My project Flashcards
(39 cards)
How are ‘pericytes’ isolated from patients?
- in the delevalle paper, muscle biopsies were taken from skeletal muscle.
- they are dissected very finely.
- fragments of interstitial tissue containing small vessels are put into a dish with megacell etc and cultured for a week
after the outgrowth of fibrobast like cells, small round ‘refractive’ cells that have bad adhesion. These cells are collected via pipetting
how common is DMD?
1 in 3500
what is the role of dystrophin protein?
it acts within a dystrophin associated protein complex (DAPC) which stabilises the sarcolemma during muscle contraction. It does this by linking the sarcomere to the extracellular ECM (laminin). An absence leads to contraction induced muscle degeneration.
what is exon skipping and how does it work?
it involves administering an antisense oligonucleotide that is complimentary to the exon containing the mutation, which hides it from the splicing machinery, forming a truncated but functional protein. It doesn’t work with large deletions or regulatory mutations.
what is the read through technique?
administer a small molecule that is able to induce a conformational chance in the mRNA structure, allowing the ribosome to skip over a mutation-induced stop codon with a single amino acid. This produces a full length protein.
what is a gene replacement therapy?
use either a vector or a cell to replace the mutated gene.
why does the size of dystrophin hinder vector candidates?
it surpasses the cloning capacity of the convetional vectors such as Adenoassociated vector and lentiviruses
why is a plasmid not feasible?
can’t be delivered to the muscle as easily- needs to be electroplated into muscle.
why is a DYS HAC a good ex vivo gene therapy approach?
they have an infinite long capacity, have a centromere, tellers, and replication origin. This means that the copy number within proliferating cells is tightly regulated.
when has a DYS hac been used successfully before?
correct a murine mesoangioblasts.
in mice, where are mesoangioblasts found?
embryonic dorsal aorta
how many cells are required for systemic treatment?
1 trillion or more
how did dellavalle identify that the human counterpart of mesoangioblasts were pericytes?
They studied the gene expression of the ‘interstitial refractive cells’ and found that they expressed pericyte markers (AP, SMA, NG2), not endothelial or myogenic markers.
how are pericytes and satellite cells positioned?
satellite cells are under the basal lamina of the muscle fibre basal lamina and persecutes are position beneath the endothelial basal lamina
how was pericytes ability to differentiate in0 myogenic cells shown in vitro?
the ALP+ cells were cocultured with mouse myogenic cells, and they fused to form hybrid myotubes and when exposed to muscle-differentiation medium.
how was the myogenic potential of parasites shown in vivo?
they injected human pericytes into mice intraarterially and they colonised downstream muscles
how did dellevalle demonstrated that human parasites could be used in dystrophin replacement therapy?
they injected human parasites into mdx mice and they were able to fuse to myotubes and stimulated dystrophin positive myofibres
what is the concept behind the reprogramming aspects of the project?
- notch pathway inhibition leads to premature myogenic differentiation and depletion of the muscle progenitor population.
- activation of notch inhibits MyoD-mediated myogenesis.
- DLL4 is expressed by developing endothelium with PDGF-BB which recruits pericytes from the surrounding mesenchyme.
- this gave rise to the theory that, during development, myoblasts may be recruited by the developing endothelium and converted into pericytes.
- they then mimicked this using murine myoblasts and treating them with DLL4 and PDGF-BB.
why was the expression of Pax3 important?
because capellari treated pax 3 + cells from E11.5 cells.
how did they show that these treated cells were pericytes?
they looked at the upregulation of pericyte markers and the downregulation of MyoD and Myf5.
- these cells were able to stabilise endothelial cells
How did capillari demonstrate that treatment in vivo could convert myoblasts to pericytes?
- they used cre and loxp do induce NICD expression in MyoD expressing cells- severely reduced skeletal muscle differentiation and increased expression of parasite markers in vessel associated cells
how has the ability of mesoangioblasts to cross the endothelial wall been shown?
murine mesoangioblasts have been used to treat a mouse model of limb gurdle muscular dystrophy via intra-arterial injection and recapitulation of the diseased muscle with normal muscle.
what are pericytes?
they support via direct contact and paracrine signalling to endothelial cells- capillaries.
what is gibson assembly?
it works by having two fragments that you want to join, you anneal primers to the end of one so that it is complimentary to the fragment you wish to anneal. AN exonuclease then chews back the 5’ end. The fragments anneal, the polymerase retranscribes back.
