splicing MO vs translation MO

[rose9999]

hi Jon,

thanks for your reply.

My boss insists on 5 mis MO. I ordered another 5 mis MO from gene tool.

I found the first 5mis MO, it does not cause CE phenotype at the low concentration , but cause curled trunk which is different from CE phenotype. I supposed it is off target effects. when I used high concentration, said 2 folds , it caused CE phenotype as i observed from AUG-MO although phenotype is not so severious as AUG-MO.

The second 5mis MO, at low does, like 1.5ng , AUG-MO at this does casued CE phenotype 100%. 5-mis at this concentration 20% embryos also caused mild CE phenotype .

IS my 5-mis MO ok?

Thanks

[jonmoulton]

Hi Rose,

The results you hope for from a five-mispair is that the five-mispair does not cause any phenotype at all -- that is, it acts like a negative control when the five mispair oligo is used at low concentration. The reason that I do not like the five mispair experiment is that often a five mispair oligo will interact with either the target mRNA or an off-target mRNA even at low concentrations. This is the outcome we hope will not occur, but with some five-mispair sequences it will occur, and this is why I recommend against using the five mispair control.

Your five mispair control is interacting with something or you would not be seeing a phenotype. I wonder if its RNA interaction is triggering p53-mediated apoptosis, as the curved-tail phenotype (ventrally curved) is a typical outcome of the p53-mediated apoptosis. Perhaps you could suppress some of the phenotype you observed by coinjecting a p53 oligo. However, if the p53-mediated apoptosis is occurring that indicates that some gene is being knocked down, so the five-mispair oligo is not working as a clean specificity control.

[rose9999]

Hi Jon,

Thank you very much