Hello All,
I am working on trying to increase the rates of successful integration of a transgene in catfish using CRISPR HDR knock-in. Right now we are using microinjection on eggs shortly after fertilization and have had great success for knock-out and have some success with HDR knock-in. Now we wanted to try to increase the integration rates for CRISPR HDR knock-in using SCR7 pyrazine and RS-1.
The question I have is has anyone tried microinjection of SCR7 pyrazine and RS-1 and if so what concentrations did you use?
The reason we want to use microinjection instead of incubation is that we have to incubate our eggs in a modified Holtfreter's solution (contains NaCl, NaHCO3, KCl, MgSO4, CaCl2, and Doxacycline) in order to prevent fungal growth and strengthen the eggs and I was not certain what effect this would have on SCR7 pyrazine and RS-1 if we add it to the solution.
Any suggestions are greatly appreciated.
Best Regards,
Hi, this may be a little late for you, but I'm currently making HDR knock-in Xenopus lines (just got it working, kind of, we're waiting to see if there is an actual line). We, and other labs have found that adding scr-7 to the oocyte culture medium increases HDR efficiency. It may be different for catfish and also for fertilised eggs apposed to oocytes, but worth a try.
Hello Sian,
Thank you for the response. We ended up not using any enhancers and got really good integration with some of our targets. However, we do have a couple of constructs/sites that still have low integration rates, so I would be interested in trying out SCR-7 to see if that helps. Do you mind if I ask what concentration you used and/or what procedure you used?
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs