LABORATORY TECHNIQUES Flashcards
PCR
Used to amplify specific region of DNA Primers complementary to region wanted to amplify Denature template anneal primers extension step - new DNA molecules
Fragment analysis - what does it do
PCR based assay - capillary electrophoresis
Sizing PCR product - detect repeat expansions
detect repeat expansion diseases
Huntington’s disease
Severe neurodegenerative disorder
Caused by CAG repeat expansion in HTT gene - 35+ = pathogenic
Expanded protein is toxic and accumulate in neurons causing cell death
Diagnosed with fragment analysis
Sanger sequencing
Cycle sequencing based on same principles as PCR
Each DNA nucleotide have different dye - determine sequence
FISH can detect
detect microscopic chromosomal abnormalities Detect large chromosomal abnormalities Extra chromosomes Large deleted segments Translocations
FISH steps
- Design fluorescent probe to chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA
- Probe binds to target
- Target fluoresces
Array CGH
Detection of sub microscopic chromosomal abnormalities
Patient DNA labelled green. Control DNA labelled red.
Depending on signal can determine if there is a dosage loss or dosage gain
MLPA
Variation of PCR - permit amplification in multiple targets
Detect abnormal copy numbers at specific chromosomal locations.
Each probe consist of 2 oligonucleotides - recognize adjacent target sites on DNA
How MLPA works
1st probe = forward primer
2nd probe = reverse primer
1. Both probes hybridise together on template DNA
2. Ligated in to complete probe
3. Amplification of probe - produce an amplified library
4. Fragment analysis of MLPA product
Next generation sequencing - disease panels
Enriching to sequence only known disease genes relevant to phenotype.
Panels expandable to include new genes as they are published
Potentially pathogenic variants confirmed by sanger sequencing
Exome sequencing - what is it
Technique used for NGS
Diagnose diseases and discovering new genes
Only interested in protein coding exons
Some pathogenic mutations are protein coding
More efficient to only sequence bits we are interested in
Exome sequencing method
Target enrichment
Capture target regions of interest with baits
- Incubate library with RNA baits - hybridisation - exon baited - purification column which recognise biotin on RNA baits so capture exon fragments and wash away unbound bits = enriched library
Potential to capture several Mb genomic regions
Tier 1 variants
Known pathogenic
protein truncating
Tier 2 variants
Protein altering - missense
introinc - splice site
Tier 3 variants
Loss of function variants in genes not on the disease gene panel
What are stem cells
Can differentiate into many different cell types
Capable of self-renewal via cell division
Provide new cells as an organism grows and replace cells that are damaged or lost.
Induced pluripotent stem cells
Made in lab - take differentiated tissue and reprogramme cell by exposure to specific set of pluripotency factors - Sox2, Oct3/4, Klf4, c-Myc = become pluripotent stem cells
Use for cell therapy and disease modelling
Adult stem cells
Rare
Supply new cells as organism grows and replace damaged cells
Ability to do this varies with organ
Tissue specific and multipotent - differentiate into subset of cell types
Embryonic stem cell
Supply all the cells of the developing embryo
Pluripotent
Derived from embryp at blastocyst stage - Reside in inner cell mass
Give rise to all 3 germ layers
Stem cell niches
Tissue specific stem cells need special supportive microenvironments = stem cell niche
Stem cell function
Interact with stem cells and regulate cell fate.
