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Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells

Nature Biotechnology (2025)Cite this article

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Abstract

In rodents, injection of haploid androgenetic embryonic stem cells (haES cells) into intact oocytes enables full-term development of offspring. The value of this method in research and genome engineering has not been replicated in ruminants because ruminant haES cells are yet to be obtained. Here we report the derivation of cow and sheep haES cells and their application to generate offspring by a method we call intracytoplasmic haES cell injection (iCHI), in analogy with intracytoplasmic sperm injection. Ruminant haES cells display characteristics of formative-state pluripotency and differentiate into the three germ layers both in vitro and in vivo. Ectopic expression of protamine in haES cells converts their nuclei into a spermatid-like structure and improves full-term development of iCHI embryos, a method we call protamine iCHI (Pro-iCHI). We also combine Pro-iCHI and prime editing to generate gene-modified cows and sheep. Overall, Pro-iCHI provides a promising approach for production of genetically modified livestock.

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Fig. 1: Efficient derivation of ruminant haES cells.
Fig. 2: Molecular characterization of ruminant haES cells.
Fig. 3: Functional differentiation analysis of ruminant haES cells.
Fig. 4: Evaluation of reconstructed embryos with ruminant haES cells.
Fig. 5: Protamine improves the development of reconstructed haES cell embryos.
Fig. 6: Application of ruminant haES cells in CRISPR studies.

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Data availability

High-throughput sequencing data generated by this study have been deposited in the GEO database under accession GSE250497. Additional supporting data can be found in Supplementary Information or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank H. Wang (IMU) for technical help. This study was supported by the National Natural Science Foundation of China (32341052 to L.Y., 32360837 to L.Y. and 32488101 to S.G.), Scientific and Technological Innovation 2030 (2023ZD0404803 to L.Y.), Inner Mongolia Open Competition Projects (2022JBGS0025 to L.Y.), Inner Mongolia Science and Technology Leading Team (2022LJRC0006 to G.L.), Inner Mongolia Science and Technology Major Projects (2022ZD0008 to L.Y., 2023KJHZ0028 to L.Y. and 2025KYPT0101 to L.Y.), Inner Mongolia Young Talents Projects (NJYT23138 to L.Y.), Inner Mongolia Natural Science Foundation (2023MS03004 to L.Y.), National Agricultural Science and Technology Project (NK2022130203 to L.Y.), Collaborative Innovation among Universities in Hohhot (XTCX202306 to L.Y.), Ministry of Education Engineering Centre Project (JYBGCSYS2022 to L.Y.), Xinjiang Uygur Science and Technology Major Project (2023A0201116 to L.Y.) and TongLiao Open Competition Projects (TL2024TW0020103 to L.Y.).

Author information

Author notes

  1. Deceased: Li Zhang.

  2. These authors contributed equally: Lei Yang, Anqi Di, Lishuang Song.

  3. These authors jointly supervised this work: Lei Yang, Shaorong Gao, Guangpeng Li.

Authors and Affiliations

  1. College of Life Sciences, Inner Mongolia University, Hohhot, China

    Lei Yang, Anqi Di, Lishuang Song, Xuefei Liu, Di Wu, Song Wang, Zhenting Hao, Lige Bu, Chunling Bai, Guanghua Su, Zhuying Wei, Li Zhang & Guangpeng Li

  2. Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Lei Yang & Shaorong Gao

  3. Key Laboratory of Animal Cellular and Genetics Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, China

    Song Wang & Zhonghua Liu

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Contributions

L.Y., A.D., X.L., L.S., D.W., S.W., Z.H., L.B., C.B., G.S., Z.W. and L.Z. performed experiments. L.Y., G.L. and S.G. designed experiments. L.Y. and G.L. wrote the manuscript.

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Correspondence to Lei Yang, Shaorong Gao or Guangpeng Li.

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Extended data

Extended Data Fig. 1 Generation of ruminant haploid androgenetic embryos.

a. Schematic overview of haSCs derivation, haploid embryos were generated by removing female pronucleus from normal diploid zygotes. ♂, male pronucleus; ♀, female pronucleus. b. Immunostaining for 5-hydroxymethylcytosine (5hmC) in bovine (left) and ovine (right) embryos. The haploid androgenetic embryos are generated by sperm injection into enucleated oocytes or by removing female pronucleus from fertilized oocytes. The diploid IVF embryos were used as controls. ♂, male pronucleus; ♀, female pronucleus; n = 3 independent experiments with similar results. Scale bar, 25 μm. c. Immunostaining for trophectoderm marker CDX2 in the bovine and ovine blastocysts on day 7 after activation or insemination. The haploid and diploid embryos are generated as described in Extended Data Fig. 1b. n = 3 independent experiments with similar results. Scale bar, 50 μm. d. Quantification for the cell numbers in the bovine and ovine blastocysts on day 7 after activation or insemination. The haploid and diploid embryos are generated as described in Extended Data Fig. 1b; ICM, inner cell mass; Data are mean ± s.d. (n = 18 IVF, n = 19 injecting, n = 21 removing (top left); n = 18 IVF, n = 19 injecting, n = 21 removing (top right); n = 22 IVF, n = 19 injecting, n = 23 removing (bottom left); n represents total embryos of three independent experiments); P values are from unpaired, two-tailed Student’s t-tests.

Extended Data Fig. 2 Derivation of ruminant haSCs from haploid androgenetic embryos.

a. List of main components in the well-known medium that support the derivation of mouse, human, and bovine diploid SCs, including 2i/LIF, mTeSR1, t2iL+Gö, 5i/L/A, ABCL, LCDM, EPSC, CTFR, and bEPSCM. The brief names of these chemicals and their respective pathways which they regulate are also shown. b. Morphology of bovine haploid androgenetic ICMs under different culture medium conditions at indicated time points. D, day; P, passage; n = 3 independent experiments with similar results. Scale bars, 200 μm. c. Representative immunofluorescence (IF) images of pluripotency factors in the indicated primary outgrowth. n = 3 independent experiments with similar results. Scale bar, 100 μm. d, e. The efficiency of outgrowth and haSCs derivation of haploid androgenetic ICMs by different culture mediums. n = 3 independent experiments with similar results. N = total number of ICMs used for each condition.

Extended Data Fig. 3 FACE medium supports ruminant haSCs long-term culture.

a. List of different combinations of small molecules used in this study. b. Summary of b-haSCs derivation efficiency from bovine haploid androgenetic ICMs by different combinations of small molecules. c. Morphology of bovine haploid androgenetic ICMs under different combinations of small molecules. Scale bars, 200 μm. d. The total cell numbers of b-haSCs during cell passaging under different concentrations of Activin-A. Cells were plated at 5 ×105 cells per well and were cultured for 4 days; Data are mean ± s.d. (n = 3 independent experiments); P values are from unpaired, two-tailed Student’s t-tests. e. Haploidy analysis in b-haSCs derived using 10 ng/mL and 20 ng/mL of Activin-A. The figure on the left is the same as Fig. 1e; n = 3 independent experiments with similar results. Note that 10 ng/mL Activin-A was beneficial for maintaining haploidy stability.

Extended Data Fig. 4 Further characterization of ruminant haSCs.

a. DNA copy number variation (CNV) analysis of ruminant haSCs at passage 25. Note that no significant genomic alternations. The results were displayed on a log2 scale. Chromosomes are arranged in numerical order and in different colours. b. Summary of karyotyping analysis of ruminant haSCs at different passages. Total, the total number of examined cells in which chromosome was successfully spread. c. Determination of the sex chromosome of ruminant haSCs by PCR assay. The primers are specific for X chromosome-specific PHEX and Y chromosome-specific ZFY genes. Normal XX and XY diploid SCs were used as controls. M, DNA marker. n = 3 independent experiments with similar results. d. Scatter-plots showing the reproducibility of buRNA-seq between different biological replicates of ruminant haSCs. The Pearson’s correlation coefficients (Cor.) are shown. e. Principal-component analysis (PCA) showing the separation of b-haSCs and bovine pre-implantation embryos. f. Scatter plot based on PCA of b-haSCs, diploid primed b-SCs, and diploid expanded b-SCs, showing that the projection position of b-haSCs is located between expanded and primed b-SCs. g. Correlation matrices showing coefficients among b-haSCs, diploid primed b-SCs, diploid expanded b-SCs, and pre-implantation bovine embryos. h. Heatmap displaying the differentially expressed genes (DEGs) among b-haSCs, primed b-SCs, and expanded b-SCs (P < 0.05, 2-fold change; left). Gene Ontology (GO) terms about the biological processes enriched in DEGs are listed (right). i. Developmental progression of pluripotency in embryos during peri-implantation. Formative pluripotency may be present at the early stages of gastrulation, according to developmental time and lineage segregation of mouse, monkey, pig, and human embryos10,71,72,73,74,75,76. E, embryonic day; ICM, inner cell mass; EPI, epiblast; HYPO, hypoblast; TE, trophectoderm. j. A uniform manifold approximation and projection representation (UMAP) of bovine peri-implantation embryos (E8 ~ E14) and b-haSCs sequenced by 10x Genomics. RNA-seq data of b-haSCs generated in this study, previously published data for bovine diploid pre-implantation embryos are from ref. 11 PMID: 24591639 (GSE52415), diploid primed b-SCs are from ref. 13 (GSE110040), diploid expanded b-SCs are from ref. 14 (GSE129760).

