Limitations of serial cloning in mammals

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
Mammals can now be cloned artificially, but it remains unknown whether they can also maintain their species through cloning. Herein, we continued serial cloning for 20 years from a single donor mouse. These re-cloned mice appeared normal and had normal lifespans, but large structural and lethal mutations accumulated in their DNA with each generation. The birth rate of serial cloning began to decline from the 27th generation, and the 58th generation was the last. When re-cloned mice from near the final generation were mated with males, their oocytes could be fertilized, but most embryos degenerated. However, a few embryos were normalized by meiosis and fertilization and developed to full term, suggesting that mammals rely on sexual rather than asexual reproduction to eliminate genetic anomalies caused by clonal reproduction.

Introduction
Asexual reproduction, including cloning, enables many species such as potatoes to thrive through efficient mass reproduction systems1,2. Even in animals, cloning from a body part, that is, producing offspring from somatic cells, is observed in a few lower species, such as pot worms and planaria3,4. However, higher animals, e.g., mammals, cannot reproduce clonally from somatic cells in nature, and the reason for this remains unknown5,6.

Until recently, mammalian cloning was not possible except in cases such as monozygotic twins7. However, following the development of nuclear transfer technology and the successful somatic cell cloning of frogs8, Dolly the cloned sheep9, and Cumulina the cloned mouse10, it has become possible to produce cloned animals even from terminally differentiated somatic cells11. This technology can be used to rescue endangered species12,13 or infertile individuals14 by increasing population numbers from somatic cells. Cloning is also thought to be useful for the large-scale production of superior-quality domesticated animals15,16, potentially making expensive meat, such as Wagyu beef, more affordable17,18.

However, mammalian cloning has not yet been put into practical use, not only due to its low cloning success rate, but also because of anomalies19 not observed in the cloning of plants or lower animals. These issues are believed to stem from failures in “reprogramming” the donor nucleus to reset the epigenetic state of differentiated somatic cells to that of a fertilized embryo; this occurs not directly due to DNA abnormalities, but the detailed mechanisms responsible remain unclear20.

If DNA abnormalities do not occur and epigenetic errors do not accumulate, mammals should be able to maintain their species through cloning. However, early studies showed that serial cloning in any mammalian species reached its limit after only a few generations21–26. This suggests that cloning or asexual reproduction may not be viable for maintaining mammalian species. Nevertheless, by improving cloning success using an epigenetic modification reagent that promotes nuclear reprogramming27, we succeeded in serially cloning up to 25 generations of mice28. Since the success rate improved slightly with each successive generation, we initially concluded that serial cloning could be continued indefinitely28. However, after continuing the experiment for 20 years, the success rate began to decline and eventually could not be sustained beyond 58 generations.

In this study, we aimed to investigate in detail what occurred in re-cloned mice after extended serial cloning, and to clarify why mammals maintain their species through sexual rather than asexual reproduction.

Results
Success rate of serial cloning up to 58 generations
We began serial cloning experiments (Fig. 1a) in January 2005. The success rate of re-cloned mice gradually increased as generations progressed (up to 15.5% in the 26th-generation), and NGS analysis showed no difference between first-generation clones and 25th-generation re-clones. Therefore, we initially believed that serial cloning could probably continue indefinitely28, and thus continued this experiment. To maintain consistent experimental conditions as much as possible, TW and SW always participated in the production of next generation re-cloned mice. Approximately 3-4 generations of re-cloned mice were produced each year. However, the cloning success rate gradually decreased with each successive generation (Fig. 1b). Although success rates varied between generations, the average success rate declined to 0.6% after 57 rounds of serial cloning, and all 58th-generation re-cloned mice died the next day. In total, more than 1200 cloned mice were produced from a single original donor mouse (Supplementary Table 1). The original donor mouse was female with an agouti coat color; therefore, all cloned mice were female and exhibited the same coat color as the donor. Whole-genome sequencing (WGS) analysis also confirmed that these clones originated from the donor mouse (see later).

Placenta and lifespan of re-cloned mice
All cloned mice have been found to possess abnormal placentas arising from epigenetic errors29,30. We therefore measured the body and placental weights of most cloned mice at the time of cesarean section. As controls, body and placental weights were also measured for mice derived from in vitro fertilized control embryos (FC), newly generated first-generation cloned mice (NG1), and 25th-generation re-cloned mice derived from the cryopreserved cumulus cells of G24 cloned mice (CryoG25). The mean body weight across re-cloned generations was ~1.88 g, slightly heavier than that of FC mice (1.3 g) but similar to other control clones (1.68–1.96 g) (Supplementary Table 2). The mean placental weight was about 0.30 g, which is two to three times heavier than that of FC mice (0.11 g), but comparable to other control clones (0.25–0.29 g). However, neither body nor placental weight increased over successive generations, suggesting that abnormalities did not accumulate. Histological examination and measurement of the area of labyrinth of these placentas showed slightly expanded the area of labyrinth zone when increased the number of serial cloning but no significant differences between NG1, CryoG25, and G50 cloned/re-cloned mice (Fig. 1c, Supplementary Fig. 1), despite expansion of the spongiotrophoblast layer, an abnormality specific to cloned mice31. However, these placentas were significantly different compared to those of FC mice. Finally, we also examined the average lifespan of randomly selected re-cloned mice across generations. The average was approximately two years (Fig. 1d, Supplementary Table 3), similar to that of the control cloned mice, suggesting no abnormality.

Effect of trichostatin A (TSA) on re-cloned mice
One possible explanation for the decreased success rate in later generations is that TSA, a known enhancer of nuclear reprogramming27, may lose its effectiveness in supporting the development of re-cloned embryos. When nuclear transfer was performed without TSA, the success rate for generating G51 clones was 1.6%, similar to that of NG1 (0.95%) and significantly lower than with TSA treatment (G51: 5.4%; NG1: 3.9%) (Fig. 2a, Supplementary Table 4). Thus, even after more than 50 nuclear transfer cycles, the somatic cell nuclei of G50 re-cloned mice retained sensitivity to TSA.

Epigenetic abnormalities in re-cloned zygotes produced by G50 cloned mice
We next examined whether the decreased cloning success was due to accumulated epigenetic abnormalities, as cloned embryos are known to differ epigenetically from fertilized embryos20,32,33. To investigate this, FC zygotes, NG1 clones, and G51 re-cloned zygotes were fixed and immunostained with antibodies targeting trimethylated histone H3 at lysines 4, 9, and 27 (H3K4me3, H3K9me3, H3K27me3), acetylated histone H3 at lysine 14 (acH3K14), and stained with DAPI. As shown in Fig. 2b, c, and Supplementary Fig. 2, the acetylation and methylation levels were similar between NG1 and G51 cloned/re-cloned zygotes, except for H3K9me3. However, both groups differed markedly from FC zygotes. These results suggest that epigenetic abnormalities did not accumulate in cloned/re-cloned embryos, at least at the one-cell stage.

In vitro development and quality of G51 cloned embryos
NG1 cloned embryos and G51 re-cloned embryos were cultured in vitro until they reached the blastocyst stage and were then compared with FC embryos. As shown in Fig. 2d and Supplementary Table 5, the developmental rate into expanded blastocysts was lower for NG1 and G51 embryos than for FC embryos. However, there were no differences between NG1 and G51 groups, suggesting that serial cloning up to 50 cycles did not impair in vitro embryo development.

To evaluate embryo quality, some blastocysts were analyzed for cell number and inner cell mass (ICM) allocation. ICM distribution did not differ clearly between groups (Fig. 2e, f, Supplementary Table 6). Although the average total cell number was significantly reduced in cloned/re-cloned embryos relative to FC embryos, no difference was observed between NG1 and G51 embryos. Interestingly, the average ICM cell number was slightly higher in cloned/re-cloned embryos compared to FC embryos.

RNA-seq analysis of G51 cloned blastocyst
To detect differences between FC embryos, NG1 clones, and G51 re-cloned embryos, we analyzed the whole-genome gene expression profiles of blastocysts using RNA-seq (Fig. 3a). Relatively well-developed blastocysts were selected, and RNA was extracted individually from each blastocyst. RNA-seq was performed, and the three groups were compared using principal component analysis (PCA) to identify gene expression differences. PCA showed that not only NG1 (green dots) and G51 (blue dots) clone/re-cloned embryos exhibited similar gene expression profiles, but these were also similar to those of FC (red dots) embryos.

Production of offspring from re-cloned mice by sexual reproduction
Although serial cloning could not continue beyond the 58th generation, the re-cloned mice remained healthy except G58, raising the possibility that subsequent generations could be produced via sexual reproduction. Therefore, FC mice, as well as G20, G50, and G55 re-cloned mice, were mated with normal male mice to evaluate pregnancy and offspring production. There was no difference in age at first delivery among the groups, and litter sizes were similar between G20 re-cloned mice (9.9) and FC mice (10.3). However, litter sizes declined sharply in G50 and G55 re-cloned mice (2.8 and 2.2, respectively) (Fig. 3b, c, Supplementary Table 7). The number of oocytes recovered from each generation was recorded when collecting cumulus cells as donor cells (Supplementary Table 8). Although superovulation was triggered by hormonal treatment, the number of oocytes did not decrease even with further generation of re-cloned mice. Therefore, the decrease in litter size was not due to a reduced number of oocytes ovulated from the re-cloned mice.

Interestingly, when the next generation (grandchildren) was produced from offspring of G55 re-cloned mice, the litter size increased to 7.0 (Fig. 3c). Moreover, histological analysis showed that placentas from the grandchildren were clearly smaller than the large placentas characteristic of cloned mice and were nearly the same size as those from fertilized embryos (Figs. 1c and 3d, Supplementary Fig. 1).

Normality of re-cloned mouse oocytes
To examine whether oocytes derived from near-final-generation re-cloned mice were of reduced quality and/or prone to fertilization or developmental failure, we collected oocytes from CryoG25 and G53 re-cloned mice and subjected them to artificial activation and diploidization for parthenogenesis (Fig. 4a). If mutations accumulate through serial cloning, diploidization can reveal homozygous or defective mutations in oocytes, allowing assessment of their effects on early development. As a result, 91.3% of parthenogenetic embryos from BDF1 control mice developed into blastocysts, while only 12.7% of embryos from G25 re-cloned mice and none from G53 re-cloned mice developed into blastocysts (Fig. 4c, e, f, Supplementary Table 9).

Next, oocytes from re-cloned mice were fertilized in vitro with sperm from normal males (Fig. 4a). Even if oocytes carried many lethal mutations, normal sperm genes could improve blastocyst development. As a result, 97.6% of BDF1 control embryos reached the blastocyst stage, compared with 39.7% from G25 re-cloned mice and 20.3% from G53 re-cloned mice (Fig. 4c, g, h, Supplementary Table 9). These results clearly indicate that the number of lethal mutations in oocytes increases with each generation of serial cloning.

