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MOLECULAR BASIS OF DISEASE > LABORATORY TECHNIQUES > Flashcards

LABORATORY TECHNIQUES Flashcards

(95 cards)

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1
Q

PCR

A 
Used to amplify specific region of DNA
Primers complementary to region wanted to amplify
Denature template 
anneal primers
extension step - new DNA molecules
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2
Q

Fragment analysis - what does it do

A

PCR based assay - capillary electrophoresis
Sizing PCR product - detect repeat expansions
detect repeat expansion diseases

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3
Q

Huntington’s disease

A

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

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4
Q

Sanger sequencing

A

Cycle sequencing based on same principles as PCR

Each DNA nucleotide have different dye - determine sequence

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5
Q

FISH can detect

A 
detect microscopic chromosomal abnormalities
Detect large chromosomal abnormalities
Extra chromosomes
Large deleted segments
Translocations
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6
Q

FISH steps

A
  1. Design fluorescent probe to chromosomal region of interest
  2. Denature probe and target DNA
  3. Mix probe and target DNA
  4. Probe binds to target
  5. Target fluoresces
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7
Q

Array CGH

A

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

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8
Q

MLPA

A

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

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9
Q

How MLPA works

A

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

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10
Q

Next generation sequencing - disease panels

A

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

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11
Q

Exome sequencing - what is it

A

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

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12
Q

Exome sequencing method

A

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

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13
Q

Tier 1 variants

A

Known pathogenic

protein truncating

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14
Q

Tier 2 variants

A

Protein altering - missense

introinc - splice site

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15
Q

Tier 3 variants

A

Loss of function variants in genes not on the disease gene panel

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16
Q

What are stem cells

A

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.

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17
Q

Induced pluripotent stem cells

A

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

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18
Q

Adult stem cells

A

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

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19
Q

Embryonic stem cell

A

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

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20
Q

Stem cell niches

A

Tissue specific stem cells need special supportive microenvironments = stem cell niche

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21
Q

Stem cell function

A

Interact with stem cells and regulate cell fate.

Protect stem cell from depletion and host from excessive stem cell proliferation

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22
Q

Generating iPSC cells

A

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

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23
Q

Stem cell tracking

A

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

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24
Q

CVS and regeneration

A

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

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25
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
26
Epicardium
Source of coronary vessels and signals promote cardiomyocyte proliferation Epicardial activation = endocardium activation - cardiomyocyte dedifferentiation - clot degrades as cardiomyocyte proliferates
27
Cardiac regeneration in non human primates
Fibrin clot does not resolve - fibrotic scar = affect cardiac function
28
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
29
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
30
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
31
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
32
Myosin thymosin β4 function
Necessary for epicardial migration, coronary vasculature and cardiomyocyte survival
33
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
34
Secreted factors of epicardium important for cardiac regeneration
FSTL1 | IPSC
35
FSTL1
expressed in epicardium - promote cardiocyocyte proliferation - lost after myocardial infarction - if restored it promotes regeneration of of pre-existing cardiomyocytes
36
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
37
Stem cell therapy for burns
Foetal fibroblasts epidermal stem cells mesenchymal stem cells IPSCs
38
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
39
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
40
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
41
IPSCs mechanism for skin
Can be differentiated into dermal fibroblasts, keratinocytes and melanocytes
42
Method of stem cell therapy to treat burns
1. Isolation and production of stem cell and selection and amplification of required phenotypes 2. Cells selected for a variety of factors including secretion of growth factors - can act as scaffold for regeneration 3. Delivered by dressings, cell spray or 3D printing of cell sheets
43
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
44
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
45
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
46
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
47
Neural stem/progenitor cells grafts (NSPC)
can integrate into site of spinal cord injury - generate neural relays across lesions that provide functional benefit
48
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
49
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
50
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 ```
51
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
52
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 ```
53
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 ```
54
Method of production of cell line cultures
Isolated from cancerous tissues OR Immortalisation of primary cultures
55
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
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Genetic manipulation to produce immortal primary cultures
Target processes that regulate cellular growth and ageing | p53, pRb, telomerase
57
Telomerase
Elongate telomeres | Telomeres shorten as cell division stops leading to apoptosis - activate p53 and pRb = cell death
58
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
59
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)
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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
61
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
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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
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Suspension cells
Cell grow suspended in liquid medium
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Cell culture contamination
Mycoplasma - common problem - bacteria lacking cell wall around membrane - resistant to antibiotics
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Cell lines cross contamination occur
Poor tissue culture techniques Cultures of multiple cell lines at one time Accidental mixing of cell lines
66
3D cultures
More physiological relevant and better represent in vivo tissue - grow cell in 3D reproduce characteristics of cell in body
67
2D cultures
monolayers have uniform access to nutrients - not the case in the body
68
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
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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
70
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
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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
72
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
73
Lipofection
Transfection technique - introduce DNA through liposomes - vesicles merge with membrane of eukaryotic cells - liposomes made with phospholipid bilayer 1. Interaction with cell membrane 2. Taken up by endocytosis 3. Release from endosome 4. Transport to the nucleus 5. Entry to the nucleus inefficient and may need mitosis
74
Electroporation
Electric field supplied to cell increase polarity - allow drug/chemical to be introduced to cells
75
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
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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
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Steps for viral infection/transduction
1. 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 2. 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
78
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
79
Flow sorting
Sorting (separating) cells based on properties measured in flow Fluorescence activated cell sorting (FACS)
80
What can a flow cytometer tell us about a sell
Relative size relative granularity/internal complexity relative fluorescence intensity
81
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
82
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
83
Basics of flow cytometry - electronics
Converted to digital values that are stored on a computer | PMT convert light into digital signal - analog digital conversion
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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
85
fluorochrome - FITC
Green - emit at 520nm
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fluorochrome - PE
Orange - emit at 580nm
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fluorochrome - PerCP
RED - emit at 620nm
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Labelling - direct
Monoclonal antibodies are preconjugate to fluorochromes
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Labelling - indirect
Unconjugated monoclonal antibodies - has a secondary antibody
90
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
91
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
92
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)
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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
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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
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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

MOLECULAR BASIS OF DISEASE (3 decks)

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