Extended Data Fig. 10: Further analysis of ruminant haSCs produced animals.
From: Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells
a. Heatmap showing the correlation of blood serum metabolomes of Pro-iCHI and IVF cattle in different replicates at 18-month-old. b. Volcano plot showing the metabolic changes between Pro-iCHI and IVF cattle at 18-month-old. The vertical and horizontal dotted lines show the cut-off of fold-change = ± 2, and p-value = 0.05 (unpaired two-tailed Student’s t-tests), respectively. c. Pie charts showing the metabolite class composition in Pro-iCHI and IVF cattle at 18-month-old. d. Heatmap showing cross-species comparison of orthologous genes in bovine and ovine fetuses produced by Pro-iCHI or IVF approach. Note that the expression level of apoptosis and degeneration-related genes was significantly increased (FC > 2, FPKM > 2) in growth-retarded Pro-iCHI fetuses compared with normal Pro-iCHI fetuses and control IVF fetuses. FC, fold change; FPKM, fragments per kilobase of transcript per million mapped reads. e. Bisulfite pyrosequencing analysis of paternally imprinted H19 DMR in bovine and ovine fetuses produced by Pro-iCHI or IVF approach. Note that all the fetuses maintained allelic-biased DNA methylation in H19 DMR. The filled and open squares represent methylated and unmethylated CpG sites, respectively. f. Schematic representation of the ePE vector used in this study. This “all-in-one” episomal plasmid contained the necessary elements of CRISPR-prime editor (PE), including Cas9-nickase and reverse-transcriptase (RT) fusion sequence, and a prime-editing guide RNA (pegRNA). The vector also contains an EF1a promoter-driven EGFP for tracking transfection efficiency. Puro, Puromycin resistance gene. g. Western blots analysis of MSTN protein in wild-type (WT) and MSTN-edited ruminant haSCs. n = 3 independent experiments with similar results. h. Episomal plasmid was decreased within haSCs over time after withdrawing puromycin drug selection. Numbers in the X-axis indicate cell passage number (passage cells every 5 days). Amp, ampicillin; Actin, beta-actin; mean ± s.d., n = 3 independent experiments; amplification cycle was maintained at 40 and the cycle threshold (Ct) values > 35 were considered as not detected (ND). i. Western blots analysis of MSTN protein in wild-type (WT) and MSTN-edited live cattle. n = 3 independent experiments with similar results. j. Sanger sequencing results of the targeting site in wild-type (WT) and MSTN-edited live cattle, the deletion sizes (Δ) are indicated. k. Genomic PCR showing the absence of exo-vector in lamb and calf produced by Pro-iCHI::ePE system. The positive and negative controls were amplified from the episomal plasmid and water, respectively. n = 3 independent experiments with similar results. Lane 1: positive control; Lane 2: sheep; Lanes 3-5: cattle; M, DNA marker.
