Looking for insertion site for driver mouse line. Publication is unclear to me

Science
1

Hello,

I am trying to figure out where a driver gene was inserted in the mouse genome. The mouse line in question is the “Tacr1-T2A-Cre” animals from this paper: “A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality” - Diagle et al. - 2018.

I am confused about where in the mouse genome the vector was inserted. Is it inserted into the Rosa26 locus, the TIGRE region, or somewhere inside the endogenous tacr1 locus? (Or none of those three options?)

I have read and re-read the paper, supplemental, as well as several papers they cite, and their supplementals. I found several passages describing knock-in methods, but I cannot seem to understand where the insertion is for the Tacr1-T2A-Cre line.

Here is an example passage that may be helpful, but I can’t seem to desipher: “All targeting vectors were constructed using gene synthesis and standard molecular cloning approaches. Knock-in driver line and Rosa26-based reporter vectors contained components that were previously described (Madisen et al., 2015; Madisen et al., 2010) … Targeting of the transgene cassettes into an endogenousgene locus or the Rosa26 locus was accomplished via standard homologous recombination using linearized targeting vector DNA orvia CRISPR/Cas9-mediated genome editing using circularized targeting vector in combination with a gene-specific guide vector
(Addgene plasmid #42230).”

Any help would be greatly appreciated!
David

2

Hello David,

This line was produced by direct knock-in of “2A-Cre” cassette into the Tacr1 locus in the mouse genome. Would that be sufficient for your purpose?

Best,
Bosiljka

3

Hello Bosiljka

This is exactly what I was looking for. For whatever reason, I though it might have been knocked into the ROSA26 locus, but then realized that does not make sense.

Thank you so much for your help!
David