
Cycle 1:
- Add sequencing reagents
- First base incorporated
- Remove unincorporated bases
- Detect signal
Cycle 2-n: add sequencing reagents and repeat
(Same as reversible terminator chemistry?)
Reversible Terminator Chemistry
All 4 labelled nucleotides in 1 rxn
Higher accuracy
No problems w/homopolymer repeats
Steps:
- Incorporation
- Detection
- Deblock: fluor cleaved/removed
(same as sequencing by synthesis (sbs)?)
Base calling from images
The identity of each base of a cluster is read off from sequential images
1x flowcell = 8 lanes 1x lane = 3 columns (rows) 1x column = 100 tiles 1x tile = 4 images/cycle = 345,600 images for a 36-cycle run
Glass Slide Array/Affymetrix Gene Chip: protocol (general)
- RNA extraction
- Reverse transcription: cDNA reaction, purfication, and labeling by IVT
- Fragmentation (heat + Mg2+)
- Hybrization (label incorporation, Cy3/5)
- Washing
- Laser scanning
(Glass Slide: scan cy5 channel + cy3 channel & overlay images)
(Affymetrix GeneChip: photolithography) - Quantify
— load into database —
computer analysis –> bioinformatics
Glass Slide cDNA Microarray: advantages/disadvantages
Hybridize two samples/chip for direct comparison of samples
Non-standardized production can affect reproducibility (although there are now many quality-controlled commercial arrays available)
Longer sequences can have cross-hybridization with other genes
Don’t necessarily need to know all the genes in the genome. Can use unsequenced ESTs, for instance.
Affymetrix GeneChip: advantages/disadvantages
Limits 1:100,000 transcripts, ~5 transcripts/cell
Internal control lane with mismatch olgionucleotide probe cells to prevent false positives.
Can hybridize only one sample/chip. No direct comparisons of 2 samples.
Standardized production tends to give good reproducibility.
Limited amount of probe sequence can be problematic, but can also be helpful in limiting cross-hybrization
ChIP-chip: protocol
ChIP: chromatin immunoprecipitation
- Add formaldehyde and sonicate DNA to ~1kb
—- 1/2 sample —-
2. Add specific antibody
3. Immunoprecipitation
4. Reverse cross links and purify DNA
5. Amplify and label with Cy5
Hybridize to microarray
- — 1/2 sample —-
2. Reverse cross links and purify DNA
3. Amplify and label with Cy3
4. Hybridize to microarray
Tiled microarray
Cover a genomic region (or whole genome) at hight coverage.
Probes are designed to cover virtually every basepair of the sequence, usually excluding only simple sequence repeats.
In this way, there is no bias toward known transcribed regions.
Probe size and spacing determines resolution of the array.
Antibody Array: protocol
- Extract proteins from 2 samples
- Label 2 samples with Cy3/Cy5 and then mix
- Incubate on the array (with antibodies)
- Scan array
Single Cell Transcriptomic Approaches (3x): protocols
1)
- Introduce cell-unique barcoded primer beads
- Intracellular RT makes barcoded cDNA beads
- Cleave barcoded cDNA from beads
- Sequence barcoded cDNAs
2)
- Introduce primers and reagents, preform RT and RCA
- RCA generates ‘rolonies’ directly in cell sections.
- Sequence rolonies directly in cell sections.
3)
- Capture RNAs on surface bound primers, perform RT
- Eliminate all cell debres, except bound cDNA
- Single molecule sequencing of cDNA
Analysis Methods
T-test
ANOVA
Mann Whitney U Test
Type I error
alpha
false positives
p-value
Single gene analysis
Molecular cloning
Bacteria are usually the host cell used for basic cloning experiments
Applications of biotechnology
Virus-resistant crop plants and livestock
Diagnostics for detecting genetic diseases and acquired diseases
Therapies that use genes to cure diseases
Recombinant vaccines to prevent disease
Biotechnology can also aid the environment through bioremediation
Definition of biotechnology
Any technique that uses living organisms or substances to make or modify a product, to improve plants, animals, or microorganisms for specific uses
Process/Workflow of bacterial cloning
Source = DNA target
Fragmentation
Ligation to linear cloning vector to for chimera
Introduce DNA into host cell
Isolate cells with cloned gene on agar plate
- suspension of bacteria plated and spead
- isolated colony dervied from single partent cell = clones
Allow to replicate and produce protein from cloned gene (binary fission)
Restriction Enzymes
Type II restriction endonucleases (most common)
- cut DNA like scissors at specific sites called restriction sites
- cut across the sugar-phosphate backbone of DNA
- DNAse cuts into random pieces, while Hae III has specific site cleavage (cut in same place)
Recognition/cleavage sites of type II restriction enzymes
Cuts usually occurs at a palindromic sequence
Homodimeric ptns to help find palindromic sites.
SmaI or HindII: produces blunt ends (more difficult to ligate together)
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends (good for adding target DNA with complement ends)
5’ GAATTC 3´
3´ CTTAAG 5´
Isoschizomers
(‘iso-sticky’)
Cut at same sequence but different end configurations
“pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.” ~ Wikipedia
Isocaudomers
Different recognition site but give same cleavage products
“pairs of restriction enzymes that have slightly different recognition sequences but upon cleavage generate identical termini… an enzyme that recognizes a slightly different sequence, but produces the same ends.” ~ Wikipedia
Meganuclease I-Sce I
Unusual restriction enzyme
Homing endonuclease I-Sce I
18-base pair sequence TAGGGATAACAGGGTAAT
4 base pair 3’ hydroxyl overhang.
Sequence will occur once in every 6.9 x 1010 base pairs.
This sequence does not normally occur in a human or mouse genome.
