Figure 1.
Design of ZFNs targeting the pig IL2RG gene and isolation of nuclear donor cells.
(A) Schematic representation of ZFNs binding to pig IL2RG. The coding and untranslated regions are indicated by gray and white boxes, respectively. A ZFN consists of a nuclease domain (Fok I) and a DNA-binding domain (zinc finger proteins), and the recognition sequences of the zinc finger proteins are underlined. (B) Flow chart for the isolation of nuclear donor cells (clone #98) for SCNT. (C) ZFN-induced mutation in cell clone #98. The upper and lower sequences represent the WT and clone #98 sequence of IL2RG, respectively. The deletion mutation and nucleotide substitution in clone #98 are indicated by a hyphen and black box, respectively. The initiation codon of IL2RG is shown in a dotted box. The ZFN-binding and ZFN-cleavage sites are double-underlined and boxed, respectively. The major transcription initiation site is indicated with a circle.
Figure 2.
Generation and analysis of IL2RG KO pigs.
(A) Cloned blastocysts transferred to recipient gilts. (B) Cloned IL2RG KO pig delivered by cesarean section at 113 d of gestation. (C) PCR genotyping for the 4 cloned piglets obtained. M: DNA marker. (D) The DNA sequence analysis of IL2RG in a cloned pig. The arrows and boxes indicate the same mutation as that of the nuclear donor cell (clone #98). (E) Western blot for IL2RG protein in the spleens of IL2RG KO pigs. β-actin was used as a loading control. M: protein standard marker.
Table 1.
in vitro development of SCNT embryos and production of IL2RG KO pigs.
Figure 3.
(A, B) The thymic phenotype in WT and IL2RG KO pigs. The white arrowheads indicate normal thymuses in WT pigs. (C, D) Histological analysis of the spleens of WT and IL2RG KO pigs. The white pulp of the spleen is indicated by a dotted white circle. Bar = 100 µm. (E) The proportion of lymphocytes in the peripheral blood (PB) of WT and IL2RG KO pigs. The data represent the means ± SD values for 4 pigs. The asterisk indicates a statistically significant difference (P<0.01) between the values for WT and IL2RG KO pigs (n = 4).
Figure 4.
Flow cytometric analysis of mononuclear cells in IL2RG KO pigs.
(A) Flow cytometric analysis of T, B, and NK cells in the peripheral blood of IL2RG KO pigs. The dot plots show CD3, CD4, and CD8 cells for the demarcation of T cell subpopulations and CD3, CD45RA, and CD16 (in the non-myeloid fraction, i.e., monocyte/granulocyte (M/G)-negative) cells for the differentiation of T cell, B cell, and NK cell subpopulations in the peripheral blood, respectively. (B) The proportion of T (CD3+) and NK (M/G−, CD3−, CD16+) cells among the mononuclear cells in the spleens of IL2RG KO pigs. The data represent the mean ± SD values of the 4 pigs obtained.