Protect stem cell from depletion and host from excessive stem cell proliferation
Generating iPSC cells
Expose differentiated cells to pluirpotency factors - Sox2, Oct3/4, Klf4, c-myc
C-myc - promote DNA replication and relax chromatin structure
Allow Oct3/4 to access target genes. Sox 2 and Klf4 co-operate with Oct3/4 to activate genes
Encode transcription factors - establish pluripotent transcription factor network leading to activation of epigenetic processes that establish pluripotent epigenome
Stem cell tracking
SC manipulated in vitro to make them easy to track once they are transplanted in vivo
Insert reporter gene = cell fluoresce
Cell transplanted back into pre-clinical models - use non-invasive long term cell tracking
CVS and regeneration
Heart attack blood supply to the heart muscle is lost - cardiac muscle dies and is not replaced - cardiomyocyte turnover is low
Fibrosis and scarring occur leading to decreased cardiac function and heart failure
Cardiac regeneration and zebrafish
Cut in heart - blood filled clot sealing injury - sealed with fibrin clot - replaced by heart muscle - new cardiac wall
Re-expression of developmental gene programmes early after injury - seen in epicardium
Epicardium
Source of coronary vessels and signals promote cardiomyocyte proliferation
Epicardial activation = endocardium activation - cardiomyocyte dedifferentiation - clot degrades as cardiomyocyte proliferates
Cardiac regeneration in non human primates
Fibrin clot does not resolve - fibrotic scar = affect cardiac function
Lymphatic and immune response in cardiac regeneration - normal/control injury response
Lymphatic response modulate immune response
Does not clear excess tissue fluid and inflammatory immune cells efficiently - oedema and inflammation - poor cardiac repair and function
Lympahtic and immune response in cardiac regeneration - modified VEGFC
Increase lymphatic response = improve clearance of tissue fluid and inflammatory cells = decrease oedema and inflammation and increase cardiac repair/function
Making cardiac lineages from IPSC
Somatic cells reprogrammed - specified to pre-cardiac mesoderm by inhibition of glycogen synthase kinase 3b - acts as a downstream switch for signalling pathways - Wnt signalling
Inhibit Wnt signalling = differentiation of cardiac progenitor cells - provision of specific signalling molecules - further differentiate cell toward specialised cardiac lineages
Transplanted IPSCs - SC transplant
- IPSC derived cardiomyocyte transplanted into primate heart (monkey)
- Differentiated IPSC cardiomyocyte injected directly into heart after myocardial infarction
- Grafted cardiomyocyte survived 12 weeks – no evidence of immune rejection
- There was electrical coupling between host and graft derived cardiomyocytes
- Improved cardiac contractile function in injected infarcted hearts at 4 and 12 weeks after transplant
- Grafted cardiomyocytes were well vascularised
- Incidence of ventricular tachycardia higher compared to controls
Myosin thymosin β4 function
Necessary for epicardial migration, coronary vasculature and cardiomyocyte survival
Myosin thymosin β4 effect
Can stimulate epicardial outgrowth and neovascularisation
Re-expression of key embryonic epicardial gene wt1 through priming Tb4 in vivo
Activated Wt1 epicardial cells give rise to cardiac progenitors in myocardial infarcted injured adult heart - could differentiate into de novo cardiomyocytes
Secreted factors of epicardium important for cardiac regeneration
FSTL1
IPSC
FSTL1
expressed in epicardium - promote cardiocyocyte proliferation - lost after myocardial infarction - if restored it promotes regeneration of of pre-existing cardiomyocytes
IPSC - secreted factors of epicardium
Produce large sheets of cardiomyocyte cells - grafting sheets onto heart can improve function - but cells do not integrate into the heart tissue.
Instead they release paracrine factors that help to regenerate damaged muscle
Stem cell therapy for burns
Foetal fibroblasts
epidermal stem cells
mesenchymal stem cells
IPSCs
Foetal fibroblasts mechanism
From embryonic stem cells
Improve skin repair due to high expansion ability, decrease immunogenicity, and intense secretion of bioactive substances like growth factors
Epidermal stem cells mechanism
Increase proliferation rate, easy access and keep their potency and differentiation potential for long periods.
Generate most skin cell types for repair and regeneration
Mesenchymal stem cell mechanism
Increase differentiation potential and certain degree of plasticity.
Migrate to injured tissues, differentiate and regulate tissue regeneration by production of growth factors, cytokines and chemokines
IPSCs mechanism for skin
Can be differentiated into dermal fibroblasts, keratinocytes and melanocytes
Method of stem cell therapy to treat burns
- Isolation and production of stem cell and selection and amplification of required phenotypes
- Cells selected for a variety of factors including secretion of growth factors - can act as scaffold for regeneration
- Delivered by dressings, cell spray or 3D printing of cell sheets
Limbal stem cells
Responsible for making new corneal cells to replace damaged ones.
If damaged - cornea cannot be repaired and affect ability of light entering the eye
Limbal stem cell therapy mechanism
Limbal SC collected from healthy donor and transplanted into damaged eye = repairs the cornea and permanently restores vision
To avoid immune rejection - only works if patient has a healthy section of limbus to collect limbal stem cells
IPSC can be induced to make corneal epithelial cells for transplant
Retinal pigment epithelium
Single later of post mitotic cells acting as a selective barrier to photoreceptor cells and the photoreceptor layer
RPE - has been made from ESC and IPSC
Spinal injury and stem cells
Somatic cell biopsy and transform these into IPSC - differentiated into neural SC implanted at site of spinal cord injury
Differentiate into variety of cells necessary for neural regeneration - neurones, oligodendrocytes, astrocytes
Neural stem/progenitor cells grafts (NSPC)
can integrate into site of spinal cord injury - generate neural relays across lesions that provide functional benefit
Calcium imaging of NSPC
Calcium imaging of MSPC grafts organise into localised and spontaneous active synaptic networks
Optogenetic stimulation of host axons produced a neuronal response in the graft and vice versa
In vivo imaging of NSPC
behavioural stimulation also elicited focal synaptic response within grafts.