Enzyme encoded by an intron in yeast mitochondria
Methylation Sensitve Restriction Enyzmes
HpaII: only cuts when non-methylated (methylation sensitive Restriction Mapping)
Isoschizomer is MspI: Does not matter whether Meth or not
Separating Restriction Fragments and Visualizing DNA
Pieces of DNA are generated by restriction enzymes can be separated, viewed and manipulated based on SIZE using gel electrophoresis.
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html
Gel Electrophoresis
The gel is submerged in a buffer solution, and DNA samples are loaded in the wells.
Electricity is applied to electrodes at opposite ends of the gel to create an electrical field in the gel and the buffer.
Pores in the agarose catch the DNA pieces and slow down movement through the gel. Supercoiled DNA moves faster than nicked DNA form II.
Sugar-P backbone is negative, runs to postive.
Agarose gel and separation
Agarose percentage = pore sizes.
- Lower percentages = larger pores, (separating large DNA)
- Higher percentages = smaller pores. [smaller pieces of DNA]
What else affects resolution?
Voltage gradient used.
Visualizing DNA in gels
Stains [ethidium bromide] added to the gel to visualize DNA.
Ethidium bromide* molecules lodge in between the bases of DNA
‘Glows’ when an ultraviolet light is used
- Intercalator: DNA gets longer!
- ITS ALSO MUTAGENIC (genotoxic)
DNA ligase for joining DNA fragments together
Enzymes that cut with staggered cuts result in complementary ends that can be ligated together.
HindIII - leaves 5’ overhangs (“sticky”)
5’ --A AGCTT--3' 3’ --TTCGA A--5’ => 5’ --AAGCTT-- 3’ 3’ --TTCGAA-- 5’
Sticky ends that are complementary (from digests with the same or different enzymes) can be ligated together.
Sticky ends that are not complementary cannot be ligated together.
Plasmids
vehicles for cloning
Plasmids are naturally occurring extra-chromosomal DNA molecules.
Plasmids are circular, double-stranded DNA.
Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.
Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.
Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.
Copy #s vary: Low (10) to High (100s)
Cloning Vectors
A cloning vector is a plasmid that can be modified to carry new genes.
Plasmids useful as cloning vectors must have:
An origin of replication.
A selectable marker (antibiotic resistance gene, such as ampr and tetr).
Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).
Easy to purify away from host DNA.
Alkaline Phosphatase: Helps prevent recircularized plasmid (Fig. 3.1)
Incorporating plasmid to bacteria
Electroporation or someing equitable
Selection and Counterselection of transformed bacteria
1) Plate with antibiotic:
Only bacteria with plasmid antibiotic resistance will survive. Original colonies will have another resistence gene. But clones will not. So will have to plate onto second plate with second antibiotic, and clone colonies will not grow.
2) LacZ with blue and white selection
Ampicillin
Inhibits cell wall formation
Inactivated by beta-lactamase
Streptomycin
Blocks protein initiation complex formation and causes misreading during translation
Inactivated by phosphotranserfase
Tetracyline
Prevents binding of aminoacyl-tRNA to 30S ribosomal subunit
Resistence gene encodes an inner cell membrane portein that passes the antibiotic out of the cell and blocks the passage of the antibiotic through the cell wall
Bonferroni correction
The traditional way to confront multiple testing problem.
Instead of p
False discovery rate (FDR or q-value)
The expected proportion of false-postives among the postive results.
At q = 0.05, 50/1000 significantly changed genes might be false postives.
Clustering approaches to analysis
Divides or groups gene/samples into groups “clusters” based on similarities and differences.
Number of groups is user defined.
Algorithms:
Hierarchial clustering/tree
Kmeans clustering
Self organising maps
Distance Metrics
Distance between 2 expression vectors (relative amounts, rather than absolute values).
proteonomics
Large-scale study of proteins, particularly their structures and functions.
SDS PAGE
The speed of migration in an electrical field depends on the dimension, form, and charge of the molecules.
For deaggregation and denaturation of the proteins, SDS, beta-mercaptoethanol or DTT, and heat is used.
SDS (strongly anionic detergent) provides negative charge to the proteins
Silver staining v. Coomassie Blue Staining
2D gel
Isoelectric focusing gel based on charge/pH.
Then equilibrate in SDS, and apply orthogonal electric field (based on size)
How to study proteonomics
Confocal microscopy
Fluorescent resonance energy transfer (FRET)
Co-immunoprecipitation
Far western blot
(Utilizes biotin modificaiton of purified bait protein probe. Prey proteins separated in-gel or transferred to membrane can be probed w/biotinylated bait. Detection w/streptividin-horseradish peroxidase conjugate + chemiluminscence)
Yeast two hybrin assay
(Fusion of yeast reporter gene that is activated every time gene of interest expressed. eg. Lac Z/beta-galatosidase)
Mass analysis/spectrometry/Matrix Assisted Laser Desorption/Ionization (MALDI)/Tandem Mass (MS/MS)
Gas chromatography
Metabolomics and profiling methods
High-throughput analysis of metabolites
Simultanous measurement of the levels of a large number of cellular metabolites
Gas chromatography/Mass-spectrometry (GC/MS)
Liquid Chromatography/Mass-Spectrometry (LC/MS)
Nuclear Magnetic Resonance (NMR) Spectroscopy
What info do we get from: DNA, RNA, Protein, Metabolites?
DNA: the ultimate potential of a cell
- What is possible
RNA: the current direction of a cell
- What appears to be happening
Proteins: the functional capabilities of a cell
- What makes it happen
Metabolites: the limiting currency of a cell
- What is happening
Metabolic Profiling Methods: Gas Chromatography/Mass-Spectrometry (GC/MS)
In GC/MS, it may be necessary to first derivatize the sample to increase metabolite stability and volatility. The derivatized mix is then fractionated by a gas chromatograph that is coupled to a mass spectrometer.