Finally, to determine whether the abnormalities originated from the oocyte nucleus (Metaphase II; MII) or cytoplasm, we performed MII replacement between oocytes from G56 re-cloned mice and fresh BDF1 mice (Fig. 4b). When the MII from one BDF1 oocyte was removed and immediately transferred into same oocyte, 68.2% of parthenogenetic embryos developed into blastocysts, confirming normality of both nucleus and cytoplasm (Supplementary Table 10). When MII from G56 re-cloned oocytes was transferred into enucleated BDF1 oocyte cytoplasm, only 8.0% of embryos developed to blastocysts. Similarly, when BDF1 MII was transferred into enucleated G56 oocyte cytoplasm, blastocyst formation was only 6.8%. These results suggest that G56 oocytes are damaged in both nucleus and cytoplasm.

Whole-genome sequencing (WGS) analysis of re-cloned mice
To investigate genomic mutations accumulated across generations, we conducted WGS on re-cloned mice from various generations. Short-read NGS-based WGS (read coverage: >30×) was performed on ten individuals from generations G6 to G57 (Fig. 5a, Supplementary Table 11). In addition, three of these individuals (G6-2, G50-1, and G57-2) underwent long-read WGS (read coverage: >20×) using Oxford Nanopore Technology long-read data.

Based on single nucleotide variant (SNV) profiles, we reconstructed the genetic lineage of each individual. As shown in Fig. 5a, yellow boxes indicate individuals analyzed by WGS, while black boxes denote estimated common ancestors. This phylogeny matched the experimental lineage, confirming that no genetic contamination occurred during the 20-year serial cloning process. SNVs accumulated over generations (Fig. 5b), with ~3700 SNVs and 80 insertion/deletions (indels; excluding repeat elements) identified from G1 to G57 (Table 1). On average, each generation acquired 69.4 SNVs and 1.4 indels. The SNV mutation signatures from G1 to G57 resembled those of naturally occurring germline mutations34 (Fig. 5c, Supplementary Fig. 3).

From G1 to G57, we identified 80–84 de novo structural variants (SVs) (Supplementary Data 1). Table 2 lists 16 large SVs (LSVs) of ≥40 kb, which are more likely to affect phenotypes. Many SVs appeared between G25 and G45. Among them, loss of one X chromosome, loss of heterozygosity (LOH) on chromosome 4 (147.5 Mb), and a translocation between chromosomes 7 and 9 are likely highly deleterious for fitness. LSVs also occurred after G45. Chromosomal abnormalities, including the X chromosome loss and two translocations, were likewise confirmed in the karyotype analysis of G54 re-cloned mice (Fig. 5e, Supplementary Fig. 4). We measured telomere length using short-read WGS and Telomerecat software 4.0.0. Although telomere length estimates varied widely and were method-dependent across samples, significant telomere shortening between G6 and G55 is unlikely (Supplementary Table 12). Nanopore long-read WGS data also revealed no evidence of extreme telomere shortening at individual chromosome ends (≥10 kb in G57-2).

To evaluate the effects of accumulated mutations, we analyzed their effects on genes. Of the 4404 SNVs and small indels identified, 0.95% were classified by SnpEff as “High” (loss-of-function) or “Moderate” (mainly missense). Dividing mutations into two groups—those from G1 to G23 during increased cloning success and those from G23 to G57 during decreased success (Fig. 1b)—revealed a significantly higher proportion in the latter group (G1–G23: 0.52%; G23–G57: 1.19%; P = 0.034) (Supplementary Table 13). Consistent with this, we assessed potentially deleterious variants using Genome Evolutionary Rate Profiling (GERP) scores, which evaluate functional constraint across coding and noncoding regions. The proportion with GERP scores ≥2 was significantly higher in the latter group (G1–G23: 2.4%; G23–G57: 4.0%; P = 0.005, Fisher’s exact test) (Fig. 5d, Supplementary Data 1, Supplementary Table 14). Mutation spectra also shifted, with transversions rising from 44.0% in G1–G23 to 47.9% in G23–G57 (P = 0.015, Fisher’s exact test) (Supplementary Fig. 3). In terms of SVs, G57 re-cloned mice showed at least 16 variants affecting exons (Supplementary Data 1). Across generations, two large deletions were identified in an adjacent region, resulting in homozygous loss of Csmd1 gene function (Supplementary Fig. 5). Considering the complete mutation spectrum, ~30 loss-of-function mutations and ~50 missense mutations had accumulated in G57 mice, even when excluding X chromosome loss (Table 1).

In this context of an extensive background mutation load, the emergence of additional deleterious mutations near the final generation, including Csmd1 disruption, may have exceeded a tolerable threshold, leading to lethality or severe developmental impairment, ultimately contributing to the collapse of cloning success (Supplementary Fig. 6). Considered together, these findings indicate that the progressive decline of serial cloning success is strongly associated with the accumulation of de novo mutations—including large SVs and harmful SNVs/indels after G23–G25—rather than the effect of any single mutation alone.

To compare mutation patterns in asexual versus sexual reproduction, we analyzed WGS data from two mouse lines subjected to 60 generations of sibling mating over ~20 years. These lines accumulated 739 and 752 SNVs, 22 and 23 INDELs and 29 and 29 SVs, respectively, with ~80% of autosomal variants being homozygous (Fig. 5b, Table 1). This corresponds to 22.3 SNVs, 0.68 INDELs and 0.91 SVs per generation, representing one-third of the SNV mutation rate in serial cloning and a slightly lower INDEL and SV occurrence frequency.

Finally, we performed WGS again on offspring produced via natural mating between G55 re-cloned mice and wild-type males (C57BL/6), as well as on their grandchildren. As expected from Mendelian inheritance, the mutations present in G55 were passed to both generations. LSVs transmitted across generations are listed in Supplementary Table 15. Notably, the translocation between chromosomes 7 and 9 in G55 was also found in G55 descendants, likely because both affected chromosomes were inherited together, preserving balance.

Methods
Animals
A three-month old BD129F1 (BDF1×129/Sv) mouse was used as the original donor animal, and cumulus cells were used as nuclear donors. This mouse was produced in our laboratory using three-way crosses between the C57BL/6, DBA/2, and 129/Sv strains. During experimentation, C57BL/6J mice were maintained at a constant temperature (25°C±1°C) under a 12-h light/dark cycle, with ad libitum access to food and water. The donor mouse that showed the highest success rate in producing first-generation cloned mice (hereafter, “G1”) was selected as the original donor and was used to initiate the serial mouse cloning experiment.

To generate subsequent generations, recipient oocytes were collected from adult BDF1 females. The surrogate pseudopregnant females used as embryo transfer recipients were ICR strain mice mated with vasectomized males of the same strain. BDF1, C57BL/6J, and ICR mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan).

For all sexual reproduction experiments, animal protocols were approved by the Animal Committee of Osaka University (Osaka, Japan; Approval No. FBS-17-006) and the Experimental Animal Care Committee of the Radiation Effects Research Foundation (RERF; Hiroshima, Japan; Approval No. 2018-02). All experiments were conducted in accordance with the 1996 National Institutes of Health guidelines and the Guidelines for Animal Experiments of the RERF. During experimentation, C57BL/6J mice were maintained at a constant temperature (22°C±1°C) under a 12-h light/dark cycle, with ad libitum access to food and water.

Housing conditions and diet were kept constant throughout the experiment. On the day of experimentation or after completion of all procedures, mice were euthanized by CO₂ inhalation or cervical dislocation. All animal experiments followed the ARRIVE guidelines and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, with approval from the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (Reference No. A29-24).

Oocyte collection
Female mice were superovulated by injection of 5 IU of equine chorionic gonadotropin, followed 48 h later by 5 IU of human chorionic gonadotropin (hCG). Cumulus–oocyte complexes (COCs) were collected from the oviducts 14–16 h later and transferred to a Falcon dish containing HEPES–CZB medium49. To disperse the cumulus cells, COCs were placed in a 50 μL droplet of HEPES–CZB medium containing 0.1% bovine testicular hyaluronidase for 3 min. Cumulus-free oocytes were washed twice and transferred to 20 μL droplets of CZB medium50 for culturing.

Collection of cumulus cells and tail tip fibroblasts
Cumulus cells were collected from donor or re-cloned mice using the same method as for oocyte collection. Fibroblasts were collected from the tail tips of G54 re-cloned mice for karyotype analysis as described previously51. Briefly, tail tips were skinned, cut into small pieces, then incubated in 5 mL DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich). After 10–14 days at 37.5°C in 5% CO₂ in air, proliferating fibroblasts were dissociated using trypsin and replated into larger dishes to expand the cell population.

Nuclear transfer
For nuclear transfer, oocytes were first transferred to a droplet (~10 μL) of HEPES–CZB containing 5 μg/mL cytochalasin B, placed under oil on the microscope stage. Each oocyte was held with an oocyte-holding pipette, and its zona pellucida was perforated by several piezoelectric pulses applied to the tip of an enucleation pipette10. The metaphase II chromosome spindle complex, visible as a translucent spot in the ooplasm, was drawn into the pipette with a small amount of ooplasm and gently pulled away until the cytoplasmic bridge was pinched off. Enucleated oocytes were then injected with a cumulus cell nucleus recovered from a 3-month-old re-cloned mouse, as described previously10. Briefly, the zona pellucida and the oocyte membrane were perforated by piezoelectric pulses, and the donor nucleus was inserted into the ooplasm. Reconstructed oocytes were transferred into CZB medium and incubated at 37°C in 5% CO₂.

Effects of nuclear transfer techniques on birth rate
During this long-term study, we improved the birth rate of cloned mice by optimizing nuclear transfer methods, such as using different histone deacetylase inhibitor (HDACi) treatments instead of TSA52,53. However, to isolate the effect of re-cloning, the standard method described above was used whenever possible. Some variations, such as in media quality or experimenter skill, were unavoidable over the study period and may have affected success rates or phenotypes. To control for these factors, we generated cloned mice from other BD129F1 donors as technical or time-matched comparisons (designated NG1). To assess the pure effect of serial nuclear transfer, cumulus cells from G24 re-cloned mice were frozen in June 2013 and thawed 8 years later to generate additional G25 mice (designated CryoG25), concurrently with G50 generation. Fertilized control (FC) mice with the same BD129F1 genetic background were also produced via intracytoplasmic sperm injection (ICSI), simulating equivalent in vitro manipulation and culture stress.

Activation of cloned embryos
Reconstructed oocytes were activated using 5 mM SrCl₂ in Ca²⁺-free CZB medium containing 50 nM trichostatin A (TSA) and 5 μM latrunculin A for 9 h27,33,54. Pseudo-pronuclear formation was verified, and embryos were cultured for either 1 day before transfer or 96 h to evaluate blastocyst development.

Embryo transfer
On the day following mating with vasectomized males, 2-cell stage cloned embryos were transferred into the oviducts of pseudo-pregnant ICR females at 0.5 days post coitum (dpc)55. On the transfer day, recipients were anesthetized by intraperitoneal injection of medetomidine, midazolam, and butorphanol. Five to eight embryos were transferred per uterine horn, and atipamezole was administered. At 19.5 dpc, offspring were delivered by cesarean section and transferred to foster mothers that had delivered naturally. Fertility was assessed by mating offspring at three weeks of age.

Intracytoplasmic sperm injection (ICSI)
To generate genetically identical but fertilized control (FC) mice, oocytes were collected from BDF1 females and sperm from 129/Sv males, producing BD129F1 offspring as with the original donor. To match cloned embryo conditions, FC mice were generated via ICSI, with equivalent in vitro manipulation and 1 day of CO₂ incubation49.

Histological analysis of placentae
Placentae from FC, NG1 (newly generated clone), G25, G50 re-cloned mice, and grandchild mice were embedded in OCT compound (Sakura Finetek, Japan). Blocks were cryosectioned at 7 μm (Thermo Fisher Scientific, USA) and mounted on Superfrost Micro Slides (Matsunami Glass, Japan). Sections were fixed in 4% paraformaldehyde for 10 min at room temperature, washed three times in PBS, incubated in 100 mM Tris-HCl (pH 9.5) for 10 min, and stained using NBT-BCIP (Vector Laboratories) for endogenous alkaline phosphatase (AP) activity. Sections were rinsed in Tris-HCl and counterstained with Nuclear Fast Red (Vector Laboratories). Trophoblasts in the labyrinth layers were detected by AP staining, and the area of AP staining-positive labyrinth layers were compared between natural, re-cloned mice and grandchild mice.

Immunostaining of re-cloned embryos at the one-cell stage
Ten hours post-activation, oocytes were fixed for 20 min at 25°C in 4% (w/v) paraformaldehyde with 0.2% (v/v) Triton X-100 (Nacalai Tesque, Kyoto, Japan). Fixed oocytes were washed thrice in PBS with 0.25% BSA (Millipore, Darmstadt, Germany) and stored at 4°C. Embryos were incubated with primary antibodies: rabbit anti-H3K4me3 (1:1000, Millipore, Darmstadt, Germany), rabbit anti-H3K9me3 (1:1000, Millipore, Darmstadt, Germany), or rabbit anti-H3K27me3 (1:1000, Millipore, Darmstadt, Germany) in PBS-BSA overnight at 4°C. Antibodies were supplemented with 0.2% (v/v) Tween (Millipore, Darmstadt, Germany). After two PBS-BSA washes (15 min each), embryos were incubated for 45 min with Alexa Fluor Cy5-labeled goat anti-rabbit IgG (1:500, Abcam, Cambridge, UK). DNA was stained with 2 μg/mL DAPI (Molecular Probes Inc., OR, USA). The largest pseudo-pronucleus per embryo was imaged using confocal microscopy (FV-1000, Olympus Corp., Tokyo, Japan). Fluorescence intensity was quantified using Olympus FluorView and NIH ImageJ.

Blastocyst immunostaining
To assess cloned blastocyst quality, cell counts were performed via immunofluorescence staining as described previously56. Briefly, blastocysts were fixed for 20 min at 25°C in 4% paraformaldehyde with 0.2% Triton X-100, washed thrice in PBS with 0.25% BSA, and stored at 4°C. Primary antibodies: anti-CDX2 mouse monoclonal (1:500, BioGenex, CA, USA) for TE cells and anti-Nanog rabbit polyclonal (1:500, Abcam, Cambridge, UK) for ICM cells. Secondary antibodies: Alexa Fluor 488-labeled goat anti-mouse IgG and Alexa Fluor 568-labeled goat anti-rabbit IgG (1:500, Abcam, Cambridge, UK). DNA was stained with 2 μg/mL DAPI (Molecular Probes, OR, USA). Blastocysts were mounted on slides, and serial z-stacks were captured by confocal microscopy (FV-1200). Cell numbers were quantified using Olympus FluorView.

Re-cloned mice oocyte quality examination
Oocytes were collected from BDF1, G25, and G53 re-cloned mice, and diploid parthenogenetic or fertilized embryos were generated to assess blastocyst development. Diploid parthenogenetic embryos were produced using the same method as that used to activate reconstructed oocytes after nuclear transfer. ICSI was performed using the same procedure as that for generating FC mice. The resulting parthenogenetic and fertilized embryos were cultured for five days, and their development to expanded blastocysts was evaluated. Next, to investigate the nucleus (metaphase II: MII) and cytoplasm of cloned mice oocytes separately, we performed MII replacement between oocytes from BDF1 and G56 re-cloned mice. This was followed by diploid parthenogenetic treatment to determine the rate of development to the blastocyst stage. MII extraction was performed using the same method as described for oocyte enucleation. However, when transferring MII to enucleated oocytes, the enucleation pipette was used to inject the MII into enucleated oocytes in cytochalasin B-containing medium, after which the hole was immediately sucked out to seal the cell membrane57. After replacing the MII, all oocytes were allowed to rest in an incubator for at least two hours before activation.

