Red fluorescent reporters for transgenic fish

[wei xia]

Dear All,

We are wondering about what kinds of red fluorescent reporters are good for making transgenic zebrafish.

Our gene’s strongest expression occurs around 24hpf by in situ results. But when we used DsRed as the reporter we could not see any fluorescence until after 44-48hpf in our transgenic embryos. The fluorescence was pretty obvious at 24hpf if transient expression was checked by plasmid injection. So we guess the late detection of DsRed in these transgenic embryos is not because of the promoter we used is not complete or something. We thought this is might because of DsRed’s long maturation half time. We are thinking about using monomeric mCherry but worry about its low brightness compared to DsRed. 

If you have any experiences of making RFP transgenic fish please share them with us!

Thanks in advance for your testimonies on this.

Wei

_______________________________________________
Zbrafish mailing list
Zbrafish@...
http://www.bio.net/biomail/listinfo/zbrafish

[Chi-Bin Chien]

Dear Wei,

Very likely your problem is due to the slow maturation of DsRed. mCherry should be better and is fairly bright. We have also had good success with TagRFP, which is extremely bright (available commercially from Evrogen). Another possibility is tdTomato, which is extremely bright. We have not used it ourselves but have heard good things from others. Don't know about maturation time.

There is an easy experiment to test whether maturation time is the issue: do a DsRed in situ on your transgenics, and see if the timecourse mimics the native gene.

best wishes,

Chi-Bin Chien

On Nov 2, 2009, at 5:19 PM, Wei Xia wrote:

Dear All,

We are wondering about what kinds of red fluorescent reporters are good for making transgenic zebrafish.

Our gene’s strongest expression occurs around 24hpf by in situ results. But when we used DsRed as the reporter we could not see any fluorescence until after 44-48hpf in our transgenic embryos. The fluorescence was pretty obvious at 24hpf if transient expression was checked by plasmid injection. So we guess the late detection of DsRed in these transgenic embryos is not because of the promoter we used is not complete or something. We thought this is might because of DsRed’s long maturation half time. We are thinking about using monomeric mCherry but worry about its low brightness compared to DsRed. 

If you have any experiences of making RFP transgenic fish please share them with us!

Thanks in advance for your testimonies on this.

Wei

<ATT00001.txt>

_______________________________________________
Zbrafish mailing list
Zbrafish@...
http://www.bio.net/biomail/listinfo/zbrafish