Generation and Dox-induction of the Tg(gfap:rtTA), Tg(TRE:GSP-Il1βmat) transgenic system (i.e. CNS/Il-1β)

Plasmids were constructed using Gateway cloning and components of the Tol2kit.^93,94 The plasmids pME-rtTA and p3E-TRE were made by inserting the Tet-On fragments from the Tet-On system (Clontech) in the appropriate Tol2 entry vectors. For p5E-gfap, the zebrafish gfap promoter was released from the pGFAP-EGFP vector and inserted into the 5’ entry clone p5E-MCS.⁵⁴ The pME:GSP-il1β^mat vector was constructed in our lab previously, as described by Lanham et al.⁵⁷

To make the pDestTol2CG2 gfap:rtTA construct, p5E:gfap, pME:rtTA, p3E:pA, and the destination vector pDestTol2CG2 were recombined using LR Clonase II Plus (Invitrogen). To make the pDestTol2CmC2 TRE:GSP-il1β^mat construct, p5E:TRE, pME:GSP-il1β^mat, p3E:pA, and the destination vector pDestTol2CmC2 were recombined in the same manner.

To generate the transgenic lines Tg(gfap:rtTA, cmlc2:EGFP) and Tg(TRE:GSP-il1β^mat, cmlc2:mCherry), the Tol2 constructs were co-injected into single-cell embryos either individually or in combination (50–100 pg total plasmid DNA) together with 20 pg of in vitro transcribed Tol2 transposase mRNA in a final volume of 1–2 nanoliters. Embryos with strong transient expression of the transgenesis markers were raised to adulthood and screened for germline transmission of the transgenes.

To induce expression of Il-1β, double transgenic gfap:rtTA; TRE:GSP-il1β^mat embryos were treated with 0.1 – 10 μg/mL doxycycline (Dox) at approximately 6 hpf. Dox (RPI) was stored at −20°C as 10 mg/mL stocks in water and added to PTU/egg water or egg water without PTU for survival analysis at the desired concentration. All experiments were performed on embryos that were heterozygous for both the gfap:rtTA and TRE:GSP-il1β^mat transgenes.