Next-generation plasmids for transgenesis in zebrafish and beyond

Abstract

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models.

pIGLET : Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish

“…pCM328 lmo2:EGFP,cryaa:Venus: pENTR5′_lmo2 upstream region (36), #383 pENTR/D_EGFP (2), #302 p3′_SV40 polyA (2), and pCM327 pDESTattB_cryaa:Venus (22) (Addgene, #68341). pRL058 elavl3:mCherry,exorh:EGFP: pENTR5′_8.7-kb elavl3 (HuC) (38), #456 pENTR/D_mCherry (2), pGD003 p3′_ubbpA (31), and pRL056 pDESTattB_exorh:EGFP. pRL045 drl:creERT2,cryaa:Venus: pCM293 pENTR5′_6.3-kb drl regulatory region (32), pENTR/D_creERT2 (Addgene, #27321) (29), pGD003 p3'_ubbpA (31), and pRL055 pDESTattB_cryaa:Venus.…”

Conserved enhancer logic controls the notochord expression of vertebrateBrachyury

“…We initially tested all six enhancer candidates in zebrafish that allows for highly efficient reporter gene activity screening in developing embryos. To test their activity within a broad evolutionary framework, we cloned the human enhancer element candidates T3, K, J, C, I, and L into reporter vectors coupled with the mouse betaE-globin minimal promoter to express the blue fluorophore mCerulean for enhancer testing in zebrafish embryos (Kemmler et al, 2023). Upon co-injection into one cell-stage zebrafish embryos together with ubi:mCherry as injection control, the human hs_T3, hs_C, and hs_I elements resulted in mCerulean expression in the developing zebrafish notochord during early somitogenesis, followed by strong, selective notochord activity in injected embryos at 24 hours postfertilization (hpf) (n=32/61, n=155/227, n=76/117; mCerulean-positive notochord/total mCherry-positive embryos) (Fig.…”

“…The reporter constructs further harbor the transgenesis marker exorh:EGFP (expression in the pineal gland, Fig. 2D-I) for precise quantification of reporter activity (Kemmler et al, 2023). After injection into zebrafish embryos and in line with the enhancer element activity at 24 hpf (Fig.…”

“…Subsequent Multisite Gateway cloning (half-volume reactions) were performed using LR Clonase II Plus (12538120, Invitrogen) according to the manufacturer's instructions (half-volume reactions) and recommended reaction calculations (Mosimann, 2022). 5' entry plasmids containing the different enhancer elements were assembled into reporter expression plasmids together with the middle entry plasmid (pME) containing the mouse betaE-globin minimal promoter expressing mCerulean (pSN001) as well as mApple (pCK068), the 3'plasmid #302 (p3E_SV40polyA), and the destination plasmid pDESTTol2A2 containing crybb1:mKate2 (pCB59) and pDESTexorh:EGFP containing EGFP expression in the pineal gland (pCK017) as transgenesis markers (Kemmler et al, 2023). Assembled vectors were verified using restriction digest and Sanger sequencing using standard sequencing primers for Multisite Gateway assemblies (Kemmler et al, 2023;Mosimann, 2022).…”

Rapid generation of single-insertion transgenics by Tol2 transposition in zebrafish

“…Development of the Tol2kit further facilitated broad adoption of Tol2 by introducing a collection of Gateway-compatible vectors containing commonly used elements such as promoters and reporters (Kwan et al, 2007). This library of compatible components has been recently thoroughly expanded (Kemmler et al, 2023). In addition, many laboratories possess their own libraries of constructs, making it very likely that Tol2 will remain the transgenesis tool of choice for zebrafish.…”