Largest knock-in via ssODN

I was wondering what the largest knock-in people have obtained using an ssDNA donor. I'm looking to make a mouse, and I'd love to knock in a FLAG-loxP cassette. Adding in the intervening region between the cassette and cut site, it would be a 75bp knock-in.

Has anyone been able to make something similar? In mice or in other models? How was your efficiency?


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Lime Sirenover 7 years ago

I don't see why this cannot be done. But are you also going to insert another LoxP site somewhere 5'? If so, how are you planning to get that done? If the entire region between the two LoxPs is not too long, have you also considered using a plasmid donor?

Also, earlier in this group someone mention his issue of always getting mutated LoxP site. You may find that interesting to read prior to conducting your experiment.

Good luck!

Terracotta Harpyover 7 years ago

I think that might be right on the edge of reasonable efficiency since you only will have ~60 bp homology arms. You might want to consider having a gBlocks dsDNA construct made since you will get longer homology arms. Please let us all know if you get correct targeting with the ssoligo as I think that could be the largest insert yet using ssoligo, The other point is I would highly recommend PAGE purification since only ~35% or so of the entire mix will be your full length ssoligo (due to poor increasinly poor coupling efficiency during synthesis).

Bronze Impover 7 years ago

This paper inserted almost 400bp using ssDNA generated using IVTRT method

http://www.nature.com/srep/2015/150805/srep12799/full/srep12799.html

Chartreuse Kelpiealmost 5 years ago

This paper has inserted more than 1.5kb KI in mouse zygotes..long ssDNA with short homology arms. IVTRT approach.

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-017-1220-4

https://www.nature.com/articles/nprot.2017.153

They used IDT ALt System crRNA and tracRNA and RNP ...

http://www.sciencedirect.com/science/article/pii/S104620231630442X

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