I am working on developing donor constructs to be used for a transgenic project where we are using CRISPR coupled with HDR to insert our gene of interest in channel catfish.
In the past we have had success using some larger promoters and terminators like opAFP (promoter 2120bps, terminator 1160bps) using other methods for insertion.
However, with testing out the new CRISPR insertions method we also want to test using a smaller insertion so that we can test the difference in success in using a single stranded oligo verse a plasmid donor.
For this we have to keep the sequence length under 2,000bps. Thus, since our gene of interest is ~500bps and we are using two 100bp homologous arms for HDR we need a promoter and terminator that is
I am not a fish geneticist, but in mammals, the human Ubiquitin promoter is only about 1.2kb and a standard polyA terminator sequence is about 135nt, but more recent publications support early termination is increased by introducing a triplet PolyA sequence (~630nt) for achieving KI via HDR, or achieving a KO using an HDR approach by adding this early terminator sequence. We are currently generating KI using homology arms at both 500bp, and as small as 40bp (PITCh system or MMEJ).
Thank you Timothy, do you have the sequence, accession number, publication that has this promoter so I can find it?
NCBI Reference Sequence: NG_027722.2
The plasmid is modified from the piggybac vector described:
Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci USA 108, 1531–1536 (2011).
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs