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We show that replication defective adenovirus can be used for localized overexpression of a chosen gene in Xenopus tadpoles. Xenopus contains two homologs of the Coxsackie and Adenovirus Receptor (xCAR1 and 2), both of which can confer sensitivity for adenovirus infection. xCAR1 mRNA is present from the late gastrula stage and xCAR2 throughout development, both being widely expressed in the embryo and tadpole. Consistent with the expression of the receptors, adenovirus will infect a wide range of Xenopus tissues cultured in vitro. It will also infect early embryos when injected into the blastocoel or archenteron cavities. Furthermore, adenovirus can be delivered by localized injection to tadpoles and will infect a patch of cells around the injection site. The expression of green fluorescent protein in infected cells persists for several weeks. This new gene delivery method complements the others that are already available. Developmental Dynamics 238:1412-1421, 2009. (c) 2009 Wiley-Liss, Inc.
Figure 3. Expression of Xenopus laevis Coxsackievirus and Adenovirus Receptors (xCAR) mRNA in embryonic and tadpole stages. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) of xCAR1 and xCAR2 in mRNA extracted from whole embryos at the developmental stages indicated. xCAR1 is expressed from gastrulation onward, while xCAR2 is expressed both maternally and zygotically. B: RT-PCR of xCAR1 and xCAR2 in mRNA extracted from stage 54-56 tadpole tissues. M, muscle; Li, Liver; H, head; S skin. C-H: Whole-mount in situ hybridizations. C,D show no probe controls at stage 25 and 31; E,F show xCAR1 at stage 25 and 31; G,H show xCAR2 at stage 25 and 31. White arrowheads in G and H indicate expression of xCAR2 in dorsal tail bud and in pronephros and nephric duct, which are greater than seen for xCAR1.
Figure 4. Adeno-green fluorescent protein (GFP) infection of Xenopus cells grown in vitro. A: Fibroblasts (f) and neuron-like cells (n) in outgrowths from explants of stage 40-44 tadpole neural tubes. B: A melanophore migrating from stage 40-44 tadpole neural tube explant (brightfield view in insert). C: Stage 45 tadpoleliver explant. Mesenchymal cells express GFP, while the underlying epithelium, stained for E-cadherin (red), does not.. D: Stage 45 gall bladder. E-cadherin is shown in red. Scale bars = 20 mu m in A, 30 mu m in B, 50 mu m in C, 20 mu m in D.
Figure 5. Adeno-green fluorescent protein (GFP) infection of Xenopus early embryos. Embryos were injected with adeno-GFP at the indicated early stage, raised to tadpoles, and photographed for GFP fluorescence at stage 45. A: Trunk and tailmuscle, injected stage 8. B: Scattered cells in liver, injected stage 8. C: Small intestine loop, injected stage 14. Bright patch on left is autofluorescence of gall bladder. D: Jaw muscles, injected stage 10.5. E: Confocal view of flattened small intestineepithelium, injected stage 14. F: Transverse confocal view of GFP (green) and smooth muscle actin (red) from small intestine, injected stage 14. G: Reverse transcriptase-polymerase chain reaction (RT-PCR) of GFP mRNA. Samples are from embryos injected with GFP RNA at two cells (top row); embryos injected with adeno-GFP as blastulae (second row); control embryos (third row); loading control (bottom row). Scale bars = 100 mu m in A-D; 50 mu m in E,F.
Figure 6. Adeno-green fluorescent protein (GFP) infection of Xenopus tadpoles at stage 54-56. A: GFP fluorescence visible in muscle and limb 5 days after injection (muscle and limb injected separately). B: Higher power of another specimen showing GFP in a limb (outlined). C: Distribution of GFP-expressing cells in the limb shown in B by anti-GFP antibody (green) and DAPI (4 prime ,6-diamidine-2-phenylidole-dihydrochloride, blue) staining. D: Tissue distribution of positive cells shown by staining with anti-GFP antibody (green) and 12/101 (anti-muscle antibody, red). Some GFP-positive cells are muscle fibers and others are connective tissue cells. Scale bars = 500 mu m.
cxadr (coxsackie virus and adenovirus receptor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anteriorleft, dorsal up.
vsig8 (V-set and immunoglobulin domain containing 8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anteriorleft, dorsal up.
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