Source data

Extended Data Fig. 5 Further analyses of ruminant haSCs about formative features.

a. The expression of typical naïve-, formative-, and primed-pluripotency genes in b-haSCs at the single-cell level. b. Heatmap displaying the expression of naïve-, formative-, and primed-pluripotency genes in b-haSCs sequenced by scRNA-seq. c. Hierarchical clustering analysis of b-haSCs, o-haSCs, primed b-SCs, and primed o-SCs based on the global transcriptome. d. RT-PCR analysis of core formative markers (DNMT3A, DNMT3B, OTX2) and pluripotency marker (OCT4) in b-haSCs, o-haSCs, and formative m-SCs. M, DNA marker. n = 3 independent experiments with similar results. e. Western blots analysis of core formative markers (DNMT3A, DNMT3B, OTX2) and pluripotency marker (OCT4) in b-haSCs, o-haSCs, and formative m-SCs. n = 3 independent experiments with similar results. f. Scatter-plots showing the reproducibility of CUT&Tag assay between different biological replicates of b-haSCs. The Pearson’s correlation coefficients (Cor.) are shown. g. UCSC genome browser views showing H3K4me3 and H3K27me3 tracks of representative formative- and primed-pluripotency genes in b-haSCs and primed b-SCs. h. Hierarchical clustering analysis of b-haSCs and primed b-SCs based on the H3K4me3 and H3K27me3 CUT&Tag signals. i. UCSC genome browser views showing H3K4me3 and H3K27me3 tracks of selected pluripotency-associated genes in b-haSCs and primed b-SCs. Note that H3K4me3 but not H3K27me3 signals exhibited high enrichment levels in both b-haSCs and primed b-SCs. j, k. Bisulfite pyrosequencing analysis of paternally (a) and maternally (b) imprinted regions in b-haSCs and o-haSCs. The filled and open squares represent methylated and unmethylated CpG sites, respectively. l. Whole genome bisulfite sequencing (WGBS) analysis of known imprinting control regions in b-haSCs. RNA-seq data of o-haSCs and b-haSCs generated in this study, previously published data for bovine primed-b-SCs are from ref. 13, PMID: 29440377 (GSE110040), diploid primed o-SCs are from ref. 44, PMID: 33065542 (PRJNA609175). The H3K4me3 and H3K27me3 data of b-haSCs generated in this study, previously published data for diploid primed b-SCs are from ref. 13, PMID: 29440377 (GSE110040).

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Extended Data Fig. 6 Ruminant haSCs harbour potency for the formation of embryoid bodies and teratoma.

a. Morphology of embryoid bodies (EBs) derived from ruminant haSCs. n = 3 independent experiments with similar results. Scale bar, 100 μm. b. RT-PCR analysis of the expression of three germ-line markers in EBs derived from ruminant haSCs. n = 3 independent experiments with similar results. c. Further immunostaining for three germ-line markers in EBs derived from ruminant haSCs, including ectoderm (NESTIN), mesoderm (BRACHYURY), and endoderm (GATA4). Scale bar, 100 μm. d. FACS analysis of haploid cells in EBs derived from ruminant haSCs at the indicated time points. e. Teratoma produced by injection of ruminant haSCs into the immunodeficient mice. f. Morphology of teratoma produced by ruminant haSCs. g. FACS analysis of haploid cells in teratoma derived from ruminant haSCs at the indicated time points. h. Summary of teratoma formation assay by using ruminant haSCs.

