Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Proper controls/design for comparing primary myoblasts?

primary myoblast variation wild type null

  • Please log in to reply
No replies to this topic

#1 assembler01

assembler01

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 48 posts
3
Neutral

Posted 21 June 2012 - 08:16 AM

We have a knockout mouse for a gene that seems to be important for muscle differentiation. It seems to cause a decrese in muscle size in the mice. When you knock it down in C2C12 cells you get fewer myotubes. We've been isolating primary myoblasts from the nulls and wild type siblings but there is a huge variation in the differentiation potential of each prep and even from passage to passage. We assumed we would be able to say that the wild types always differentiate well and the nulls don't but often the wild type don't. Sometimes the nulls do. The technique doesn't seem reproducible. We've talked with other labs and they note the procedure isn't reproducible itself, so I wonder if we can even make that comparison.

I wonder if anyone has thought of this before. How can we control for the variability? Could we try something like combining the cells from a null and a wild type in the same dish so they are exposed to the same densities and secreted growth factors? Maybe we could differentiate them and then stain for our knocked out protein to compare? Would that even be a proper control or might there be too many differences in the isolation such that it's still not a fair comparison?

Could we isolate nulls and somehow transfect some with wild type protein? I heard it is extremely difficult to transfect primary myoblasts though. Then we also have the potential problem of overexpression of our protein.

Thanks for any ideas.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.