Optimal number of plates for (improved) MosSCI integration screening?

Model Organism: Worms Methods & Reagents
1

I am currently making several MosSCI lines, and I was wondering how many plates people typically expand their worm lines onto for Phsp::peel-1 negative selection. I have expanded some of my array lines onto five 10 cm plates, and so far I still haven’t been able to get any integrants.

2

I don’t think you need to expand anything. You just let your original P0 plates with F1s and F2s starve, then heat shock. I don’t recall the need to ever do anything, although the older protocol included some rounds of chunking. In my experience, I need 25-50 P0 plates that produced non-Unc lines in order to get 1-2 integrants, half of which will be correct. https://sites.google.com/site/jorgensenmossci/protocols

3

Kevin is right. In our experience, there is no benefit to expanding array animals for several generations. Insertions happen in the F1/F2 generation but not in later generations (presumably because the transposase is silenced in the array).

The number of injections you need to do to get an insertion also tends to vary quite a bit. We’ve heard from several people that the higher the quality of the DNA you inject, the more F1 rescued animals you get and the higher insertion frequency you will get. See for example Moris Maduro’s comment in WBG:
http://www.wormbook.org/wbg/articles/volume-19-number-1/omission-of-rnase-and-addition-of-ethanol-precipitation-improve-transgenesis-using-spin-column-purified-dna/

Although we’ve mostly used miniprep DNA for injection, we are testing right now if midipreps of the co-injection markers improve the insertion frequency.

4

Unfortunately, I’ve already tossed my P0 plates, so it appears that I may be better off injecting more worms. I also have been using Maduro’s no RNAse/EtOH precipitation protocol (I found this to dramatically increase success rates). Do you deposit injected worms onto 5 cm or 10 cm plates, and how many worms per plate?

5

I’ve had good luck putting one injected P0 animal per 6cm NGM plate, but I’ve not tried omission of RNase. My regular injections aren’t as good as they used to be, so maybe I will.