RNA-seq of re-cloned blastocysts
Five blastocysts were sampled from each of the following groups: FC mice, NG1 cloned mice, and G50 re-cloned mice. Samples were frozen immediately in liquid nitrogen and stored at –80°C until further analysis. RNA-seq libraries were prepared using the SMART-seq Stranded kit (Takara Bio, Shiga, Japan) and sequenced as 150-base single-end reads on the DNBSEQ-G50RS system (MGI Tech., Shenzhen, China). Sequence data were then mapped against the mouse reference genome sequence (GRCm39/mm39) using STAR58. We then calculated the “transcripts per million” value for each gene using RSEM59. To extract transcripts that often exhibited upregulation in blastocysts greater than two fold, we then calculated the fold change of each signal in all individuals within each group (i.e., NG1 and G50). These were then compared with the mean signal in the FC; the mean difference was then assessed using the DESeq2 R package60.

WGS and mapping
Genomic DNA was extracted from spleen samples using smart DNA prep (m) (Analytik Jena) and Qiagen Genomic tip DNA extraction kits (QIAGEN). For short-read WGS, paired-end libraries were prepared with DNA samples without PCR amplification using a TruSeq DNA PCR-Free Library Prep Kit (Illumina). All libraries were then sequenced on NovaSeq 6000 or X plus platforms (Illumina) to obtain paired-end 151-bp reads. Short-read sequence data were then mapped to the mouse reference genome (UCSC mm10) using BWA-MEM version 0.7.1761 with the “-M” option. This mapping was also processed with Picard version 2.18.26 (broadinstitute.github.io/picard) to remove PCR duplicates. In the analysis of sib-mating mouse lines, we utilized the ancestral genome data (F0 mice) from our previous paper (Ancestor ConA2 male-female mice). For long-read sequencing, libraries were prepared from G6-2, G50-1, and G57-2 using the SQK-LSK114 kit (Oxford Nanopore) and sequenced on a PromethION with FLO-PRO114M flow cells in high-accuracy mode. Base calling was performed with Guppy version 6.4.2 and Dorado version 7.2 (https://community.nanoporetech.com/). Base-called reads were mapped to the mouse reference genome (UCSC mm10, excluding chrY) using minimap2 version 2.21 and version 2.2862 with the parameters “-ax map-ont --MD.”

Identification of de novo mutations
Genomic variants were called from short-read bam files using GATK HaplotypeCaller version 4.1.0.063 with a minimum base quality of 20. For accurate detection of somatic mutations, effective whole-genome coverage (EWC) regions were defined per sample based on the following criteria: (i) MQ60 read depths were between 50% and 300% of the chromosome-specific peak coverage; (ii) MQ60 reads comprised at least 80% of mapped reads at each site; and (iii) a minimum base quality of 20 was confirmed using SAMtools version 1.964. Only shared EWC regions were used for mutation analyses (excluding SVs), covering 76.4% of the diploid genome for serial nuclear transfer lines, and 78.6% for sib-mating mouse lines (Supplementary Table 11). All de novo mutations were identified from filtered variants using the following criteria: (1) located within EWC regions; (2) multiallelic variants excluded; (3) variants with 0.35 > AF ≥ 0.03 in G45, G57-1, and G57-2 but < 0.03 in others were excluded to remove NovaSeq X Plus–specific noise; (4) minimum VAF = 0 in any clone; (5) Fisher strand bias (FS) < 23; (6) variants with AF ≥ 0.06 and < 0.2 detected in ≥3 samples excluded; (7) max VAF ≥ 0.3 for SNVs and ≥ 0.25 for INDELs in G6, G25, G45, G50, G55 and G57. We also used the following INDEL-specific filters: (8) exclude variants in homopolymers (>3 bp) or short repeats (e.g., di-, tri-, tetra-, penta-, or hexa-). (9) Only manually confirmed variants in IGV were retained. (10) Finally, we retained variants that met either of the following: (i) VAF < 0.10 in G6-1; or (ii) VAF < 0.10 in G6-2 and, in G6-1, VAF ≥ 0.30 for SNVs or VAF ≥ 0.25 for INDELs. In the analysis of sib-mating mouse lines, we retained variants that satisfied the following: VAF < 0.06 in both the F0 male and F0 female, and—on autosomes—VAF ≥ 0.30 for SNVs or ≥ 0.25 for INDELs in F60 or F62; on sex chromosomes, VAF ≥ 0.80 in F60 or F62.

SV calling using short- and long-read data
SV calling of short-read data was conducted using Manta version 1.6.065, RUFUS version 1.066, and Delly version 0.8.667. SV calling by Manta was conducted using the following criteria: 1) The number of split reads for the “ALT” allele (SRA) was > 3 in a mouse. 2) The minimum SRA number of other mice was zero. SV calling by RUFUS was conducted using the following criteria: 1) “SVTYPE” was described in the INFO column of the VCF file, or 2) the length of the “REF” allele or the “ALT” allele was > 40 bp. SV calling by DELLY was conducted using the following criteria: 1) In all cases the number of variant reads (i.e., DV or RV) was > 3 for a mouse. 2) The minimum number of variant reads in other mice was 0.

