Fig. 1.

Incorporating the small mouse beta-globin minimal promoter into transgenic reporters. (A) Workflow of reporter generation or enhancer testing with Multisite Gateway recombination and Tol2 transgenesis. An enhancer fragment is cloned into a p5E vector and Gateway- or restriction enzyme-cloned into an expression construct composed of a 5′ fragment, a middle entry mouse beta-globin minimal promoter:fluorophore/KalTa4/creERT2, 3′ SV40 polyA and a transgenic marker (optional). The resulting Tol2 expression construct is injected along with Tol2 transposase-encoding mRNA into one-cell stage zebrafish, randomly integrated into the genome and screened to establish transgenic lines. Schematic created with BioRender.com (license through CU Anschutz). (B) Schematic of mBmp4^ECR2_minprom:EGFP containing the distal mouse Bmp4^ECR2 element, the mouse beta-globin (Hbb-bt) minimal promoter driving EGFP and the SV40 polyA. (C-G) mBmp4^ECR2 reporter expression in a variety of zebrafish tissues linked to endogenous bmp4 expression. (C) Lateral view of a stable transgenic line of mBmp4^ECR2_minprom:EGFP at the 20-somite stage; asterisk marks the developing heart; arrowhead indicates the olfactory bulb. (D-F) Lateral (D,E,G) and ventral (F) views of 3 dpf stable mBmp4^ECR2_minprom:EGFP transgenic larva (D) show expression in the heart (asterisk) and pectoral fin (arrowhead), as well as in muscles and fins (D,E), parts of the head musculature (F), and in the mesothelium surrounding the yolk, the trunk musculature and fin fibroblasts (G). (H,I) The mHbb-bt minimal promoter has little to no background activity in transient injections. (H) Schematic of expression vector T7_minprom:mCerulean using the bacterial phage T7 promoter (inactive in zebrafish), followed by the mHbb-bt minimal promoter and mCerulean ORF as a reporter. An eye lens-specific cryaa:Venus cassette in cis was used as a transgenesis control. (I,J) Representative 2 dpf embryo of a control or reference injection repeat with T7_minprom:mCerulean, imaged for mCerulean (I) and with Venus as a reference for successful injection (J). There is no detectable mCerulean expression (representative among 136 individually injected embryos in this replicate). Scale bars: 500 μm (C,D,I,J); 200 μm (E-G).