31-35 Other Flashcards Preview

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Flashcards in 31-35 Other Deck (19):
1

31

What different constructs are there for gene therapy?

  • 1. Gene replacement/substitution - microdystrophin
  • 2. Reduce gene expression – RNA interference
  • 3. Modifying protein translation: Exon skipping – antisense oligonucleotides
  • 4. Genome editing/repair – CRISPR/Cas9 can cut DNA
  • 5. Interventions to compensate in spite of disease - follistatin blocks myostatin

2

31

What is a bottom-up approach to vectors?

What is a top-down approach to vectors?

  • Start safe - enhance effectiveness
  • Start effective - enhance safety

3

31

What are lentiviral vectors?

What are adeno-associated viral vectors?

  • Bigger to deliver more construct, integration
  • Smaller for easier delivery, no integration

4

32

Where do trunk and limb muscles come from?

Where do head and neck muscles come from?

  • Mesoderm --> somites --> myotome
  • Some muscles e.g. tongue from somites
  • Not from somites - Extra-ocular muscles Jaw muscles Facial muscles

5

32

What are the stages of myogenesis?

  • EPC - totipotent
  • MPC - multipotent
  • Myoblast - proliferative
  • Myotube - differentiated
  • Myofibre - peripheral nuclei and specialised structures

6

32

What transcription factors are expressed in myogenesis?

  • Pax3 and Pax7 - 3 for limb (and satellite of trunk), 7 for satellite, need for ESC-->MPC and regulation of MRF
  • Myogenic regulatory factors (MRFs) - initiate myogenesis on own
    • Primary - MyoD and Myf5 - expression of myogenin in myoblasts
    • Secondary - Myogenin and MRF4 - need both for differentiation and muscle-specific genes (MyHC)
  • MEF2 and SRF - contribute to differentiation and maturation

7

32

What are growth factors?

What growth factors are involved in myogenesis?

  • Induce intracellular signalling cascades
  • IGF-1: ↑ proliferation, ↑ differentiation
  • LIF, IL-6: ↑ proliferation, ↔ differentiation - myoblasts
  • FGF, HGF: ↑ proliferation, ↓ differentiation - myoblasts
  • TGF-β1 (Myostatin): ↓ proliferation, ↑ differentiation - myotubes
  • Insulin, IGF-2: ↔ proliferation, ↑ differentiation - myotubes

8

32

What miRNAs are involved in myogenesis?

  • MyomiRs - regulated by MRFs
  • miR-1/206 inhibits follistatin for differentiation
  • miR-133 inhibits SRF for proliferation

9

33

What are the different theories for satellite cell self renewal?

Theory A - Asymmetric cell division - original divides one populates the nicheone is activated and proliferates more likely

Theory B - Return to Quiescence - original activated and proliferates one returns to the niche

10

33

What are the other sources of adult stem cells?

Mesoangioblasts - blood-vessel associated from embryonic, prefer large

Pericytes - blood-vessel associated from post-natal, prefer small

AC133+ cells - blood and skeletal muscle derived Bone marrow-derived stem cells

Muscle-derived stem cells (MDSC) - muscle interstital space, CD34 and Sca1 positive

Side population (SP) cells - in skeletal muscle and bone marrow associated with vasculature, efflux Hoechst

11

33

How has transplantation been attempted in cell therapy?

  • Myoblast transplantation - poor survival, migration and regeneration
  • Satellite cell transplantation - poor survival and migration, need regenerating environment
  • Myofibre-associated satellite cell transplants - protects against age-related muscle degeneration in mice
  • Mesangioblasts - systemic delivery, but difficult to purify and poorly defined cell population

12

34

What are the different levels of trancription regulation?

  • Binding of transcription factors to 5' promotor region
  • Binding of RNA polymerase to TSS
  • Epigenetic regulation

13

34

How does epigenetic regulation of transcription occur?

  • DNA methylation
  • Histone methylation
  • Histone acetylation

14

34

How does DNA methylation occur?

How is it facilitated and mediated?

How is it related to metabolism?

How are satellite cells involved?

  • Methylation of cytosine base of CpG dinucleotide - often at promotor leading to repression, SAM-->SAH
  • DNA methyl transferases and DNA demethylases - a-ketogluterate
  • Serine from glycolysis to 1 carbon cycle --> SAM for DNMT and a-ketogluterate from TCA for TET
  • Increased DNA methylation in activated

15

34

How does histone methylation occur?

How is it facilitated and mediated?

How is it related to metabolism?

How are satellite cells involved?

Methylation of lysine (or arginine) in tail - tight chromatin to prevent binding

Histone methyl transferases and lysine demethylases (LDH and JHDM)

Serine from glycolysis to 1 carbon cycle --> SAM for DNMT and a-ketogluterate from TCA for JHDM

More H3K27me3 when move from quiescence to proliferation

16

34

How does histone acetylation occur?

How is it facilitated and mediated?

How is it related to metabolism?

How are satellite cells involved?

  • Acetylation of lysine - loose chromatin to allow binding
  • Histone acetyl transferases and histone deacetylases
  • Acetyl-CoA for HAT NAD+ for SIRT
  • Activation - more glycolysis, less NAD+ for SIRT

17

35

What does caloric restriction do to MuSCs?

Glycolysis to oxidative phosphorylation, better transplantation efficiency

18

35

What are the different models to study myogenesis?

Culture of single fibres - niche, can study first division

FACS isolation of MuSCs - large numbers of cells for RNA and protein

C2C12 cells - easy and cheap, but biologically differ

19

35

What is the relationship between mitochondria and specification?

What does galactose do?

  • Specification leads to more mitochondria
  • Decreases specification
    • Reduced metabolic capacity
    • Decreased mitochondria
    • More asymmetric divisions
    • More Pax7