The mass spectrometer scans the peaks emerging from the GC column at frequent intervals (~1 sec) and so acquires the mass spectrum of each peak, from which peaks can be identified and quantified. Mass spectrometry ‘weighs’ ionized individual molecules and their fragments. Molecules are identified from their fragmentation pattern and ‘weights’ (mass/ charge ratios – m/z values), with the help of mass spectra libraries, and can be quantified from peak size.
Metabolic Profiling Methods: Liquid Chromatography/Mass-Spectrometry (LC/MS)
In LC/MS (also termed high performance liquid chromatography, HPLC/MS) the samples are not derivatized before analysis and an HPLC instrument is used for separation. LC/MS is more suitable than GC/MS for labile compounds, for those that are hard to derivatize, or hard to render volatile. LC/MS is less developed than GC/MS. A closely related method is capillary electrophoresis (CE)/ MS.
Metabolic Profiling Methods: Nuclear Magnetic Resonance (NMR) Spectroscopy
Advantages of NMR over MS: - NMR does not destroy the sample - NMR can detect and quantify metabolite because the signal intensity is only determined by the molar concentration - NMR can provide comprehensive structural information, including stereochemistry Many atoms have nuclei that are NMR active, but most NMR data are collected for 1H and 13C since these are present in all organic molecules.
The main weakness of NMR is low sensitivity relative to MS. It is therefore less suited for analysis of trace compounds. As the natural abundance of 13C is only 1.1%, 13C-NMR is less sensitive than 1H-NMR. Recent developments have considerably increased sensitivity, making it less of a problem
Restriction Fragment Length Sizes(predicted)
If 25% A, 25% T, 25% G, 25% C and Random Distribution of Nucleotides (probability of given base is 0.25), then Distance between cut sites is equal to 4^n bases (n = #bp in recognition site)
n = 4 --> 256 bp n = 6 --> 4096 bp n = 8 --> 65.5k bp
Clone Screening: Hybridization, Immunological
Hybridization:
Make specific DNA probe
Hybridization screen (Southerns with colony lifts).
- Denature/anneal
Immunological:
Expression based screening
Requires mono-specific Antibody
Lysis
Infection by phage produces many progeny and breaks open (lyses) the host bacterium
Lysogeny
After infection, the phage DNA integrates into the host genome and resides there passively
- No progeny
- No lysis of the host
- Can subsequently lyse (lysogeny)
ELEMENTS of Lysogeny
The phage genome integrated into the host bacterial genome is a prophage.
- Genome: 50kb but 20kb can be replaced.
Packaging requires 50kb (and COS or cohesive ends). >50kb cannot package and
Cosmids
Combine properties of phage l and plasmid vectors
Manipulation of Prokaryotic Gene Expression
Major goal in biotech applications is over expressed gene products
Promoter considerations
Regulated?
Constitutive?
mRNA considerations
ATG start/termination
Stability
Secretion/signal seq.
Protein stability considerations
Purification strategies
Oxygen supply (often limited)
O2 limited solubility
High yield growth limits
Stationary phase protease
Plasmid copy # considerations Metabolic load considerations Biofilm issues Secretion into periplasm Gram negatives only
Optimal System Defined (prokaryotic gene expression)
MCS + 3 different ORFs
Strong regulatable promoter
Good selectable markers
Ori
HR site to get integration into host chromosome
Secretory signal seq.
Removable (cleavable) fusion tag
- Promotes stability
- Speeds purification
IMPORTANTLY: optimize for system being studied and is platform dependent
Regulatable promotors
E. coli lac and trp (tryptophan) operons
the tac promoter, which is constructed from the –10 region (i.e., 10 nucleotide pairs upstream from the site of initiation of transcription) of the lac promoter
pL promoter from bacteriophage λ
gene 10 promoter from bacteriophage T7
Recognition of promotor by E. coli RNA polymerase holoenzyme
The T7 gene 10 system
Requires T7 RNA Pol
T7 RNA Polymerase induced by IPTG (Lac operon)
T7 gene 10 promoter engages T7 RNA Pol VERY robustly
Gives significant mRNA yiels
Lac operon
LacZ gene produces beta-galactosidase, which turns lactose into allolactose.
Lac repressor bound to operator when lactose is low
cAMP/CAP (activator) bound to CAP box when glucose is low
RNA pol increases transcription beyond basal when low glucose, but high lactose
Promoter Improvements
Generally thought that the spacer seq b/t -35 and -10 regions of the bacterial basal promoters not very critical- (spacing for sigma factor/RNA Polymerase optimal interplay)
Mutations in the spacer found to contribute to better promoter activity.
Spacer G+C content changed to more A+T
Intrinsic “strength” of promoter greatly enhanced.
New Commercial E. coli Strains
Rosetta™ host strains are BL21
Enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli.
Codon Engineered…
tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid.
Rosetta strains provide for “universal” translation
Otherwise limited by the codon usage of E. coli.
The tRNA genes are driven by their native promoters.
In Rosetta(DE3)pLysS, the rare tRNA genes are present on the same plasmids that carries the T7 lysozyme gene.
Gene Dosage Manipulations
Increasing Gene dosage = increases yield
Virus do this sort of genetic ‘trickery’
pCP3 an example of ultra-high yield plasmid
TS Origin: At 28o the cI repressor (genomic) binds to keep copy # low
At 42o repressor is off: activates Origin and copy # increases 10 fold
Fusion Proteins
Foreign genes often degraded
Avoid by in frame fusion with native protein
Insert target gene into vector for ‘in frame’ expression of the foreign gene
Fusions Help in Purification using Affinity Column Chromatography
Same strategy: Eu or Pro systems. Immunoaffinity columns can work well Problem: Elution can denature the protein His tags work great (N- or C-terminal) Bind to Nickel Columns Wash free unbound Elute with imidizole (side chain of histidine) >90% recovery >100 fold purification in one step. Also works with denature proteins.