Thus, neural progenitor grafts can form functional synaptic subtle networks whose activity patterns resemble intact spinal cord
Primary cell culture characteristics
Cell derived from tissues Finite lifespans Can grow in 2D or 3D Cells can divide or differentiate Good for personalised medicine
Method of isolation of primary cells
Cells allowed to migrate out of an explant
Mechanical (mincing, sieving, pipetting) an enzymatic dissociation
EXCEPTION - HSC - do not need disaggregated - they already are
Density centrifugation
Disadvantages of primary cells
Inter-patient variation - cannot reproduce results Limited - finite lifespan Difficult molecular manipulation Phenotypic instability Aberrant expression of some genes Variable contamination
Cell line culture characteristics
Immortalised cells limited number of cell divisions Phenotypically stable, define dpopulation Limitless availability Easy to grow, good reproducibility Good model for basic science
Method of production of cell line cultures
Isolated from cancerous tissues
OR
Immortalisation of primary cultures
How is Immortalisation of primary cultures achieved
Spontaneously from prolonged culture - multiple ill defined mutations transformed phenotype
Through genetic manipulation - artificial transformation of healthy primary cells
Genetic manipulation to produce immortal primary cultures
Target processes that regulate cellular growth and ageing
p53, pRb, telomerase
Telomerase
Elongate telomeres
Telomeres shorten as cell division stops leading to apoptosis - activate p53 and pRb = cell death
How can we inhibit function of tumour suppressor proteins, or introduce telomerase to alter a cell’s capability for finite number of divisions?
Viral oncoproteins
Large T antigen of SV40 binds to p53 and pRb = prevent p53 and pRb from carrying out its function
HPV - oncoproteins = E6 and E7
E6 target p53 for degradation. E7 bind to Rb inactivating it.
Telomerase gene introduced to target primary cell
How do we introduce telomerase into cells
Plasmid with telomerase and neomycin resistance and then transfect
2. Add neomycin - so only plasmid survive - colonies selected
(Neomycin is an example of gene we want to add)
Conditions for growth culture
Handle under aseptic conditions - grow on tissue culture treated plastic flasks/dishes
Maintained in warm humidified atmosphere - 37 degrees. 5% CO2
Grow cell in supplemented medium - nutritent, pH, growth factors for survival
Indicators and pH - phenol red results
Phenol red = pH indicator
Red = neutral - pH = 7.0
Purple = basic pH = 7.4-7.6
Yellow = acidic pH =6.8
Adherent cells
Cell grow attached to surface
Needs trypsin - break down protein - dissociate cells from vessels to transfer them to another flask
Growth limited by surface area
Suspension cells
Cell grow suspended in liquid medium
Cell culture contamination
Mycoplasma - common problem - bacteria lacking cell wall around membrane - resistant to antibiotics
Cell lines cross contamination occur
Poor tissue culture techniques
Cultures of multiple cell lines at one time
Accidental mixing of cell lines
3D cultures
More physiological relevant and better represent in vivo tissue - grow cell in 3D reproduce characteristics of cell in body
2D cultures
monolayers have uniform access to nutrients - not the case in the body
Spheroid
Made from cell lines
There is a necrotic core, a layer of cells proliferate - gradient of O2 and nutrients
Represent single/partial tissue components
resemble cell organisation
Difficult to maintain long time due to gradient of oxygen
Organoid
From primary cell - can organise themselves and make mini organs
Usually hollow
Derived from stem cells
multiple cell lineages
Long term culture
patient derived organoids allow the study cancer drug resistance
Organoid advantages
Gene expression as in vivo - 87% phenotype and genotype similarity
cell-cell communicaiton re-established
cells orientated in same way as tissue
ideal platform for individualised therapeutic screening
Organoid limitations
Limited amount of tissue in some cases e.g. prostate
Organoids in same culture are heterogenous
Absence of immune cells in culture system
Unable to mimic in vivo growth factor/signalling gradients
What is transfection
Process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab e.g. plasmid, CRISPR/Cas9 complex
Lipofection
Transfection technique - introduce DNA through liposomes - vesicles merge with membrane of eukaryotic cells - liposomes made with phospholipid bilayer
- Interaction with cell membrane
- Taken up by endocytosis
- Release from endosome
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis
Electroporation
Electric field supplied to cell increase polarity - allow drug/chemical to be introduced to cells
Nucleofection
Combination of electroporation and lipofection
Increase efficiency particulary of non dividing cells - open pore on cell and nuclear membrane.