Next, we performed SV detection using long-read data to recover mutations missed in short read analyses. Mutation candidates were then extracted from G6-2 and G50-1 using sniffles version 1.0.1268 with the parameter: “-s 3.” VCF files of SV calls from each sample were merged using SURVIVOR version 1.0.769 with the following parameters: max distance between breakpoints was 1000 bp and “types must match.” The merged VCF file was then used to perform a “force call” of SVs using sniffles. Force-called VCF files of each sample were again merged using SURVIVOR. Structural variants were called using the following inclusion criteria: (1) SVTYPE is TRA or SVLEN is ≤ −40 or ≥ 40, (2) The number of reads supporting alt is 0 for G6-2, > 5 for G50-1, and/or G57-2, and (3) depth is > 10 and < 100 for both G6-2 and G50-1. All candidates were then manually inspected by IGV, including short read results. Using this procedure five new de novo genes were detected. Three of these could be linked to the generation in which they arose using short read sample information. Finally, we note that this analysis was not limited to EWC regions.

Mutation analysis
The genetic lineage of each re-cloned mouse was inferred from its SNV profile. Assuming a constant number of SNVs per generation, lineage divergence points were identified. By excluding regions affected by LOH, large deletions, or X chromosome loss, we constructed a consistent phylogenetic relationship.

Mutational signature analysis and visualization of single-base substitutions were carried out using SigProfilerAssignment70 (https://cancer.sanger.ac.uk/signatures/assignment/app/), with the parameters set as follows: Reference genome = “mm10/GRCm38,” Variant class = “SBS.” Reference mutational signatures = “COSMIC v3.4.” Type of study = “Whole-Genome Sequencing”70. Functional annotations were performed with SnpEff version 4.3t71. De novo mutations were further annotated using GERP scores72. GERP score data derived from the mouse mm9 reference was obtained from the UCSC website (http://hgdownload.soe.ucsc.edu/gbdb/mm9/bbi/All_mm9_RS.bw).

Telomere length evaluation
Telomere length was assessed using WGS data from two G6 individuals, two CryoG25 individuals, and two G50 individuals by Telomerecat (version 4.0.0)73 using the default parameters. Although we performed whole-genome sequencing on either the Illumina NovaSeq 6000 or NovaSeq X Plus platforms, only datasets produced on the NovaSeq 6000 were used for telomere length analysis in order to ensure methodological consistency and avoid potential platform-specific biases.

Karyotyping of G54 re-cloned mice
Chromosomes from tail-tip cells of two G54 re-cloned mice (G54-1 and G54-2) were analyzed by spectral karyotyping using fluorescence in situ hybridization (SKY–FISH) chromosome painting techniques (SCAS Ltd., Tokyo, Japan).