Fusion Purification Systems
Histidine tail
- size: 6-10 aa
- ligand: Ni2+
- eluction conditions: imidazole
Strep tag
- size: 10 aa
- ligand: streptavidin
- elution condition: iminobiotin
GST tag
- size: 26 kDa
- ligand: glutathione
- elution condition: reduced glutathione
Flag tag
- size: 8 aa
- ligand: specific monoclonal antibody (MAb)
- elution condition: EDTA or low pH
c-Myc
- size: 11 aa
- ligand: specific MAb
- elution condition: low pH
Codon Engineering
Improving translation efficiency
- Proper ribosome binding sites, secondary structure of mRNA are factors
Cloned target gene has codons rarely used by host cell (E. coli)
- Insufficient supply of tRNAs reduces yields
Corrected by:
Homologous expression systems (Eukaryote match)
Change target gene sequence (codon optimize)
Host cell engineered to over express rare tRNAs
Increasing Protein Stability
Half life of proteins: Highly variable (few min to many hr)
- Ptn is turned over (synthesized, utilized, degraded)
- “Intrinsic” survival time
- Influenced by disulfide bonds, N-terminal residues
- Ideally you want low turnover rates
Internal domains caninduce breakdown
- PEST: (pro, glu,ser, thr)
- Can change (mutate)But may affect ptn!
Overcoming Oxygen (O2) Limits
Coli requires aerobic growth for optimal yields
Goal is to gain a large biomass = high yield of ovr.exp. Gene product.
BUT: in Stationary phase things get ugly
- Severe limits in O2 availability
- Results in high protease activity
To overcome: protease deficient strains are used
Biofilm Limits
Bacteria grow in masses on surfaces (biofilms) with alginate = increases resistance to phage, antibiotics, hostile agents, etc,
But: Bad for biotech applications
- Limits yields, biomass production, limits protein over expression
Corrected by mutating biofilm genes (colonic acid, pilus genes)
DNA Integration Strategy
Plasmid imposes a metabolic “load”
- High copy #s are a drain on resources
- Slow growth limited biomass
- Must maintain positive counterselections (antibiotics… costly in fermenter productions)
- Also: if constructs are released into environment
Usually: antibiotic genes must be excised if going into humans
- In large scale ups: plasmid loss can occur (bad!)
DNA Integration by Homologous Recombination
Use a genomic site that can be disrupted without harming host
Can also strive to multi-copy gene dosage (more difficult)
In most cases: removal of antibiotic gene required for engineered bacteria released into environment (bioremediation vectors).
Removal of marker gene with lac/cre.
Secretion Considerations
Pass through inner cytoplasmic membrane into periplasm (or released in some cases)
- Periplasm targeting ideal (enhances recovery and concentrates the product).
Usually need to add signal peptide to N-terminus.
Generally difficult in Gram negatives
Can engineer cells to be ‘permeable’
Using a bacteriocin pathway cascade; 2 plasmid system
Nucleic Acids as Therapeutic Agents
- Genes
- Antisense RNA and oligonucleotides
- Ribozymes
- Aptamers
- siRNA or RNAi
The major limitation of the practical utilization of nucleic acids as therapeutic agents is delivering these compounds to inside their target cells
Acquired Diseases that are candidates for gene therapy
Cancer: liver, brain, pancreas, breast, kidney cells
Neurological diseases: neurons, glial cells, schwann cells
Cardiovascular: arteries, vascular endothelia walls
Infectious diseases: T-cells, liver, macrophages
Consider somatic vs germline gene therapy; the later is currently banned. Note that gene therapy is limited to somatic cells and disorders that are caused by a single gene.
Gene Therapy: Severe Combined ImmunoDeficiency (SCID)
How is ADA deficiency treated? There are no real cures for ADA deficiency, but doctors have tried to restore ADA levels and improve immune system function with a variety of treatments:
• Bone marrow transplantation from a biological match (for example, a sibling) to provide healthy immune cells
• Transfusions of red blood cells (containing high levels of ADA) from a healthy donor
• Enzyme replacement therapy, involving repeated injections of the ADA enzyme
• Gene therapy - to insert synthetic DNA containing a normal ADA gene into immune cells
Two types of gene therapy: Ex vivo v. In vivo
• Ex vivo:
cells are removed from the body, the gene of interest is inserted into them, the cells are cultured to increase cell numbers, and they are returned to the body by infusion or transplantation (time consuming and expensive)
• In vivo: a gene is introduced directly into specific cells within the body (quick and inexpensive), but targeting certain cells (e.g., bone marrow stem cells) is difficult
Vectors used to deliver genes in Human Gene Therapy
- Retroviruses
- Adenoviruses
- Adeno-associated viruses
- Herpes simplex virus
- Liposomes/Lipofection
- Naked DNA/Plasmid DNA
Introduction of Genes
Direct DNA Injections Calcium Phosphate DEAE Dextran Liposomes Cationic Lipids Electroporation
Ballistic DNA Injection
Particle bombardment, microprojectile gene transfer (gene guns)
Invented for DNA transfer to plant cells
Fully applicable to mammalian cells
Plasmid DNA is precipitated onto 1-3 micron sized gold or tungsten particles.
Discharge: helium pressure, or high-voltage electronic
DNA vaccines
Antiviral and antibacterial (traditional vaccines are better when available)
Cancer immunotherapy
- Passive: to increase the pre-existing immune response to the cancer
- Active: initiates an immune response against an unrecognised or poorly antigenic tumor
Liposomes
DNA inside lipids
Cationic liposomes
Positively charged lipid droplets can interact with negatively charged DNA to wrap it up and deliver to cells
Positively charged lipid heads transverse cell membrane
Lipofectin, lipofectamine, lipofectase….
