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Fig. 3.

Fig. 3. From: Site-specific integrase-mediated transgenesis in mice via pronuclear injection.

GFP expression in embryos carrying a single copy of pHb9-GFP transgene site-specifically integrated in H11. (A) Schematic representation of the generation of H11P3-pHb9-GFP allele. After site-specific integration of the plasmid pBT366 (SI Appendix, SI Materials and Methods), the bacterial backbone (BB) was removed by crossing to the GFP-FLPo transgenic line (SI Appendix, SI Materials and Methods). The embryos that inherited only the Hb9 allele but not the Flpo transgene were tested for GFP expression. (B) GFP expression in a whole-mount representative embryonic day 11 embryo containing the H11P3-pHb9-GFP allele. A WT littermate is also shown (Right). (Scale bar, 1 mm.) (C) Immunofluorescence of sections from embryonic day 11 spinal cords at limb level with anti-GFP signal in green, anti-Hb9 signal in red, and DAPI in blue. Insets: Magnified bottom left portions of each image containing Hb9-positive nuclei. (Scale bar, 100 μm.)

Bosiljka Tasic, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7907.
Fig. 1.

Fig. 1. From: Site-specific integrase-mediated transgenesis in mice via pronuclear injection.

Schematic summary for site-specific ϕC31 integrase-mediated transgenesis via pronuclear injection in mice. A single or three tandem attP sites were knocked into the Hipp11 (H11) or Rosa26 loci via homologous recombination in ES cells (Left; for details, see Fig. S1). Mice homozygous for one of the modified loci (H11 is shown as an example) served as embryo donors. A mix of DNA and in vitro transcribed ϕC31 mRNA was injected into a single pronucleus of each zygote. The integration of plasmid bacterial backbone (BB) that decreases the transgene expression was avoided either by injecting a minicircle DNA with a single attB site (top branch; in this case, ϕC31 catalyzes a typical integration reaction), or by injecting plasmid DNA where the gene of interest (e.g., GFP) was flanked by two attB sites (bottom branch; in this case, ϕC31 catalyzes a recombinase-mediated cassette exchange reaction). Right, Middle: Green fluorescence of a representative transgenic F0 embryo and the corresponding PCR results that indicate site-specific insertion. The red numbers for the PCR results correspond to the numbers on the H11P3-pCA-GFP transgene scheme below; the same PCR tests can be used for the transgene above. The particular embryo shown was obtained by cassette exchange. Inset: Bright-field image of the same embryo.

Bosiljka Tasic, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7907.
Fig. 2.

Fig. 2. From: Site-specific integrase-mediated transgenesis in mice via pronuclear injection.

GFP expression in animals carrying site-specific pCA-GFP transgenes introduced by ϕC31 integrase-mediated transgenesis. (AD) Representative fluorescence images from embryonic day 11 embryos and adult livers, hearts, and cerebella of N1 or N2 transgenic animals corresponding to the genotypes and schematics of transgenes shown (Upper). Embryos or same tissues were imaged under identical conditions, except that “5×-exp” designates fivefold longer exposure time than for the rest of the images in the same column. Whole-mount embryos were imaged for GFP fluorescence; corresponding bright-field images of each embryo are also shown (Insets). The livers and hearts are represented by epifluorescence images of 10-μm sections stained only by DAPI (blue). The green signal is GFP fluorescence. The cerebella are represented by confocal images of sections stained by anti-GFP antibody (green), anti-calbindin (red) for Purkinje cells, and DAPI (blue). Two Purkinje cells labeled by asterisks appear negative for GFP. ϕC31 attL and attR are the product of recombination of an attP (black arrows) and attB site (purple) and are therefore half black and half purple. Half circles represent FRT5 sites. Half white/half black triangle represent λ-integrase attB site created during minicircle production. pSV40, SV40 promoter. pVASA, VASA promoter. U, unique sequence. pCA, CMV enhancer and β-actin promoter. G, GFP. pA, polyA signal. BB, plasmid bacterial backbone. (Scale bars: 1 mm for embryos, 100 μm for tissue sections.) (E) Average fluorescence in arbitrary units (AU) in the GFP channel for liver sections from animals of genotypes shown below (no int., no integration; represents wt, H11P3NVϕ/wt, H11P3/wt, and H11P/wt genotypes). Each dataset is represented by mean ± SD. The numbers of individual animals and founders analyzed for each genotype are listed below the genotypes. When samples from multiple founders were combined to obtain an average, each founder was represented by the same number of animals except in the case labeled by a spade. The fluorescence intensities differ significantly among the groups by one-way ANOVA [F(3, 50) = 84.09, P < 0.0001]. Tukey's post-hoc test was used for pair-wise comparisons (ns, not significant; *P < 0.05 and ***P < 0.001).

Bosiljka Tasic, et al. Proc Natl Acad Sci U S A. 2011 May 10;108(19):7902-7907.

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