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