Journal: Science advances

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

doi: 10.1126/sciadv.adn6603

Figure Lengend Snippet: Fig. 1. Zebrafish pIGLET14a and pIGLET24b landing sites enable high-quality transgenics. (A and B) Schematic of pIGLET system workflow. Transgenes with attB- containing pDEST or MCS backbones are coinjected with phiC31 integrase mRNA into pIGLET14a or pIGLET24b zebrafish (A). Integration is depicted with both attB- containing pDEST (pCM268, pRL055, pRL056, pCK122, and pCK123) (top) or MCS (pRL092 and pRL093) (bottom) backbones (A). The 5′ and 3′ transgene integration boundaries can be confirmed via PCR and sequencing (B). (C and D) Genomic locations of both landing sites. (E to J) Validating pIGLET14a and pIGLET24b with F2 drl:EGFP, lmo2:EGFP,cryaa:Venus and elavl3:mCherry,exorh:EGFP. (E and F) Crossed to Tol2-based drl:mCherry, p14a.drl:EGFP and p24b.drl:EGFP show analogous reporter activity in lateral plate mesoderm (LPM; 10 to 12 ss green fluorescence). Note faint brain expression in p14a.drl:EGFP (E, white asterisk) common with Tol2-based drl reporter trans- genics with strong expression. (G and H) Crossed to Tol2-based lmo2:Switch (dsRed2, magenta), p14a.lmo2:EGFP and p24b.lmo2:EGFP show analogous, overlapping activ- ity in endothelium at 20 hpf. EGFP expression is more consistent/complete compared to dsRED2 in Tol2-based lmo2:Switch (G and H, white asterisk). (I and J) p14a. elavl3:mCherry and p24b.elavl3:mCherry show complete, consistent reporter expression in the central nervous system at 72 hpf, akin to previous Tol2-based elavl3 trans- genics. The in cis exorh:EGFP transgenesis marker indicated with white arrowhead. Note common autofluorescence in blood, kidney, and yolk (I and J, white asterisk). F2 heterozygous embryos are depicted (E to J). Scale bars: 100 μm (E), 250 μm (G), 250 μm (I), and 50 μm.

Article Snippet: All plasmids were created with the Multisite Gateway system with LR Clonase II Plus (Life Technologies, catalog no. 12538120) according to the manufacturer’s instructions and original Tol2 Kit information (2, 31), assemblies were described below, and vector ratios were calculated using the Multisite Gateway Excel Spreadsheet (68): pCM318 drl:EGFP: pCM293 pENTR5′_6.3- kb drl regulatory region (32), #383 pENTR/D_EGFP (2), #302 p3′_SV40 polyA (2), and pCM268 pDESTattB (22) (Addgene, #68313). 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. pRL069 hsp70l:Switch2,cryaa:Venus: pDH083 pENTR5′_hsp70l:loxPStop- loxP (69), #763 pENTR/D_mApple (31), pGD003 p3′_ubbpA (31), and pRL055 pDESTattB_cryaa:Venus. pRL050 Hs_enh9Bwt:min- mCherry,exorh:EGFP: pRL049 pENTR5′_Hs_enh9Bwt, pCK006 pENTR/D_min- mCherry (31), pGD003 p3′_ubbpA (31), and pRL056 pDESTattB_exorh:EGFP. pRL052 Hs_enh9BT>G:min- mCherry, exorh:EGFP: pRL051 pENTR5′_Hs_enh9T > G, pCK006 pENTR/D_min- mCherry (31), pGD003 p3′_ubbpA (31), and pRL056 pDESTattB_exorh:EGFP. pCK086 Hs_I:min- mCerulean, exorh:EGFP: pENTR5′_Hs_I (58), pSN001 pENTR/D_min- mCerulean (31), #302 p3′_SV40 polyA (2), and pRL056 pDESTattB_exorh:EGFP. pCK085 Hs_IdelTbox:min- mCerulean, exorh:EGFP: pCK075 pENTR5′_Hs_IdelTbox (58), pSN001 pENTR/D_min- mCerulean (31), #302 p3′_SV40 polyA (2), and pRL056 pDESTattB_exorh:EGFP.

Techniques: Sequencing, Activity Assay, Fluorescence, Expressing, Marker