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[assembler01]

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.