Technology is protected under patent
Different solution and protocols are used for each cell type
Viral infection/transduction
Exploit the mechanism of viral infection
High transfection efficiency
Retrovirus, adenovirus, but most commonly lentivirus is used
Target cells needed to express viral receptor to work
there are safety aspects to consider
Steps for viral infection/transduction
- Production of virus - universal cell line in contact with vectors containing viral protein and with gene of interest. Translate information and produce cell virus particles - stay in supernatant
- Infection of eukaryotic cell of interest - with the supernatant. Supernatant contains virus and gene of interest - infect cells of interest and deliver material to cells
What is flow cytometry
Technique which simultaneously measure several physical characteristics belonging to a single cell in suspension
This is done by light scatter and fluorescence
definition = measure properties of cell in flow
Flow sorting
Sorting (separating) cells based on properties measured in flow
Fluorescence activated cell sorting (FACS)
What can a flow cytometer tell us about a sell
Relative size
relative granularity/internal complexity
relative fluorescence intensity
Basics of flow cytometry - fluidics
Cell in suspension - flow single file
Inject sample into a sheath fluid - passes through small orifice
Sample fluid flow in a central core that does not mix with the sheath fluid - LAMINAR FLOW
Hydrodynamic focusing - intro of large volume leading to small volume
Basics of flow cytometry - optics
An illuminated volume where they scatter light and emit fluorescence that is collected, filtered
Single wavelength of light - coherent light - 488nm
Forward light scatter - proportional to size
90 degree light scatter proportional to granularity
Overlap of emissions - mirrors/filters restrict amount of light hitting PMT to differentiate fluorescence
Basics of flow cytometry - electronics
Converted to digital values that are stored on a computer
PMT convert light into digital signal - analog digital conversion
Fluorescence
Occurs when laser hit fluorochrome and it is excited at 1 wavelength and goes back to unexcited state it emits fluorescence at higher wavelength
fluorochrome - FITC
Green - emit at 520nm
fluorochrome - PE
Orange - emit at 580nm
fluorochrome - PerCP
RED - emit at 620nm
Labelling - direct
Monoclonal antibodies are preconjugate to fluorochromes
Labelling - indirect
Unconjugated monoclonal antibodies - has a secondary antibody
Propidium iodide
Most commonly used fluorescent dye
Undergo dramatic increase in fluorescence upon binding DNA, requires permeabilisation of plasma membrane.
Intensity of PI is proportional to the amount of DNA
How does PI work
PI cannot normally cross the cell membrane
If the PI penetrates the cell membrane - it is assumed to be damaged.
Cells that are brightly fluorescent with the PI are damaged or dead
Can differentiate between viable and non-viable cells
Methods detecting apoptosis
By staining with dye PI
Phosphatidyl serine, can be detected by incubating the cells with fluorescein labeled annexin V, and PI (cells not fixed)
By staining with 7-aminoactinomycin D (Cells not fixed)
Cell sorting
Cells are viable and sterile
Machine sees a cell that satisfies the criteria at that point where the laser hits the stream - machine wait till it get to end of stream
Nozzle vibrates - break off cell droplets - droplets have charge and they are pulled by deflection plates into tube
Applications of flow cytometry and cell sorting
Immunophenotyping leukaemia and lymphomas - panel of monoclonal antibodies - leukaemia to be classified
Quantification of CD4/CD8 in diagnosis of HIV
Measurement of cell proliferation
Assessment of transfection efficiency
Study cell cycle
Blood as liquid biopsies can detect
- Circulating endothelial cells – early detection of heart attacks
- Cells that detach from tumours. Circulating tumour cells
- Cell free nucleotides – released from cells in apoptosis/necrosis or inflammation or tumour developed - er rate of cell free nucleotides
- Extracellular micro-vesicles – micro RNA – micro RNA 105 promote breast cancer metastases – can be early detection
- Exosomes