Source data

Extended Data Fig. 7 Ruminant haSCs harbour potency for intra- and inter-species chimaeras.

a. Schematic illustration for producing chimeric blastocysts. Ten EGFP-labeled ruminant haSCs were microinjected into an 8-cell stage embryos, and the chimeric embryos were in vitro cultured until the blastocyst stage. ICM, inner cell mass; TE, trophectoderm. b. Representative phase contrast and fluorescence images showing the incorporation of EGFP-labeled b-haSCs in mouse, bovine, and ovine blastocysts. Scale bar, 50 μm. c. Representative phase contrast and fluorescence images showing the incorporation of EGFP-labeled o-haSCs in mouse, bovine, and ovine blastocysts. Scale bar, 50 μm. d. The percentage of chimeric blastocysts with o- or b-haSCs contribution to inner cell mass (ICM). n = 3 independent experiments. e. Summary of the intra-species chimaeras forming efficiencies. The EGFP-labeled b-haSCs and o-haSCs were microinjected into bovine and ovine 8-cell stage embryos, respectively. The cattle-cattle and sheep-sheep chimeric embryos were transferred into surrogate mothers, and analyzed chimaeras at E40 and E30, respectively. N, the total number of fetuses analyzed for each condition. f. Representative phase contrast and fluorescence images showing EGFP-labeled b-haSCs or o-haSCs contributed to indicated organs of cattle-cattle and sheep-sheep chimaeras at E40 and E30, respectively. g. Upper panel: representative phase contrast and fluorescence images showing EGFP-labeled b-haSCs and o-haSCs contributed to the gonads of cattle-cattle and sheep-sheep chimaeras at E40 and E30, respectively. Lower panel: FACS analysis of DNA content of the EGFP-positive and EGFP-negative cells within the chimeric gonads. Note that the EGFP-labeled b-haSCs (but not o-haSCs) contributed to the gonads. Scale bar, 500 μm. h. Summary of EGFP-labeled b-haSCs or o-haSCs contributed to the gonads in the E40 cattle-cattle and E30 sheep-sheep chimaeras, respectively. N, the total number of fetuses analyzed for each condition. i. Summary of the inter-species chimaeras forming efficiencies. The EGFP-labeled b-haSCs or o-haSCs were microinjected into 8-cell stage mouse embryos, and the cattle-mouse or sheep-mouse chimeric embryos were transferred into surrogate mouse mothers, and analysed chimaeras at E18.5. N, the total number of fetuses analysed for each condition. j. Representative images of eye colour in the cattle-mouse and sheep-mouse chimaeras at 2-day-old. The albino mice were used as hosts. k. Representative phase contrast and fluorescence images showing EGFP-labeled b-haSCs or o-haSCs contributed to indicated organs of cattle-mouse and sheep-mouse chimaeras, respectively. l. Left panel: immunohistochemistry images showing EGFP-labeled b-haSCs or o-haSCs contributed to different tissues in the cattle-mouse and sheep-mouse chimaeras at 2-day-old. Organ sections were stained with anti-GFP antibody. Right panel: FACS analysis of DNA content of the EGFP-positive or EGFP-negative cells within the different tissues. n = 3 independent experiments with similar results. Scale bar, 200 μm.

Extended Data Fig. 8 Ruminant haSCs harbour competency for oocyte fertilizing.

