Hi all,
I did 40 injections with my target plasmid DNA +myo2:mCherry+pBS a total 100ng and had 48 red F1s . However, they did not give rise to any lines. I had one that was too faint, which didn’t give red progeny thereafter. Any inputs on why this might be the case? Chances to increase no. of lines?
What’s the concentration of myo2m:mCh in your mix? That construct is slightly toxic, so if there’s too much it could select against maintenance of the array. We use it at 2 or 2.5 ug/mL.
Thanks for the reply. I used 2ng/uL conc so it appears within the suggested range. Any thoughts?
Maybe other elements of your array are toxic? Do you get good, stable transmission if you inject myo2::mCh by itself?
Another thought is to make sure you are targeting the correct region of the gonad. You want to aim for the “dust” between the syncytial nuclei, which is an easy visual marker of the rachis. It is possible to get some F1s even if you’re injecting the wrong part of the gonad, but you’ll struggle to get transmission if you’re not targeting that region.
It could be other elements too. I don’t know yet. I haven’t tried just the myo2::mCherry by itself, will try that out. Thank you. I didn’t know that you could still get some F1s and not have lines. Do you happen to have a good visual for targeting the right area of the gonad?
I am new to microinjections
When trying the myo-2:mCherry by itself, remember to bulk up the injection mix with other DNA - there is some correlation between DNA concentration injected and frequency of transmission (not a strong correlation, not useful to get super high transmission or anything, but there are a lot of anecdotal reports that if DNA concentration is too low then transmission is very low). You can use 1 kb ladder or any other popular DNA molecular weight marker, or you can use vector DNA (Bluescript, for example).
And, yes, it’s very common for transgenic F1s to not give lines. Even when things go really well a minority of F1s - maybe 1/4 if you’re lucky - will give lines.
Ah…Okay. Thanks.
I don’t have a good video myself, but check YouTube and wombook. I’m sure there are some good ones out there. Basically you just want to focus through the germline; you’ll see a cylinder with nuclei on the outside. In the middle is some dusty-looking or granular material; put the tip of your needle right in the middle of there. It’s normal to struggle with injections at first - this is a technique that takes some practice to get good at. Keep at it and don’t be discouraged.
Thank you very much!
One of my favorite features of Pmyo-2::FP as a co-injection marker is mosaicism. It is really easy to tell if an F1 is mosaic in the pharynx. In my experience those animals are much less likely to transmit than those with a solidly fluorescent pharynx.
My favorite training mix is rol-6(d) (pRF4) at 50 ng/ul and Pmyo-2::GFP at 20 ng/ul, with pBS up to a total of 100 or 200. (GFP is much less toxic than mCherry in the pharynx: mCherry aggregates.) Easy to spot rollers, and the mosaicism is useful.
That’s an interesting notion. I assume that’s because the pharynx is composed ~1/3 of cells from the AB lineage and ~2/3 of cells from the P lineage?
It would be interesting if loss in pharyngeal cells the in the AB lineage versus the P lineage (which gives rise to ectodermal pharyngeal cells and also to the germline) correlates with whether the transgene establishes a line.
Could also just reflect general instability of that given array, and so pharyngeal mosaicism is a bad bet.
Never followed it up quantitatively. Just observed it enough anecdotally that I am quite confident of it.