Development ability of nuclear transfer embryos derived from pZCpTG-PFFs and WT-PFFs.

<p>The embryo numbers are the total numbers counted from three independent experiments, and the percentage values in the third and fourth columns are shown as mean ± S.D.</p><p>*<i>p</i>>0.05 compared with WT-PFF; no significant difference.</p

Co-expression of the four fluorescent proteins in the fibroblast isolated from multi-transgenic piglets.

<p>(A) Fibroblasts isolated from the ear tissue of a representative animal (Piglet 10) observed under a confocal microscope using appropriate filters. The scale bar represents 10 µm. (B) The fluorescence intensities of the four fluorescent proteins were measured. The results are shown as mean ± S.D.</p

Maps of the pTGZC (A) and pZCpTG (B) vectors.

<p>Maps of the pTGZC (A) and pZCpTG (B) vectors.</p

Fluorescence in living multi-transgenic piglets.

<p>Florescence on the noses and hooves of multi-transgenic piglets was directly observed using goggles. (A) Bright light. (B) tdTomato fluorescence. (C) EGFP fluorescence. (D) ECFP fluorescence. A WT piglet was used as negative control.</p

Co-expression of the four fluorescent proteins in PFFs and blastocysts.

<p>(A) PFFs transfected with the linearized pZCpTG vector were observed under a confocal microscope using appropriate filters. The scale bar represents 10 µm. (B) Several reconstructed embryos derived from the pZCpTG PFFs were cultured until they reached the blastocyst stage. The blastocysts were observed under a fluorescence microscope using appropriate filters.</p

Genotype identification of multi-transgenic piglets.

<p>(A) Positions of the two primer pairs for genotype identification. Genomic DNA from Piglet 2, 3, 4, 6, 7, 9, 10 was extracted, whereas ZC (B) and TG (C) bicistronic cassettes in the genome were detected by PCR using appropriate primers. Lane 1, DNA marker; lane 2, positive control; lane 3, negative control; lane 4–10, genomic DNA from Piglet 2, 3, 4, 6, 7, 9 and 10.</p

Expression profile analysis of four fluorescent proteins in various tissues by RT-PCR.

<p>Total mRNA was extracted from various tissues of Piglet 7. The mRNA levels of the target genes (tdTomato, EGFP+ECFP and ZsYellow1) were determined by RT-PCR. The results are shown as mean ± S.D.</p

image_4.JPG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

image_1.JPEG

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p

table_1.DOCX

<p>Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.</p