Generation of a transgenic Xenopus tropicalis that expresses SB 10 transposase. (a) Schematic of the pCAGGS-SB 10;γcRFP construct used to develop SB (SB 10) transposase-expressing transgenic frogs. The two transgenes were cloned in a tail-to-tail orientation. Not to scale. (b) Red lens in the right eye of an adult F1 transgenic frog from outcross of founder CAGGS-SB 10;γcRFP 2 M. The border of the eye is indicated by the dashed white line. (c) Southern blot analysis of genomic DNA harvested from RFP-positive and control animals indicated integration of multiple copies of the CAGGS-SB 10;γcRFP linear transgene. The DNA was digested with Bam HI and the blot was probed with a radiolabelled SB 10 cDNA probe (see schematic (a)). (d) RT-PCR analysis of SB 10 expression in tadpoles. SB RNA was detected in RFP-positive tadpoles (+RFP) but not in RFP-negative (-RFP) progeny from CAGGS-SB 10;γcRFP 2M. RNA from a wild-type tadpole was used as a negative control (St. 15). A mock reverse transcription reaction, without added RT, with RNA harvested from an RFP-positive tadpole (+RFP(-RT)) was used as a negative control. Primers for X. tropicalis α-actin were used as a control for RNA recovery. (e) Western blot analysis of SB transposase expression in tissues harvested from adult transgenic frogs. A monoclonal antibody to SB was used to demonstrate abundant transposase expression in the testis and liver of RFP-positive adults, but not in the RFP-negative siblings. Protein lysates prepared from tadpoles injected with SB 10 mRNA at the one-cell stage were prepared at stage 15 (control lane). The blots were stripped and re-probed with a monoclonal antibody that recognizes Xenopus α-actin. PCR: polymerase chain reaction; RFP: red fluorescent protein; RT: reverse transcriptase; SB: Sleeping Beauty.