Liposome advantages v. disadvantages
Advantages: Stable complex Can carry large-sized DNA Can target to specific cells (eg. in lung-delivery) Does not induce immunological reactions Cheaper then viruses
Disadvantages: Low transfection efficiency (100-1000 times more plasmid DNA needed for the same transfer efficiency as for viral vector) Transient expression Inhibited by serum Some cell toxicity
Liposomes are rapidly cleared from the circulation largely taken up by the liver macrophages
How to overcome it?
Liposome surface ligands decrease degradation (monosialoganglioside or PEG)
Immunoliposomes for active targeting
Antibodies to intracellular myosin target liposomes to infarcted areas of heart
Antibody against tumor specific molecules will target them to tumors
Cystic fibrosis gene therapy
January 1995. Results of intranasal CFTR-liposome spaying in CF patients. 12 patients, Temporary relief in 20% of patients. Maximum on day 3, faded away on day 7. No immune reasctions
Integrated transgenes v. non-integrated transgenes
Integrated
- stable expression = may provide cure
- random insertions into heterochromatin = can by inactivated
- random insertions inton euchromatin = can disrupt important host genes
Not integrated
- for episomes (plasmids) random mutagenesis not an issue
- expression is transient
- repeated treatments necessary
Retrovirus:
characteristics
- 63%
- insert size = 8kb
- integration = yes
- production > 10^6 cfu/mL
- administration = ex vivo
- expression = long
- express level = moderate
- immune = few
- safety concerns = insertional mutagenesis
- enveloped
- genetic material = RNA
- tropism = dividing cells only
- main limitations = only transduces dividing cells, integration might induce oncogenesis in some applications
- main advantages = persistent gene transfer in dividing cells
Adenovirus: characteristics
- 16%
- insert size = ~30kb
- integration = no
- production > 10^11 cfu/mL
- administration = ex/in vivo
- expression = transient
- express level = high
- immune = extensive
- safety concerns = inflammatory response
- non-enveloped
- genetic material = ssDNA
- tropsim = broad
- main limitations = capsid mediates a poten inflammatory response
- main advantage = extremely efficient transduction of most tissues
Adeno-associated virus:
characteristics
- 2%
- insert size = 4kb
- integration = rare
- production > 10^12 cfu/mL
- administration = ex/in vivo
- expression = pot. good?
- express level = moderate
- immune = ??
- safety concerns = inflammatory response
- non-enveloped
- genetic material = ssDNA
- tropism = broad, with possible exceptions of haematopoietic cells
- main limitations = small packaging capacity
- main advantages = non-inflammatory, non-pathogenic
Nake DNA/liposomes:
characteristics
- 13%
- insert size = unlimited
- integration = rare
- production = unlimited
- administration = ex/in vivo
- expression = transient
- immune = none
- safety concerns = none? toxic?
Lentivirus:
characteristics
- enveloped
- genetic material = RNA
- packaging capacity = 8kb
- tropism = broad
- inflammatory potential = low
- vector genome forms = integrated
- main limitations = integration might induce oncogenesis in some applications
- main advantages = persistent gene transfer in most tissues
HSV-1:
characteristics
- enveloped
- genetic material = dsDNA
- packaging capacity = 40kb-150kb
- tropism = strong for neurons
- infammatory potential = high
- vector genome forms = episomal
- main limitations = inflammatory; transient transgene expression in cells other than neurons
- main advantages = large packaging capacity; strong tropism for neurons
Virus as Vectors
Any virus can potentially be used to express foreign genes
Different viruses are better suited for different kinds of uses
Integration may be important, such as in many gene therapy uses
Larger viruses can express more and larger foreign genes, but are more difficult to manipulate
The cis-acting promoters for genome replication and packaging must be understood
The Ideal Vector for Gene Transfer
- High concentration of virus allowing many cells to be infected or transduced
- Convenience and reproducibility of production
- Ability to transduce dividing and non-dividing cells
- Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome
- A transcriptional unit that can respond to manipulation of its regulatory elements
- Ability to target the desired type of cell
- No components that elicit an immune response
Recombinant Vaccinia virus expression vector
Bacterial plasmid w/TK gene flanking sequences and foreign gene (FG)
- -> insertion into bacteria
- -> undergoes homologous recombination = linear DNA
- -> integrated into wt vaccinia virus (innoculated into bacteria)
- -> results in recombinant vaccinia virus
- -> BUdR selecetion for TK- cels (against TK+ cells = no FG)
Adenoviral Vectors:
adavantages v. disadvantages
Advantages:
Higher titer
Efficient transduction of nondividing cells
Disadvantages:
Toxicity
Immunological response
Transient transfection
– Topically administered Adenovirus anyway will move to other tissues, that produces distant toxic effects, especially in the liver (where virus is cleared)
– adenoviral receptors are less common in airway epithelium and cancer cells –> needs escalating doses –> more toxicity
Adenovirus:
partical structure
- Nonenveloped particle
- Contains linear double stranded DNA
- Does not integrate into the host genome
- Replicates as an episomal element in the nucleus
Generation of non-replicating adenovirus expression vector
Adenovirus vector DNA: E3 deleted, expression cassette inserted
Transfect adenovirus vector DNA into complementing cell line that expresses the E1A gene
Inserted into complementing cell line -- Attachment via CAR (MHC class I molecule coxsackievirus-adenovirus receptor), internalization via integrins
Vector DNA packaged into virion particles
Infect target cell
Invasion into new cell
– Adenoviral particles are disrupted in endosome
Production of expression cassette
Adeno-associated virus (AAV) :
advantages v. disadvantages
Advantages:
– does not stimulate inflammation in the host
– does not elicit antibodies against itself
– can enter non-dividing cells
– integrates successfully into one spot in the genome of its host (on chromosome 19 in humans).
