slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
PTRM Student Presentation Dan Wang 10-9-2008 PowerPoint Presentation
Download Presentation
PTRM Student Presentation Dan Wang 10-9-2008

Loading in 2 Seconds...

play fullscreen
1 / 18

PTRM Student Presentation Dan Wang 10-9-2008 - PowerPoint PPT Presentation


  • 113 Views
  • Uploaded on

PTRM Student Presentation Dan Wang 10-9-2008. Gentamicin treatment promotes dystrophin expression in mdx myotubes. Immunohistochemistry of myotubes from primary cell culture from muscles

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'PTRM Student Presentation Dan Wang 10-9-2008' - xiang


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

PTRM Student Presentation

Dan Wang

10-9-2008

gentamicin treatment promotes dystrophin expression in mdx myotubes
Gentamicin treatment promotes dystrophin expression in mdx myotubes
  • Immunohistochemistry of myotubes from primary cell culture from muscles
  • Dystrophin was detected by mAb to the C-Ter of dystrophn, followed by a rhodamine-conjugated anti-mouse igG
dosage dependent manner
Dosage-dependent manner

There is a window of gentamicin dosage in which misreading can lead to the restoration of full-length dystrophin synthesis.

gentamicin treatment can protect mdx muscle from contraction induced injury
Gentamicin treatment can protect mdx muscle from contraction-induced injury

Eccentric contraction protocol

mdx EDL (extensor digitorum longus)muscles

G: gentamicin

Gi200: injection, 200% dose equivalent

GP: infusion pumps

D: DHB treatment

F: females

reduction in the damaged sarcolemma
Reduction in the damaged sarcolemma

Procion orange (fluorescent dye) can enter muscle cells only through membrane disruptions.

Mice were subjected to eccentric contractions in the presence of Procion orange.

gentamicin treatment protected muscle from injury through the restoration of dystrophin
Gentamicin treatment protected muscle from injury through the restoration of dystrophin

dystrophin

-sarcoglycan

Tibialis anterior (TA) muscle cross-sections

gentamicin treatment increased both dystrophin and sarcoglycan levels
Gentamicin treatment increased both dystrophin and -sarcoglycan levels

C57 controls

mdx muscle (untreated)

mdx muscle (Gi200)

mdx muscle (Gi50F)

mdx muscle (GP200)

Immunoblot analysis of TA muscles for dystrophin and -sarcoglycan.

ptc124 suppresses nonsense stop codons in vitro
PTC124 suppresses nonsense stop codons in vitro

HEK293 cells stably transfected with LUC-190 gene

HeLa cell-free extract containing synthetic LUC-190 mRNA

Differences in transcript levels?

PTC124

ptc124 treatment rescued the dystrophic phenotype in muscles of the mdx mouse
PTC124 treatment rescued the dystrophic phenotype in muscles of the mdx mouse

a. Force per cross-sectional area of treatment

b. Eccentric contraction assay

c. Serum creatine kinase changes

d. Western blot of quadriceps and TA muscles

ptc124 demonstrates little off target activity at the level of transcription or mrna stability
PTC124 demonstrates little off-target activity at the level of transcription or mRNA stability
  • Levels of 22 transcripts changed significantly
    • Not NMD substrates
    • Low expressing cellular mRNAs
conclusions
Conclusions
  • Gentamicin and PTC124 induced readthrough of premature stop codons both in vitro and in vivo
  • Full-length, functional proteins (dystrophin) were produced after treatment (10-20% of WT)
  • Dystrophic phenotype was partially rescued in the mdx mouse
  • Optimized dosage and regimen
  • PTC124 has no observed effect on normal stop codons
discussion
Discussion
  • Efficiency of readthrough at premature stop codons
    • Stop codon context (other genetic diseases caused by nonsense mutations)
    • Dosage effect
    • Derivatives of compounds
  • Rescue of the disease phenotype
    • How much protein is needed?
    • Any problem with the inserted amino acid?
    • Combination with other approaches
  • Side effect
  • Mechanism of PTC124 activity
    • Understand how the cells can distinguish a premature stop codon from a normal one.