Tol2 spearheaded by
@koichi_kawakami
has transformed #zebrafish transgenesis. Vector sets to assemble transgenes with (relative!) triggered a prolific jump in tools, including
* https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/dvdy.21343

* https://anatomypubs.onlinelibrary.wiley.com/doi/10.1002/dvdy.21354

* https://liebertpub.com/doi/10.1089/zeb.2016.1321

What are we adding now?

2/n

1) Minimal promoter vectors🧬🐁

#Enhancers need a minimal promoter to drive transgenes.
To test regulatory elements, we have been using the mouse beta-globin minprom.

We coupled this tiny (129 bp!) promoter in Gateway pME vectors with fluorophores, creERT2, and Gal4.

3/n

The mouse beta-globin minprom plays well with various enhancers we & others have tested over the years. Try it in your constructs!

...and thx @ojtamplin for introducing me to this🙏

Here's a downstream enhancer from mouse Bmp4 (PMID: 19159624) as minprom:EGFP in zebrafish:

4/n

2) mCerulean🔷 & mApple🍎 vectors
#EverythingIsGFP

To expand ease of use of diverse fluorophores, we made Gateway vectors with the blue mCerulean - our current lab favorite!

Here @aburger2009's isolated human HOXP regulatory region driving mCerulean (& mKate2 lens marker!)
5/n

@BlockInTheBack pointed us to the bright, well-behaved red mApple🍎 as alternative to mCherry, dsRED2, et al - and together we built a suite of mApple Gateway vectors.

Here nicely applied by @TheLovelyLab to label pharyngeal endoderm membranes - give it a try!

#AnmAppleADay
6/n

3) p2A as transgene labels

@kwantourage invested into tools to fluorescently label proteins of interest - cloning a beautiful suite of 3' Gateway vectors with viral p2A + diff. fluorophores & localizations.

Fuse your protein of interest with a C-terminally detaching color!
7/n

4) exorh as new transgenesis marker

A good transgenesis marker to track your construct is key.
As field, we have been mainly using heart (myl7) and eye lens (crystallin a/b). Highly instrumental tools.

But can we expand on these?

Enter the pineal gland.
8/n

The pineal sits on top of the head, forms early in development, stays visible throughout life...and it is distinct from the heart & eye (duh).

Encouraged by @PatrickBlader, we got the regulatory region of the zebrafish exorh gene as isolated here:
https://pnas.org/doi/full/10.1073/pnas.232444199

9/n

Cassie Kemmler coupled the exorh promoter with different fluorophores and generated Tol2-based pDest vectors for Multisite Gateway - all validated as stable transgenics.

exorh-based pineal fluorescence comes up in late somitogenesis - easy to sort throughout zebrafish life:
10/n

exorh-carrying backbones have become the main Tol2 vectors in our lab over the past years for all sorts of lines: CreERT2 drivers, KalTA4 lines, loxP Switches, you name it.

Easy to sort & combined with other transgenesis markers for complex multi-transgene experiments!

11/n
(hope that gif works...)

...and it's not just for zebrafish - enter #cavefish!

Sylvain Bertho in
@NicolasRohner's lab used exorh-based Tol2 backbones in #Astyanax:
both surface fish and eye-less cavefish express the reporter - hello new transgenesis marker!

#collaborate #TeamFish

12/n

In sum, we present a collection of new tools applicable for transgenesis in zebrafish, cavefish, and potentially lots of other species.

We are currently depositing the main plasmids
@Addgene.

Pls get in touch if you are interested in any plasmids already!
#ShareYourTools

13/n

Big thanks and congrats again to all involved, feat.
@aburger2009

@BlockInTheBack

@kwantourage

@TheLovelyLab

@NicolasRohner
& Affolter labs
@CUAnschutz

@CUDevBio

@UUtah

@ScienceStowers

@biozentrum
🐟🧬🗺️

14/n

And:
None of this work would have been possible without amazing prior work in the #zebrafish field, and the generous exchange of reagents & ideas.

Thanks to everyone who supported this work over the years - and good luck for your future clonings!

#GetCloning #MakeMoreTransgenics

/fin