• All viral genes removed
• Safe
• Transduction of nondividing cells
• Stable expression
Disadvantages:
• Small genome limits size of foreign DNA
• Labor intensive production
• Status of genome not fully elucidated
Adeno-associated viral vectors
- AAV is a simple, non-pathogenic, single stranded DNA virus dependent on the helper virus (usually adenovirus) to replicate.
- It has two genes (cap and rep), sandwiched between inverted terminal repeats that define the beginning and the end of the virus and contain the packaging sequence.
- The cap gene encodes viral capsid proteins and the rep gene product is involved in viral replication and integration.
- It can infect a variety of cell types and in the presence of the rep gene product, the viral DNA can integrate preferentially into human chromosome 19.
Application of rAAV in Gene Delivery
Retrograde Viral Delivery of IGF-1 Prolongs Survival in a Mouse ALS Model
Brian K. Kaspar, Jer nia Lladó, Nushin Sherkat, Jeffrey D. Rothstein and Fred H. Gage
Science, Vol 301, Issue 5634, 839-842 , 8 August 2003
To produce an AAV vector
- The rep and cap genes are replaced with a transgene.
- The total length of the insert cannot exceed 4.7 kb, the length of the wild type genome.
- Production of the recombinant vector requires that rep and cap are provided in trans along with the helper virus gene products.
- The current method is to co-transfect two plasmids, one for the vector and another for rep and cap into cells infected with adenovirus.
- Interest in AAV vectors is due to their integration into the host genome allowing prolonged gene expression.
Retroviral vectors:
- Retroviral vectors are based on Moloney murine leukemia virus (Mo-MLV) which is capable of infecting both mouse and human cells.
- The viral genes, gag, pol and env, are replaced with the transgene of interest and expressed on plasmids in the packaging cell line.
- Because the non-essential genes lack the packaging sequence, they are not included in the virion particle.
- To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone is removed.
- Transcription could be under the control of LTRs or enhancer promoter elements might be engineered in with the transgene.
- The chimeric genome is then introduced into a packaging cell, which produces all of the viral proteins, such as the products of the gag, pol and env genes, but these have been separated from the LTRs and the packaging sequence.
- Only the chimeric genomes are assembled to generate a retroviral vector.
- The culture medium in which these packaging cells have been grown is then applied to the target cells, resulting in transfer of the transgene.
Retroviral vectors:
Limitations
- A critical limitation of retroviral vectors is their inability to infect nondividing cells, such as those that make up muscle, brain, lung and liver tissue.
- The cells from the target tissue are removed, grown in vitro and infected with the recombinant vector, the target cells are producing the foreign protein are then transplanted back into the animal (ex vivo gene therapy).
- Problems with expression being shut off, prolonged expression is difficult to attain.
- Expression is reduced by inflammatory interferons acting on viral LTRs, as the retroviral DNA integrates, viral LTR promoters are inactivated.
- Possibility of random integration of vector DNA into the host chromosome.
Cancer Gene Therapy: Principle
- Cells of tumor often interconnected by cytoplasmic bridges and pores
- Introduce expression vector into some tumor cells
- Gene expressed converts prodrug into lethal compound
- “Shared” with other interconnected tumor cells
Lentiviral Vectors:
- Belong to the retrovirus family but can infect both dividing and non-dividing cells.
- They are more complicated than retroviruses, containing an additional six proteins, tat, rev, vpr, vpu, nef and vif.
- Human immunodeficiency virus (HIV) has been disabled and developed as a vector for in vivo gene delivery.
- Low cellular immune response, thus good possibility for in vivo gene delivery with sustained expression over six months.
- No potent antibody response
Ideal Vectors for Gene Therapy
Sustained production or regulated expression of the target gene
Regulation of the gene expression
Location of the expression
Level of the expression
Switch on or off
Regulatable systems
- TRS, the tetracycline regulatable system
- PRS, the progesterone regulatable system
- ERS, the ecdysone regulatable system
- RRS, the rapamycin regulatable system
The tetracycline transactivator system
Schematic outline of the tet-regulatory system. The system can be designed to either activate (Tet-ON) or repress (TetOFF) expression of a gene. Tet-OFF: The transactivator is composed of the repressor (tetR) of the Tn10 Tcresistance operon of Escherichia coli and a C-terminal operon of VP16 that functions as a strong transcription activator. tTA binds in the absence of tetracycline (but not in the presence) to an operator sequence (tetO) and activates the transcription. The Tet-ON system is identical but here the TetR is modified by a 4-amino acid change (rtetR) to convey the reverse phenotype.
Gossen, M., S. Freundlieb, G. Bender, G. Muller, W. Hillen, and H. Bujard. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766-9
How to Deliver and Express Transgene in Target Cell or Tissue?
A.Vector targeting - Change tropism
B. Transcriptional targeting – Tissue- or cell- specific promoters
Tissue-Specific Promoters/Enhancers
CMV immediate/early gene
- target tissue: neuron and other tissue
Rous sarcoma virus long terminal repeat
- tartget tissue: neuron and astrocytes
Myelin basic promoter/enhancer
- target tissue: oligodendrocytes and schwann cells
Neruon-specific enolase
- target tissue: gray matter in CNS
Platelet-derived growth factor beta-chain
- target tissue: whole brain
Beta-glucaronidase
- target tissue: brain
CMV-beta-globin exon 2 hybrid
- target tissue: rat dopaminergic neruons
CMV enhancer-beta-actin promoter
- target tissue: every tissue except erythrocytes and hair
Tetracycline/doxycline-regulated
- target tissue: inferior colliculs of the brain
Human glial fibrillary acidic protein
- target tissue: brain
Blocking RNA
- Many human disorders e.g. cancer and inflammatory conditions (virus, parasites) are often caused by overproduction of a normal protein.
