Darius Balciunas

Last but not least, thank you to my lab in Vilnius, especially Edita Bakūnaitė, Justas Lazutka and @MigleKalvaityte for making this event happen.

Thank you to our corporate sponsors Animalab, Linea Libera and Thermo Fisher Scientific Baltics. (4/5)

Yesterday we wrapped up a workshop "Genome editing in zebrafish: knock-ins and their applications". A huge THANK YOU to EVERYONE who joined us for very rigorous and stimulating scientific discussion throughout the workshop! (1/5)

Rapid generation of single-insertion #transgenics by #Tol2 transposition in #zebrafish. This study describes modifications to Tol2-mediated transgenesis that facilitates the generation of multiple independent single-insertion F1 transgenic lines doi.org/10.1002/dvdy.7…

@chrmosimann @ScienceAdvances @CUAnschutz @CUDeptofPeds @Rob_Lalonde89 @aburger2009 Congratulations!

"Speed" and unreliability of #SASWIFI makes me think #SK903 is dragging behind a phone line from the nineties. Quite an experience.

@filodelbene @ramialison_lab @sonkelab @danionella Congratulations!

🎉 Exciting News! 🎉 I am absolutely thrilled to announce that I've been awarded a HFSP grant to embark on a brand new project decoding the invisibility superpower in fish! Together with @ramialison_lab and @sonkelab we will study the basis for transparency in @danionella

@YanivElkouby Horrible but sadly not surprising anymore.

Unbelievable… Wynnewood, PA, was our home for six years during my postdoc at UPenn. A beautiful place to raise your kids. This is just awful.

@sumeetpalsingh8 I was not trying to argue against I-SceI. It can be great in some scenarios like various "bow" applications you highlighted. With regard to Tol2, I believe one should only use single-copy lines in publications but I am clearly in a minority.

@Balciunas_Lab We could perform clonal analysis with I-SceI based lines: nature.com/articles/s4146… Here, 4-OHT concentration and recombination needed to be reproducible between animals. With Tol2, the multiple loci lead to high variability between siblings, and single copy could be weak/mosaic.

1/8 Are you interested in knocking genes out in adult zebrafish? If so, you should read our new preprint! Our new ~ubiquitous ubbR:CreERT2 driver enables gene knockout in adult #zebrafish hearts with efficiency approaching 100%. Yes, we quantified it. biorxiv.org/content/10.110…

@chrmosimann @sumeetpalsingh8 There is no practical way to prove near-100% efficient recombination in every possible cell type. When induced at 6 hpf and assessed at 3 dpf by qPCR on DNA, our driver gives better excision of ubi:Swi GFP than ubi:CrERT2 you went to great lengths to demonstrate was ubiquitous.

@YanivElkouby Thank you!

@Balciunas_Lab Congrats Darius and team! 🍾 As always, impressive genetic engineering from your lab!

@chrmosimann @sumeetpalsingh8 That is why we quantify excision at DNA level. It is certainly NOT perfect, but it does tell us that excision occurs in absolute majority of cells in a complex tissue like the adult ventricle.

@chrmosimann @sumeetpalsingh8 We have not explicitly - would be fun to do!

@sumeetpalsingh8 As for I-SceI vs Tol2, my impression is that with I-SceI one often - if not mostly - gets a concatemer in instead of single copy. Am I wrong?

@Balciunas_Lab In our experience, the ubb promoter is weak in adults, but 9.8kb actb2 promoter or medaka actb promoter retains strong activity in adult tissues. (Fig 2 here onlinelibrary.wiley.com/doi/10.1111/dg…) And transgenesis with I-SceI has less positional effects than Tol2.

@sumeetpalsingh8 Thank you for bringing your paper to my attention! We have other transgenics using the same carp actb2 that are super strong. Something about that particular transgene is just "funny". Even lens RFP is weaker. We re-made the plasmid, sequenced everything, and re-made transgenics.

8/8 - While we did not do a proper comparison our observations suggest that it is best to place the two loxP sites close together when making a #floxed allele. Similar observations have been made in other systems.

7/8 - Carp bAct2 did not seem to work at all in our docking site (line tpl102 @ZFINmod). However, its enhancer still boosted the expression of ubb. Is that strange or not?