Fig. 5

1 The process and characterization of CTFR-bESCs. In panel A, bright-field images and AP staining are shown, illustrating the typical colony morphologies of CTFR-bESCs. It is important to note that the feeder layer in the images is negative for AP staining. The passages shown are P3 (passage 3) and P24 (passage 24). The scale bars in the images represent a length of 50 μm. Panel B displays immunofluorescence (IF) staining for various markers, including SOX2, POU5F1, GATA6, and CDX2. The top row shows bovine blastocysts at a magnification of 20 × objective, while the middle and bottom rows show CTFR-bESCs. Panel C presents the results of expression analysis for markers specific to different lineages: ICM (inner cell mass), TE (trophectoderm), and PE (primitive endoderm). The analysis was performed using RNA-seq, and the samples include two independent CTFR-bESC lines (P10), two independent pools of whole blastocysts (10 each), and two lines of bovine fibroblasts. The color scale indicates expression levels, ranging from red (high expression) to green (low/no expression). In panel D, representative images exhibit H&E staining of histological sections obtained from teratomas generated by CTFR-bESCs. These teratomas contain tissues from all three germ lineages: ectoderm, endoderm, and mesoderm. The magnification used for these images is 10 × . 2 The pattern of histone methylation in CTFR-induced pluripotent stem cells (CTFR-bESCs). In part (A), the transcriptional status of genes containing H3K4me3, H3K27me3, or bivalent domains is depicted. Genes with an RPKM (Reads Per Kilobase Million) value of 0.4 or higher are considered expressed, while genes with an RPKM value below 0.4 are considered nonexpressed. The bar plot inside the figure shows the average RPKM values ± SEM (Standard Error of the Mean) for expressed genes, while the x-axis displays the average RPKM values ± SEM for all genes (both expressed and nonexpressed). In part (B), the functional characteristics of genes containing H3K4me3, H3K27me3, or bivalent domains are presented. The figure displays the top 10 Gene Ontology (GO) terms associated with these genes. The bar plot represents the negative logarithm (base 10) of the P-value for selected GO terms related to biological processes, as determined by DAVID (Database for Annotation, Visualization, and Integrated Discovery). In part (C), a snapshot of the genome browser is provided, showing specific genes associated with H3K4me3, H3K27me3, or bivalent domains. The genes are listed for each category, such as TGFBR1, FGF8, SALL4, TRIM8, SBDS, and TAF8 for H3K4me3; OOEP, REC8, SLITRK4, LRRC4B, ARRX, and CSNB1 for H3K27me3; and WNT2, WNT7A, MATN2, CHL1, MSX2, and ETV4 for bivalent domains. These genes are associated with three distinct GO terms. The start of each gene is indicated by a black arrow in the genome browser snapshot. 3 The molecular characteristics of CTFR-bESCs, indicating their state of primed pluripotency. In panel A, the expression levels of specific markers for naive and primed pluripotency were analyzed using RNA-seq, and the results are represented using red (expressed genes with RPKM ≥ 0.4) and green (nonexpressed genes with RPKM < 0.4) color-coding. The data shown are the means of two independent biological replicates. Panel B provides snapshots from a genome browser displaying the histone methylation profiles of markers associated with primed and naive pluripotency in CTFR-bESCs. Panel C displays genome browser snapshots of H3K4me3 and H3K27me3 marks on key pluripotency genes (POU5F1, SOX2, NANOG, SALL4) in CTFR-bESCs. 4 The potential applications of CTFR-bESCs (Chimeric Trained Functional RNA-blastocyst-derived Embryonic Stem Cells) in genomic selection. In part A, the efficiency of deriving CTFR-bESCs is evaluated using different plating methods (whole blastocyst, mechanical isolation of inner cell mass [ICM], and immunosurgery-derived ICM) and various embryo sources (in vitro maturation [IVM]-in vitro fertilization [IVF], ovum pick-up [OPU]-IVF, somatic cell nuclear transfer [SCNT], and Holstein and Jersey breeds). The derivation efficiency is measured by calculating the percentage of blastocysts that successfully produce a stable CTFR-bESC line at the third passage (P3) in relation to the total number of embryos seeded using each method. Part B presents a schematic diagram illustrating the strategy of utilizing CTFR-bESCs for genomic selection. This approach aims to produce animals with superior genetic value through a highly efficient process involving CTFR-bESC derivation and somatic cell nuclear transfer (NT). The diagram demonstrates the potential of using CTFR-bESCs to select desirable genetic traits and generate animals with enhanced genetic characteristics. Part C highlights that CTFR-bESCs generated from different sources can serve as nuclear donors for cloning. This suggests that CTFR-bESCs derived from various embryo sources can be utilized in the cloning process to produce genetically identical copies of an organism. Reprinted from with [87] permission from the PNAS