Novel vectors for functional interrogation of Xenopus ORFeome coding sequences

Abstract

The current Xenopus ORFeome contains~10,250 validated, full-length cDNA sequences without stop codons from Xenopus laevis and~3,970 from Xenopus tropicalis cloned into Gateway-compatible entry vectors. To increase the utility of the ORFeome, we have constructed the Gateway-compatible destination vectors pDXTP and pDXTR, which in combination can control the spatial and temporal expression of any open reading frame (ORF). pDXTP receives a promoter/enhancer of interest, which controls the spatial expression of a doxycycline-inducible transcription factor rtTA. pDXTR receives an ORF of interest, which is controlled by a tetracycline response element enabling temporal control of ORF expression via rtTA activation by simple addition of doxycycline to the rearing water at any desired time point. These vectors can be integrated into the genome via well-established microinjection-based SceI, tol2, or phi-C31 transgenesis procedures and contain fluorescence reporters to confirm transgene integration. Cell-autonomous verification of ORF expression occurs via red nuclear fluorescence due to an mCherry-histone H2B fusion protein that is cleaved from the ORF during translation. Function of all essential features of pDXTP and pDXTR has been experimentally validated. pDXTP and pDXTR provide flexible molecular cloning and transgenesis options to accomplish tissue-specific inducible control of ORF expression in transgenic Xenopus. K E Y W O R D S doxycycline, ORFeome, rtTA, tetracycline response element, Xenopus transgenesis

Endosome-Mediated Epithelial Remodeling Downstream of Hedgehog-Gli Is Required for Tracheoesophageal Separation

“…For Xenopus transgenesis, the I-SceI meganuclease method was used as described (Sterner et al, 2019). The Tg(hhex;rtTA; TRE:dnRab11a-GFP) construct was generated as follows.…”

“…The -1.2Kb X.laevis hhex.L promoter (Rankin et al, 2011) and the dNRab11a-GFP fusion coding sequence (Kim et al, 2012) were PCR amplified gel purified and TOPO-TA cloned into the pCR8 Gateway entry vectors (Thermo Scientific #K250020). Gateway LR Clonase II Plus enzyme (Thermo Scientific #12538120) was used in standard recombination reactions according to manufacturer's instructions to transfer the hhex.L promoter into the pDXTP and dNRab11-GFP into the pDXTR transgenesis plasmids (Sterner et al, 2019).…”

“…Embryos were cultured at 13 C for the first two hours after injection and subsequently at 18 C to 23 C degrees thereafter. Transgenic embryos were selected based on GFP fluorescence in the eye, which becomes visible during early tailbud stages (Sterner et al, 2019). In half of the embryos the transgenes were activated by the addition of doxycycline hyclate (Sigma #D9891) at a final concentration of 50mg/mL culture buffer.…”

Endosome-Mediated Epithelial Remodeling Downstream of Hedgehog/Gli Is Required for Tracheoesophageal Separation

“…Embryos were cultured at 13 0 C for the first two hours after injection and subsequently at 18 0 C to 23 0 C degrees thereafter. Transgenic embryos were selected based on GFP fluorescence in the eye, which becomes visible during early tailbud stages (Sterner et al, 2019). In half of the embryos the transgenes were activated by the addition of Doxycycline hyclate (Sigma #D9891) at a final concentration of 50ug/mL culture buffer.…”