Transgenic Xenopus laevis embryos can be generated using phiC31 integrase

Abstract

Bacteriophage phiC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding phiC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken beta-globin 5' HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using phiC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of phiC31 integrase in other systems as well.

Figure 1

Co-injection of ϕC31 integrase with CMV-GFP reporter plasmid enables detection of GFP expression in Xenopus embryos in stage 46 embryos. Embryos were anesthetized and the tails curled around toward the head for photo documentation. Images a--f are images of embryos viewed under fluorescent light passing through a 535 nanometer limit GFP emission filter. In every case the insert shows a brightfield image of the embryo. (a) Stage 46 Xenopus embryo injected 5 pg of pEGFPB2. (b-d) Stage 46 Xenopus embryos injected with 1ng of integrase mRNA and 5 pg of pEGFPB2. Depending on the embryo we noted GFP signal in either dorsal muscle (b), eyes and neural tissue (c), or in the gill structures of the embryos (d). (e) Stage 39 Xenopus embryo injected with 100pg pEGFPB2 reporter plasmid. (f) Stage 46 Xenopus embryo injected with 100pg pEGFPB2 reporter plasmid.

Figure 2

Southern blot analysis of treated embryos indicates insertion of the GFP reporter plasmid into the embryonic genome. Markers in kilobase pairs are indicted on the left side of the blot. Lane c is linearized pEGFPB2, lane 1 and 2 used DNA isolated from the embryos in figure 1 in panel b and c. Lane 3 and 4 are from embryos transgenic for the insulated crystallin lens-GFP reporter and lane 5 is from an embryo transgenic for insulated CMV-GFP reporter.

Figure 3

Co-injection of ϕC31 integrase with insulated CMV-GFP or crystallin-lens-GFP reporter plasmid generated Xenopus embryos with tissue appropriate expression. . Images (a-f) are images of embryos viewed under fluorescent light passing through a 535 nanometer limit GFP emission filter. In every case the insert shows a brightfield image of the embryo. (a-c) are stage 46 embryos assaying the expression of insulated CMV-GFP reporter (a) is non-injected, (b) is the insulated CMV-GFP reporter without co-injection of ϕC31 integrase (c) is an embryo co-injected with insulated CMV-GFP and ϕC31 integrase. (d-f) are stage 42 embryos assaying the expression of insulated crystallin-lens-GFP reporter (d) is non-injected, (e) is the insulated crystallin lens-GFP reporter without co-injection of ϕC31 integrase (f) is an embryo co-injected with insulated crystallin lens-GFP reporter and ϕC31 integrase. The two grayscale inserts represent photographs of the eye using either brightfield or fluorescence of the embryos eye taken through a 10× objective on a Zeiss Axioplan 2 microscope.