- Theoretically a small ss nucleic acid can hybridize to a specific gene or mRNA and diminish transcription or translation. (antisense RNA)
- An oligonucleotide (oligo) that binds to a gene and blocks transcription is an antigene.
- An oligo that binds to mRNA and blocks translation is called an antisense oligo or antisense RNA.
- Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific mRNA for degradation.
Inhibition of translation of specific RNA by antisense nucleic acid molecules
Inhibition of translation of specific mRNAs by antisense (AS) nucleic acid molecules.
A. An antisense cDNA is cloned into an expression vector and the construct is transfected into a cell, where the antisense RNA is synthesized. Antisense RNA hybridizes to target mRNA, and translation is blocked.
B. An antisense oligonucleotide is introduced into a cell, and after it hybridizes into a cell, and after it hybridizes witht he target mRNA, translation is blocked.
Ribozymes
RNA molecules that act as enzymes are called ribozymes. • Earliest known examples: RNase P Group I and II introns Ribosomes Hammerhead ribozymes
- Principal reactions: RNA transesterification RNA cleavage (hydrolysis of phosphodiester bonds)
- Substrate aligned into the active site using a guide sequence which is complimentary to the substrate
- All ribozymes depend absolutely on the assumption of correct 3dimensional structure for activity
Transesterification
Transesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester.
Ribozymes: RNase P
1.The RNA component of bacterial Rnase P has 350-400 nucleotides. It has:
• a specificity domain and
• a catalytic domain.
- Bacterial RNase P contains a single protein subunit of about 120 amino acid residues.
- Zn & Mg needed as cofactor
Ribosome as a Ribozyme
The three-dimensional structure of the large (50S) subunit shows that formation of the peptide bond is catalyzed by the 23S RNA (& 28S RNA) molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the tertiary structure of the RNA.
Hammerhead ribozyme
The hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule.
The minimal catalytic sequence active consists of three base-paired stems flanking a central core of 15 conserved nucleotides.
Hammerhead ribozymes play an important role as: • therapeutic agents
• biosensors, and
• its applications in functional genomics and gene discovery
Hairpin ribozyme
- The hairpin ribozyme of plant viruses is 50 nucleotides long, and can cleave itself internally, or, can cleave other RNA strands in a transesterification reaction.
- The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis.
- Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases when the 2’ OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G.
Biological application: Possible gene therapy would be the hairpin and the hammerhead ribozyme against viral RNA.
SELEX :Systematic Evolution of Ligands by EXponential enrichment (protocol)
- Random nucleotide sequence
- Transcription
- Folded RNA aptamers with 5’ and 3’ flanking regions
- Add target molecules = aptamer-target complex
- Dissociate aptamer-target complex
- Reverse transcribe selected aptmers
- PCR amplify aptamer cDNA
- Rescreen selected aptamers against target molecules to find high-affinity aptamers
Aptamer = oligonucleotide that binds well to proteins, aa, etc
(eg. VEGF receptor = contains both a receptor-binding domain and a heparin-binding domain. VEGF stimulates growth of new blood vessels in senescing retinal pigment epithelial cells; however, when the blood vessels don’t form properly, there is scarring and loss of vision in the macular region of the retina. An aptamer that binds to VEGF is injected into the eye and suppresses age-related macular degeneration.)
Interfering RNAs
Addition of dsRNA to animal cells reduces expression of the gene from which the dsRNA sequence is derived
This has been termed RNA interference or RNAi, and occurs naturally. RNAi may protect animals and plants from viruses, and may also be an important regulatory mechanism.
Following introduction of dsRNA into a cell, it is cleaved into ssRNA 21-23 nt long. These oligos bind to and cleave mRNA. Transfection of mamalian cells in culture with duplexes of 21 nt RNA is also effective.
(Overview of the process of RNA interference. Following introduction of dsRNA into a cell, the Dicer complex binds to the RNA and cleaves it to a siRNA containing approximately 21bp. The antisense strand becomes part of the RISC complex, directing the cleavage of the complementary mRNA. A short hairpin RNA encoded on a plasmid may be used instead of dsRNA.)
miRNA v. siRNA
miRNA
- mRNA gene + Pol II?
- -> Pri-miRNA - Orosha
- Ran-GTP/Exportin5
- Dicer –> miRNA:miRNA duplex
- Helicase
- RISC –I native expression
siRNA
- Exogenous dsRNA, transposon, virus… –> long dsRNA
- Dicer
- Dicer
- Dicer –> siRNA duplexes
- Helicases
- RISC –I exogenous dsRNA, transposon, virus…
Post-transcriptional Cleavage of mRNA,
Translational repression of the mRNA,
Transcriptional silencing
(how obtained, in short)
Post-transcriptional Cleavage of mRNA
- extensive complementary in coding region or UTR
Translational repression of the mRNA
- short complementary segments in 3’-UTR
Transcriptional silencing
- Interaction w/DNA
- Active chromatin + Histone methylation –> silent chromatin
Expression of siRNAs
U6 or H1 Expression cassette
+ Pol III –> short hairpin RNA
– processing –> siRNA duplex
RNA comparisons
siRNA
- Direct cell insertion of siRNA
- Nuclear viral replication/Intracellular antiviral transgene expression (Pol II/III) –> shRNA –> siRNA
- Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> siRNA
- —> + RISC –> RNAi-mediated cleavage of target RNA
Antisense oligonucleotide
- Direct cell insertion of antisense oligonucleotide + RNase H –> antisense oligonucleotide-mediated translation inhibition or cleavage through RNase H activation
Ribozyme
- Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> ribozyme
- —> ribozyme-mediated cleavage of target RNA
Viral protein
Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> viral protein
—-> decoy RNAs sequester essential viral proteins or cellular cofactors
Conjugation of cholesterol and siRNA
Choleserol is coupled through the 5’ -OH of teh sense strand of the siRNA.