a. Summary of the pseudo-polar body (PPB) extrusion efficiencies in iCHI and Pro-iCHI approaches. Note that the female pronucleus and male pseudo-pronucleus could be efficiently formed in the reconstructed iCHI embryos (110 of 388 in cattle, 110 of 517 in sheep) and Pro-iCHI embryos (110 of 374 in cattle, 110 of 466 in sheep) after extrusion of the PPB, respectively. Data are mean ± s.d. (n = 388 iCHI, n = 374 Pro-iCHI (cattle); n = 517 iCHI, n = 466 Pro-iCHI (sheep); n represents total embryos of three independent bovine and ovine iCHI experiments, and five independent bovine and ovine Pro-iCHI experiments; the source data are provided in Supplementary Table 2); P values are from unpaired, two-tailed Student’s t-tests. b. Immunostaining for 5-methylcytosine (5mC) of the bovine and ovine iCHI embryos at the indicated stage. The IVF embryos were used as control. ♀, female pronucleus; ♂, male pronucleus or pseudo-pronucleus; n = 3 independent experiments with similar results. Scale bar, 25 μm. c. Quantifications of H3K4me3, H3K9me3, and H3K27me3 immunostaining analysis in different pronucleus of ruminant iCHI, Pro-iCHI, and IVF embryos. ♀, female pronucleus; ♂, male pronucleus or pseudo-pronucleus. d, e. Immunostaining for H3K4me3, H3K9me3, and H3K27me3 of the ruminant iCHI embryos after injection of different mRNA (a). Quantifications of immunostaining results in different embryos (b). The KDM5B, KDM4D, and KDM6A encoding the H3K4me3, H3K9me3, and H3K27me3 demethylases, respectively. The in vitro transcription vectors KDMs tagged C-terminally with the hemagglutinin (HA) epitope, which allowed us to track the KDMs proteins in embryos, without the use of specific antibodies. The embryo injected with a blank plasmid vector was used as the negative control. ♀, female pronucleus; ♂, male pronucleus or pseudo-pronucleus; Scale bar, 25 μm. f. Preimplantation development of ruminant iCHI embryos. The iCHI embryo was injected with mRNA as indicated. The KDM5B, KDM4D, and KDM6A encoding the H3K4me3, H3K9me3, and H3K27me3 demethylases, respectively. Data are mean ± s.d. (n = 388 iCHI, n = 219 KDM5B, n = 261 KDM4D, n = 253 KDM6A (top); n = 517 iCHI, n = 338 KDM5B, n = 307 KDM4D, n = 253 KDM6A (bottom); n represents total embryos of three independent experiments; the source data are provided in Supplementary Table 2). j. FACS analysis of haploid cells in ruminant haSCs at 24 h after KDM5B, KDM4D, and KDM6A transfection, respectively. The haSCs were transfected with blank plasmid vector was used as the negative control.

Extended Data Fig. 9 Further characterization of ruminant iCHI embryos.

a. Representative phase contrast and fluorescence images of ruminant haSCs at 0‐, 24-, 48‐, and 72‐h post‐protamine-induction (top panel). The bottom panel shows higher-magnification images. Note that after 48 h protamine-induction, the cell nuclei acquire a spermatid-like structure, detach from the culture dish, and float in the medium. After 72 h of protamine-induction, the protaminized cells showed signs of degeneration. These haSCs harbour the doxycycline (dox)‐inducible PRM1-EGFP vector. Scale bars: in top, 100 μm; in bottom, 20 μm. b. Quantifications of spermatid-like cells in ruminant haSCs at 0‐, 24-, 48‐, and 72‐h post‐protamine-induction. Data are mean ± s.d. (n = 3 independent experiments); P values are from unpaired, two-tailed Student’s t-tests. c. Heatmap and pileup of CUT&Tag signal for the protamine deposition in b-haSCs at 0‐, 24-, 48‐, and 72-h post‐protamine-induction. Each row of the heatmap is a genomic locus and rows are sorted from highest peak intensity to lowest. d. Representative images of dissected bovine uteri isolated from surrogate mothers after day-65 of iCHI embryo transfer. e. Summary of the morphometric measurements of bovine conceptuses at E75 produced by Pro-iCHI or IVF techniques. Data are mean ± s.d. (n = 5 IVF, n = 2 Normal, n = 12 Retarded; n represents total embryos of three independent experiments); P values are from unpaired, two-tailed Student’s t-tests by comparison with IVF group. f. Representative images of dissected ovine uteri isolated from surrogate mothers after day-65 of iCHI embryo transfer. g. Summary of the morphometric measurements of ovine conceptuses at E65 produced by Pro-iCHI or IVF techniques. Data are mean ± s.d. (n = 5 IVF, n = 17 Retarded; n represents total embryos of three independent experiments); P values are from unpaired, two-tailed Student’s t-tests by comparison with IVF group. h. An image of stillborn ovine Pro-iCHI conceptus isolated from surrogate mother after day-150 of embryo transfer.

Extended Data Fig. 10 Further analysis of ruminant haSCs produced animals.

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.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Discussion and Tables 1–8.

Reporting Summary

Supplementary Data 1

Unprocessed gel for Supplementary Fig. 1b,d.

Supplementary Data 2

Unprocessed gel for Supplementary Fig. 7d.

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Source Data Extended Data Fig. 4

Unprocessed gels for Extended Data Fig. 4c.

Source Data Extended Data Fig. 5

Unprocessed gels and western blot for Extended Data Fig. 5d,e.

Source Data Extended Data Fig. 6

Unprocessed gels for Extended Data Fig. 6b.

Source Data Extended Data Fig. 10

Unprocessed gels and western blot for Extended Data Fig. 10g,i,k.

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Yang, L., Di, A., Song, L. et al. Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02832-4

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