Figure 1.
Transgene construct and the NLS-I-SceI molecule.
A: The schematic structure of p2IS-UBC-eGFP vector. IS site: the inversely flanking I-SceI recognition sequence; the black bar indicates the position of the probe used for Southern blot assay. B: The schematic structure of NLS-I-SceI molecule. C: The in vitro transcribed NLS-I-SceI mRNA. polyA+: the mRNA with polyA tail; polyA-: the mRNA without polyA tail. D: The expected working principle of NLS-I-SceI-mediated transgenesis.
Figure 2.
The NLS-I-SceI molecule was capable of cutting circular p2IS-UBC-eGFP plasmids in porcine parthernogenetic embryos.
A: Detection of uncut I-SceI site and eGFP CDS by PCR in the embryos cytoplasmically injected with circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNA and only with circular p2IS-UBC-eGFP plasmids. I: embryos cytoplasmically injected with circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNA; II: embryos cytoplasmically injected only with circular p2IS-UBC-eGFP plasmids. B: The levels of uncut I-SceI site relative eGFP CDS detected by qPCR in the injected embryos. C: The eGFP CDS copy numbers in the injected embryos. *: statistical significance.
Figure 3.
Transfer of DNA fragments from cytoplasm into nuclear by NLS-I-SceI molecule in porcine parthernogenetic embryos.
The activated porcine MII oocytes (1-cell parthernogenetic embryos) stained with Hoest33342 were cytoplasmically injected with Cy3-labelled DNA fragments plus NLS-I-SceI mRNA, and the localization of DNA fragments were observed under LSCM at 16 and 24 h post microinjection respectively. In control groups, the embryos were injected with Cy3-DNA fragments included into the native I-SceI endonuclease digestive reaction system or only with Cy3-DNA fragments. A: The structure of Cy3-labeled DNA fragments. B: The localization of Cy3-DNA fragments co-injected with NLS-I-SceI mRNA. C: The localization of Cy3-DNA fragments co-injected with the native I-SceI nuclease. D: The localization of Cy3-DNA fragments injected alone. Red fluorescence: the Cy3-DNA fragments; Blue fluorescence: the chromosomal DNAs.
Figure 4.
Transgene expression in cytoplasmically injected mouse embryos.
A: Mouse eggs cytoplasmically injected with 30 ng/µL of circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNAs at different concentratiions. Controls A-C were the control groups injected with 30 ng/µL circular plasmids included into the native I-SceI endonuclease digestive reaction system (control A), linearized plasmids (control B) or circular plasmids (control C). B: The dynamics of transgene expression in embryos subjected to cytoplasmic microinjection with circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNA or pronuclear microinjection only with circular p2IS-UBC-eGFP plasmids.
Figure 5.
Transgene expression and detection of uncut I-SceI site and eGFP CDS by PCR in cytoplasmically injected porcine embryos.
A: Transgene expression in the porcine embryos cytoplasmically injected with circular plasmids (p2IS-UBC-eGFP) plus NLS-I-SceI mRNA, circular plasmids included into the native I-SceI nuclease digestive reaction system and circular plasmids only. B: Detection of uncut I-SceI site and eGFP CDS by PCR in the cytoplasmically injected porcine embryos as described in A.
Figure 6.
Quantitative analysis of uncut I-SceI site and eGFP CDS by qPCR in cytoplasmically injected porcine embryos.
A: The eGFP CDS copy numbers in the cytoplasmically injected porcine embryos as described in Fig. 5. B: The uncut I-SceI site levels relative to eGFP CDS in the cytoplasmically injected porcine embryos as described in Fig. 5. *: statistical significance.
Figure 7.
Genetic screen of transgenic mice derived from cytoplasmically microinjected eggs.
A: Screen of transgenic founder mice by PCR. M: DL2000 DNA marker; 1–10: the founder mice derived from cytoplasmic microinjection with circular p2IS-UBC-eGFP plasmids (30 ng/µL) included into the native I-SceI nuclease digestive reaction system; 11–23: The founder mice derived from cytoplasmic microinjection with circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNA (30 ng/µL each). B: Screen of transgenic individuals of F1 offspring derived from transgenic founder mice by PCR. M: DNA marker; 1–8: Genomic DNA samples of F1 individuals. C: Genetic screen of transgenic founder mice by Southern blot assay. M: DNA molecular weight marker II; 1: plasmids; 2–7: genomic DNA samples of founder mice; 8: negative control (wild-type mouse genomic DNA). D: The transgenic mice exhibiting in vivo fluorescence derived from breeding between transgenic individuals over three consecutive generations. The arrow indicates the founder mouse detected to be transgenic by both Southern blot and PCR screen.
Figure 8.
Genetic screen of transgenic pigs derived from embryos cytoplasmically microinjected with circular p2IS-UBC-eGFP plasmids plus NLS-I-SceI mRNA.
A: In vivo fluorescence in founder pigs. B: Screen of transgenic founder pigs by PCR. M: DNA marker; 1–4: genomic DNA samples of 1–4# founder pigs; 5: positive controls (wild-type pig genomic DNAs containing p2IS-UBC-eGFP plasmids); 6: negative control (wild-type pig genomic DNA). C: Southern blot assay of transgenic founder pigs. M: DNA marker (1KB DNA Ladder); 1: positive control (plasmids); 2: wild-type pig genomic DNA as negative control; 3–6∶1–4# founder pigs. D: Southern blot analysis of F1 offspring individuals derived from founder pig 1#. M: DNA molecular weight marker II; 1: plasmid as positive control; 2–8: the F1 offspring individuals. E: Southern blot analysis of genomic DNAs extracted from different organs of founder pig 1#. M: DNA molecular weight marker II; 1: positive control (plasmids); 2: skin; 3: heart; 4: liver; 5: spleen; 6: lung; 7: kidney; 8: wild-type pig genomic DNA as negative control.