Following injection of the complex into mice, cholesterol facilitates uptake of the siRNA into specific tissues and silencing of apolipoprotein B (which is involved in cholesterol metabolism).
The antisense strand becomes part of the RISC complex and specifices where the mRNA is to be cleaved.
In mice, the target mRNA was decreased significantly in the liver and the jejunum; thereby reducing serum cholesterol levels.
Nonpathogenic E. coli to deliver siRNA
The bacterium was engineered to produce the protein invasion which permits E. coli to enter beta1-integrin-(+) mammalian cells as well as the gene HlyA that encodes listerolysin O which permits the short hairpin RNAs (shRNAs) synthesized by the bacterium to be released inside the mammalian cell. This technique may be used to target and kill specific cancer cells (in culture and live mice).
Atelocollgen-siRNA complex
Negatively charged siRNAs bind to postively charged atelocollagen (subunit size ~300 kDa; from calf dermis following digestion with pepsin in acid).
The complex facilitates delivery of siRNAs and protects them against nuclease digestion.
Used to deliver siRNA to injected mice.
Fab fragments as siRNA delivery
A single chain Fab fragment directed against a mammalian cell surfect (breast cancer) protein is fused to the positively charged polypeptide protamine which binds non-covalently to negatively charged siRNAs.
The Fab fragment acts to deliver the siRNA to specific cancerous cells.
A two chain Fab fragment has also been used to deliver siRNA.
Used in culture and by injection directly into tumours.
Chimeric RNA molecules with aptamer-binding site + siRNA
Chimeric RNA molecule consisiting of an aptamer (binds to a specific antigen) and an siRNA.
Gene doping
Non-therapeutic use of gene therapy to enhance athletic performance
Erythropoietin = inc. RBC level
Myostatin KO = inhibits muscle growth inhibition
Insulin-like growth factor overexpression = inc muscle size/strength
Vascular endothelial growth factor overexpression = induce formation of new blood vessels
Restriction enzymes are great products for recombinant microbes(E. coli)
- $350 million in annual RE sales in 2007
- Some microbesare difficult orexpensive to grow in culture
- Strategy:clone the gene for the RE from a given microbe and express it in E. coli (along with the corresponding modification [methylase] gene for protection of the E. coli DNA)
- E.coli is simple to grow
Synthesis of Commercial Products by Recombinant Microorganisms:
Cloning and selecting gene for PstI restriction enzyme
- pBR322 plasmid + HindIII –> linear DNA
- P. stuartii DNA + HindIII –> fragments
- Mix
- T4 DNA ligase
- Cloned DNA
- Transform into E. coli
- Grow in liquid culture
- Infect with bacteriophage lambda
- Only lysis-resistant colonies will grow
What do you do if you want folded recombinant protein?
Use yeast instead of E. coli
Smallmolecules for recombinant microbes
Small molecules are also great products for recombinant microbes (often E. coli)
• Ascorbic acid (Vitamin C)
• Amino acids (e.g. Glutamic acid for production of the flavor enhancer MSG)
• Antibiotics, novel antibiotics, and polyketide antibiotics
• Note: in all of these cases, one needs to clone the genes encoding the enzymes making these metabolites in order to createor alter
a biochemicalpathway
(MSG) Monosodium Glutamate
MSG has been produced by three methods: hydrolysis of vegetable proteins with hydrochloric acid to disrupt peptide bonds (1909–1962); direct chemical synthesis with acrylonitrile (1962–1973), and bacterial fermentation (the current method). Wheat gluten was originally used for hydrolysis because it contains more than 30 g of glutamate and glutamine in 100 g of protein. As demand for MSG increased, chemical synthesis and fermentation were studied..[The polyacrylic fiber industry began in Japan during the mid-1950s, and acrylonitrile was adopted as a base material to synthesize MSG..
Biopolymers are also great products for recombinant microbes
- Xanthan gum production in Xanthomonas compestris (genetically engineered to grow on whey, a lactose-‐rich byproduct of cheese production)
- Melanins
- Animal adhesive proteins (from the blue mussel)
- Rubber (from the rubber plant Hevea brasiliensis)
- Biodegradable plastics(polyhydroxyalkanoates)
- Note that in all of these cases, one needs to clone thegenes encoding enzymes inorder tocreateor alter a biochemical pathway
Synthesis of Commercial Products by Recombinant Microorganisms:
biodegradable plastic from Alcaligenes eutrophus
The enzymes responsible for the production of this biodegradable plastic were cloned from Alcaligenes eutrophus and transferred to E. coli to make even more of this biodegradable plastic.
Synthesis of Commercial Products by Recombinant Microorganisms:
Cloning and selecting gene for DdeI restriction enzyme and Dde I methylase
- Cloned DNA w/ 2x HindIII cut sites and 1x DdeI cut site
- Transformation into E. coli
- Isolate plasmid (Midi prep probably)
- Digest with DdeI
- Transformation
- -> Methylated DdeI cut site/no digestion = transformants
- -> Cut DdeI site = no transformants
Synthesis of Commercial Products by Recombinant Microorganisms:
E. coli lacZ and lacY genes in Xanthomonas campestris
Engineering of the E. coli lacZ (encoding β-galactosidase) and lacY (encoding lactose permease) genes for constitutive expression in Xanthomonas campestris
Recombination:
- p^lac - lacZ - lacY –
- p^Xc - X. campestris gne –
- ——->
- p^Xc - lacZ - lacY –
