Generation of an endogenous porcine Oct4-2A–tdTomato reporter.

<p>(A) Schematic of the method in generating porcine Oct4-2A–tdTomato KI allele. The genes Cas9 and sgOct4 were constructed in a single vector. The homologous arms of the donor vector are indicated as left arm (950 bp) and right arm (1350 bp). PCR primers (P1, P2 and P3) used for PCR genotyping are indicated by blue and red arrowheads. (B) Red fluorescence in porcine Oct4-2A–tdTomato reconstructed blastocysts derived from SCNT embryos. The porcine fibroblast colonies C51, C95, and C98 were reprogrammed to become highly efficient tdTomato-expressing blastocysts. Scale bar, 100 μm. (C) PCR genotyping using the primers indicated in the diagram detected the KI colones and embryos. PCR revealed wild type band (0 bp) and the 2A-tdTomato KI band (1000 bp) using primers P1/P2. PCR revealed the WT band (2300 bp) and the 2A-tdTomato KI band (3900 bp) using primers P1/P3. (D) The targeted porcine blastocysts remained tdTomato positive in the ICM (arrow), whereas disappeared in trophoblast cells (arrowhead), after 5 days cultured in ES medium. Scale bar, 50 μm. (E) The targeted porcine blastocysts differentiated into tdTomato negative colonies after being cultured for 15 days. Scale bar, 50 μm. (F) Q-PCR compared the endogenous Oct4 mRNA levels of porcine gene-target blastocysts cultured for 5 and 15 days. PFFs were used as negative control and porcine blastocysts without being cultured were used as positive control.