Figure 1

Generation of Tg[Cca.actb:GCaMP6s](ihb371Tg) transgenic zebrafish. (A) Schematic of the pTol2-Cca.actb-GCaMP6s construct. ITR-L and ITR-R, left and right inverted terminal repeats of Tol2 transposon; Cca.actb promoter, promoter of the common carp ?-actin (actb) gene. Directions of the arrows and arrow heads are the same as the transgenic elements they represented. The transgenic elements were shown in different color for clear discrimination. (B) PCR screen of positive P0 individuals. M: DNA marker; 1 to 12: fish number. (C) Transgene copy number of the positive F1 individuals detected by qPCR assay. The x-axis represents fish number. (D) Southern blotting confirmed a single copy of the transgene in the genome of F1 transgenic zebrafish. The genomic DNA was digested by XcmI and HindIII, respectively. (E) Cloning flanking sequences of the transgene through genome walking. L1 to L3: products of the first to third round of PCR reactions using primers located on ITR-L; R1 and R2: products of the first and second round of PCR reactions using primers located on ITR-R. (F) Schematic of the integration site of the transgene in zebrafish genome. The insertion site is 88 kb downstream of the mamdc2b gene and 16 kb upstream of the apba1b gene on chromosome 10. The arrows indicate primers used for genotyping. (G) Genotyping of the transgenic zebrafish. PCR assays using primer pairs amplifying both ends of the transposon demonstrate the integrity of transgene cassette. This transgenic fish line was submitted to the China Zebrafish Resource Center (CZRC) under the accession number ihb371.