Generation of Knock-In Pigs Carrying Oct4-tdTomato Reporter through CRISPR/Cas9-Mediated Genome Engineering

<div><p>The porcine pluripotent cells that can generate germline chimeras have not been developed. The Oct4 promoter-based fluorescent reporter system, which can be used to monitor pluripotency, is an important tool to generate authentic porcine pluripotent cells. In this study, we established a porcine Oct4 reporter system, wherein the endogenous Oct4 promoter directly controls red fluorescent protein (RFP). 2A-tdTomato sequence was inserted to replace the stop codon of the porcine Oct4 gene by homogenous recombination (HR). Thus, the fluorescence can accurately show the activation of endogenous Oct4. Porcine fetal fibroblast (PFF) lines with knock-in (KI) of the tdTomato gene in the downstream of endogenous Oct4 promoter were achieved using the CRISPR/CAS9 system. Transgenic PFFs were used as donor cells for somatic cell nuclear transfer (SCNT). Strong RFP expression was detected in the blastocysts and genital ridges of SCNT fetuses but not in other tissues. Two viable transgenic piglets were also produced by SCNT. Reprogramming of fibroblasts from the fetuses and piglets by another round of SCNT resulted in tdTomato reactivation in reconstructed blastocysts. Result indicated that a KI porcine reporter system to monitor the pluripotent status of cells was successfully developed.</p></div

Phenotypes of somatic mutation.

<p>(A) Fibroblasts from the cloned pigs were analyzed by FACS with FITC-conjugated GS-IB4 lectin staining. All three pigs were proven to be α 1,3-Gal-negative. (B) Porcine ear fibroblasts stained with FITC-conjugated GS-IB4 were observed under fluorescence microscopy. The original PFFs of BMI were used as the positive control. The scale bar represented 150 μm. (C) The major tissues were cryosectioned and stained with FITC-conjugated GS-IB4. The scale bar represented 250 μm. (D) Immunofluorescence assay. Fibroblasts from cloned pigs were stained with Gal-α1,3Gal-specific mAb M86. The original PFFs of BMI were used as the positive control. The scale bar represents 50 μm. (E) Western blotting for cells of KO piglets and WT pig. β–actin was used as the internal control. (F)Complement-mediated lysis for double KO fibroblasts and WT pig cells. Data shown here are the average of three trials. HIA–HS was used as the negative control. BSA was used as the reagent control.</p

Schematic of TALENs targeting the porcine GGTA1 locus and the activity assay.

<p>(A) Schematic of TALEN Set#1. TALEN repeat domains are colored differently to designate the identity of the repeat variable di-residue (RVD). The relationship between RVDs and the cognate-targeted DNA base is NI = A, HD = C, NN = G, NG = T. (B) Schematic of TALEN Set#2. (C) Detection of TALEN activity using luciferase SSA recombination assay. Luciferase activity was increased by 13.1- and 2.2-fold for TALEN Set#1 and Set#2 respectively compared with the control.</p

Analysis of the KI fetuses.

<p>(A) PCR genotyping detected the KI fetuses by using primers indicated in the diagram. 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. (B) TdTomato was expressed, with varying degree of fluorescence, in PGCs of genital ridges isolated from fetuses. (C) TdTomato was expressed in the genital ridges of target fetuses, but not in other tissues, such as intestines. tdTomato was not expressed in WT genital ridges. Bright field image of the tissue is shown on the lower right corner of each image. Scale bar, 1 mm. (D) PFFs cultured in vitro did not express tdTomato. Scale bar, 100 μm. (E) tdTomato was expressed in blastocysts after another round of SCNT using fibroblasts isolated from pOct4-2A-tdtoamto KI fetuses as nuclear donors. WT blastocysts did not express tdTomato. Scale bar, 100 μm. (F) Dome-like colonies formed 10 days after transfection did not express tdTomato. Scale bar, 50 μm. (G) RT-PCR was used to identify the transcription of endogenous and exogenous Oct4 in piPS-LCs. All colonies expressed exogenous Oct4 but not endogenous Oct4.</p

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.</p