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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.
Figure 1
pDXTP and pDXTR workflow and plasmid diagrams. (a) Work flow overview. (1 and 2) a promoter of interest (POI) from a gatewayâ€compatible promoter plasmid (e.g., from pTransgenesis) and an ORF from the Xenopus ORFeome are cloned into pDXTP and pDXTR, respectively. (3 and 4) these transgenesis plasmids are injected into eggs to make mosaic founders that may be analyzed in the F0 generation. (5) transgenic lines may be characterized and then crossed with each other to obtain doubleâ€transgenic offspring capable of tissueâ€specific and inducible control of ORF expression. (b) pDXTP plasmid diagram. pDXTP receives promoters via restriction enzyme or gateway cloning, and these promoters control expression of the doxycyclineâ€responsive transcription factor rtTA. (c) pDXTR plasmid diagrams. pDXTR receives ORFs from the Xenopus ORFeome housed in pDONR223 vectors via gateway cloning. The ORF is under inducible control by virtue of the tetracyclineâ€responsive element (TRE) upstream of the ORF. Gateway cloning puts the ORF in frame with peptidase recognition sequence T2A and mCherry and Xenopus histone H2B. Both pDXTP and pDXTR plasmids contain transgenesis (Tg) cassettes capable of phiC31, tol2, and SceI methods of transgenesis. The three black dots in pDXTP and the four black dots in pDXTR represent chicken HS4 insulator sequences to protect against undesired enhancer activity from the genomic insertion site and from the promoter controlling rtTA. The gray boxes represent multiple cloning sites to insert promoters, exchange N†or Câ€terminal tags, and transgenesis reporter cassettes. pDXTP has the transgenesis reporter cassette driving green fluorescent protein (GFP) in the lens of the eye from the gamma crystallin promoter (CRY), and pDXTR has the transgenesis reporter cassette driving mCherry fluorescent protein in the lens of the eye. The rtTA in pDXTR is not expressed unless a promoter is cloned into the MCS upstream of it
Figure 2
Diagrams of transgenesis cassettes and ORF N†and Câ€termini. (a) the transgenesis cassettes in pDXTP and pDXTR provide the options for phiC31 integrase, SceI meganuclease, and tol2 transposase methods of transgenesis. The chicken HS4 insulator sequence reduces the influence an integration site may have on the integrated transgenic DNA. The loxP/lox2272 and FRT/FRT3 recombination sequences allow for transgenic animals with these sequences to serve as target sites for future integration of donor plasmids with matching lox and FRT sites using CRE and FLP recombinases. (b) Just upstream of the 5′ gateway recombination site (attR1) where ORFs enter pDXTR are restriction enzymes sites available for addition of Nâ€terminal tags. (c) The 3′ gateway recombination site (attR2) maintains the reading frame of the ORF with the T2Aâ€mCherryâ€histone H2B Câ€terminal tag. Restriction enzyme sites 3′ of the ORF enable replacing the builtâ€in Câ€terminal tag with a STOP codon or a custom tag
Figure 3
Functional analysis of the cloning and transgenesis features in pDXTR. (a) Diagrams of transgenesis plasmids pCAR:TRDNâ€GFP (inducible) and pDXTRâ€GFP. (b) pDXTRâ€GFP was cloned and injected into eggs from a wildâ€type female crossed to a male transgenic for the pCARâ€TRDN construct. Due to absence of transgenesis markers, the presence of the pCAR:rtTA transgene can be readily detected only by doxâ€induction of TRDNâ€GFP (data not shown). Both tadpoles were treated with 50 μg/mL doxycycline for 24 hr. The tadpole on the left is singly transgenic for pDXTRâ€GFP as evidenced by red eye fluorescence and no GFP expression in muscle from TRDNâ€GFP. The tadpole on the right has both constructs as evidenced by red eye fluorescence and strong green fluorescence in the cytoplasm. Top images represent merged bright field and red fluorescence, and bottom images represent fluorescence with green filter. (c) Higher magnification of the muscle cells from the boxed region in (b) shows green, red, merged fluorescence images. Each green rod represents a single, multinucleated muscle cell with nuclei labeled with mCherryâ€histone H2B
Figure 4
Functional analysis of gatewayâ€cloned promoter and ORF in pDXTP and pDXTR. (a) Using gateway cloning, the Ef1α promoter from pTransgenesis was cloned into pDXTP, and bmp4 from the Xenopus ORFeome was cloned into pDXTR. (b) Exemplar embryos coâ€injected with both constructs show normal phenotype in the absence of Dox and show reduced and absent head structures when treated continuously with 50 nM Dox starting at early cleavage stages
