Figure 1.
Identification of the pRosa26 locus.
(A) A diagram of the pRosa26 locus on chromosome 13. (B) Expression of pRosa26 ncRNA in various tissues relative to b-actin by Q-PCR. Error bars are mean±SD.
Figure 2.
GFP expression driven by the pRosa26 promoter.
(A) Southern blot of the transgenic cell lines. Expected bands of 0.8 kb were detected after Eco47III/HindIII digestion. (B) Flow cytometry analysis of the transgenic cell lines. (C) GFP expression in the transgenic PFFs detected by Western blot. (D) GFP expression in transgenic PFFs over a long term culture up to 55D detected by Q-PCR. (E) GFP expression in transgenic cloned embryos detected by Q-PCR. (F) pR26 sequence of Congjiang minpig and Yorkshire pig. There is a lack of GGC in pR26 sequence of Congjiang minpig compared to Yorkshire pig. (G) DNA methylation status of pR26 in transgenic PFFs and cloned blastocysts. The methylation status was detected by the bisulfite sequencing. Methylated and non-methylated CpG dinucleotides of each clone are illustrated with closed and open circles, respectively.
Figure 3.
EF1a-GFP targeting in the pRosa26 locus.
(A) Schematic representive of the EF1a-GFP targeting vector and a segment of the pRosa26 locus. SA, splice acceptor. The blue dashed line indicates the band size (1.8 kb) in the 5′ arm PCR analysis using F and R primer set. (B) 5′ arm PCR analysis of targeted cell clone. M, DNA marker. P6, PPNT6 plasmid. WT, wild type DNA. (C) GFP expression in correctly targeted cells (200×). (D) GFP expression in cloned blastocysts from targeted cells (100×). (E) DNA methylation status of EF1a in pRosa26 targeted cells and cloned blastocysts. The methylation status was detected by the bisulfite sequencing. Methylated and non-methylated CpG dinucleotides of each clone are illustrated with closed and open circles, respectively.
Table 1.
The targeting efficiency of GFP and Fst.
Table 2.
The development of GFP-KI cloned embryos in vitro.
Figure 4.
MyoP-Fst targeting in the pRosa26 locus.
(A) Schematic representive of the MyoP-Fst targeting vector and a segment of the pRosa26 locus. SA, splice acceptor. The red and black dashed lines indicate the knockin (1.2 kb) and WT (3.0 kb) band sizes expected after XhoI/XbaI digestion in the Southern blot. The yellow dashed line indicates the Fst band size (1.85 kb) expected after HindIII/BamHI digestion in the Southern blot. The blue dashed line indicates the band size (1.8 kb) in the 5′ arm PCR analysis using F and R primer set. (B) Southern blot of cloned pigs. 5 correctly targeted pigs were confirmed. (C) Fst expression in ear of 3 Fst-KI pigs by Western blot. (D) Fst expression relative to b-actin in various tissues of Fst-KI pig by Q-PCR. (E) Fst expression in various tissues of Fst-KI pig by Western blot. (F) DNA methylation status of MyoP in various tissues of Fst-KI pig. (G) Fst-KI pigs. 130731-1 and -2 are marked with *. WT, wild type.
Table 3.
The development of Fst-KI cloned embryos in vivo.