Statistical analyses
Most serial cloning experiments for each generation were repeated at least twice. Other experiments were independently performed at least three times by two to three experimentalists, and similar results were obtained regardless of the experimenter. Rates of embryo development, offspring birth, and body and placental weights were evaluated using chi-squared tests. Fluorescence intensity was analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests as implemented in GraphPad Prism (GraphPad Software, USA). Principal component analysis (PCA) was performed using the DESeq2 R package. For all statistical tests, a value of P < 0.05 was considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wakayama and collaborators demonstrate that re-cloning mice up to the 58th generation is harmful. They illustrate how DNA\nmutations can accumulate over time. The evidence is compelling. The experiments assessing the developmental potential\nof oocytes from cloned females are outstanding.\nMinor issues:\nThe increase in cloning efficiency up to the 26th generation is notable. Authors should elaborate on possible explanations or\nat least mention in the discussion that this is an area for future research. They mention in the discussion section possible\nbeneficial mutations, but have they analyzed the sequencing data to identify which ones?\nOn the production of offspring from cloned mice, G50 and G55 had litter sizes of 2.8 and 2.2, respectively. Authors should\nclarify whether the reproductive function of these females was examined beyond oocyte quality. Specifically, they should\naddress ovarian reserve and reproductive hormone levels.\nAuthors should avoid stating in the discussion section that ‘re-cloned mice retained normal fertility,’ as their results show that\nonly G20 re-cloned mice had normal fertility in terms of litter size.\nThe discussion about the Red Queen Hypothesis doesn’t add to the manuscript; however, Muller’s ratchet theory is\nappropriate.\nThe authors should also mention that Trichostatin A could contribute to mutation rates. Literature shows that TSA can\nreactivate endogenous retroviruses and transposable elements, and it may also interfere with DNA repair mechanisms. The\nreviewer is not overly concerned about this due to the short exposure time; however, the authors should include this point in\nthe discussion section.\nTwo issues in the methods section\n1- The authors mention the isolation of fibroblasts. What were they used for?\n2- Explain what ‘CB’ means, cytochalasin B?", "reviewer_2": "Reviewer’s comments\nManuscript Title: Limitations of serial cloning in mammals\nJournal: Nature communications\nManuscript ID: NCOMMS-25-89349-T\nDate of review: Nov. 17, 2025\nOverall Comments:\nFirst of all, I admire the work by the authors very much. It’s an extraordinary work, lasting for 20 years, cloning/recloning the\nanimals for 58 generations. Simply amazing!\nAs the authors state in their Discussion part, this work is the first empirical demonstration that mutational meltdown, as\npredicted by Muller’s ratchet, can indeed occur in mammals. In my opinion, this alone makes this work deserving to be\npublication in Nat Comm or any other journal.\nI have some minor comments:\n1. Figure 1 b and d, please mark the actual year, e.g., 2005, 2025 below each generation. In particular, I am interested to see\nif COVID years have any impacts?\n2. Any changes in technician throughout years? Any major changes in the cloning protocols?\n3. Any insights onto the roles of enucleated oocytes? Mitochondria?\n4. Line 310, why does this finding provide “a plausible explanation for why sexual reproduction has been maintained\nthrough evolution”? I suggest delete this.\n5. Line 333, find another example other than “pet loss syndrome”.\n6. Line 225, in Supplementary Table 9, the telomere lengths are labeled around 5 kb, which is much shorter than the typical\n10 to 50 kb length in mice. Please check if the values are correct.\n7. More regarding telomere lengths. I know authors published findings in earlier generations of these recloned animals that\ntelomeres do not get shortened with increased generations. I wonder if the “shortest” telomeres may have contributed to the\ncollapse of cloning success. In other words, do authors have any telomere length data at individual chromosome level?\n8. It’s interesting that late generation cloned embryos lose their “responsiveness” to epigenetic remodeler TSA. Does this\nsuggest mutational changes related to histone deacetylase?\n9. Line 106, It will be good if an example could be presented to show how to measure the labyrinth zone and\nspongiotrophoblast layer.\n10. Line 111, The “lifespan-related abnormalities”. Not very clear. Figure 1D shows that the lifespan of re-cloning mice is\nsimilar to those of control cloned mice.\n11. Line 122, in the epigenetic staining experiment, Figure 2B and 2C do not clearly demonstrate the “abnormality” of\nepigenetics in nuclear transferred embryos. On the other hand, the Extended data Figure 2 has higher quality results and\nbetter demonstrates the statement in the text. I recommend switching the figures or reorganizing the way to demonstrate the\nresults.\n12. Line 143, In Figure 2e and f, specify the markers that are used for staining, in addition to labeling them as ICM or TE.\n13. Line 157, I did not see any discussion of “fusion transcripts” abnormality in recloned embryos in the main text. Please\ncheck if the statement needs to be clarified or removed.\nAgain, I admire this work very much. Regardless of authors’ answers to my above questions, their work showcased that\nsomewhere along the asexual reproduction course, disastrous events would happen, which provides an explanation why\nsexual reproduction is dominant in mammals.", "reviewer_3": "The paper by Wakayama et al. not only highlights the limitations of cloning technology but also offers numerous biological\ninsights into areas such as evolutionary biology and mutational genomics. The detailed genomic analyses, including whole-\ngenome sequencing (WGS), serve as extremely valuable records. It is also intriguing that cloning efficiency began to decline\naround the G25 milestone. Throughout the paper, the logic is sound, and the experimental results support the conclusions.\nThe experimental methods are also generally accepted methods.This paper is descriptive in nature and simply consists of a\ncompilation of clone's data, but it represents the only long-term serial clone data available.\nThe major weakness of this study is that its reproducibility cannot be evaluated by other laboratories. Therefore, it is\nessential to demonstrate that clone experiments using BDF1 cumulus cells as standard donors in the authors’ laboratory\nhave maintained consistent cloning efficiency. While it is not necessary to provide data for every generation, please include\na summary of the efficiency of these standard clones at several time points over the past 20 years.Such stability of cloning\ntechnology ensures the reliability of this research.\nThere are several areas in which clarity and impact could be improved in the manuscript. Nevertheless, most of the points\nraised below are minor and are intended to strengthen the paper rather than to challenge its main conclusions.\nLine 106: Please show data of measurement of the spongiotrophoblast layer, which depicts the cloned placental phenotype.\nLine 197: It is interesting that G56 oocytes exhibited damage to both the nucleus and cytoplasm. Given the genes involved in\nthe mutation, is it possible to speculate what aspect of the cytoplasm is affected?\nLine 251: The author cites Csmd1 disruption as an example causing deleterious effects, but knockout of this gene appears\nto manifest abnormalities after birth. Are there no other examples causing “deleterious effects” to embryonic/fetal\ndevelopment?\nP305: Please cite a recent paper that reported a DNA repair mechanism in oocytes (\nhttps://pubmed.ncbi.nlm.nih.gov/41241715/ ) and discuss the involvement of this ability in the reproductive performance of\nthe cloned mice."}, "author_response": "Point-by-point responses\nReviewer #1 (Remarks to the Author):\nWakayama and collaborators demonstrate that re-cloning mice up to the 58th generation\nis harmful. They illustrate how DNA mutations can accumulate over time. The evidence\nis compelling. The experiments assessing the developmental potential of oocytes from\ncloned females are outstanding.\nWe thank the reviewer for the comprehensive analysis of the presented evidence and\nhelping to improve the quality of this report. As suggested, appropriate corrections were\nmade throughout the manuscript.\nMinor issues:\nThe increase in cloning efficiency up to the 26th generation is notable. Authors should\nelaborate on possible explanations or at least mention in the discussion that this is an\narea for future research.\nThank you for your important suggestion.\nAlthough this point is briefly mentioned in the main text, further information is\nprovided in the revision as suggested.\n(Original text: Line 299–302): During this period, adaptive mutations may have\ntransiently enhanced cloning performance or somatic cells that were easily\nreprogrammable and capable of generating viable re-clones may have been\npreferentially selected, thereby creating a positive selection effect.\nAdditional sentence:\nLine 292\nThe build-up of harmful variants appears to have outweighed adaptive effects, such as\nthe positive selection effect around G25, resulting an irreversible decline in birth rates.\nLine 354\nYet, these findings demonstrate that practical application of cloning of mammals\nrequires a deeper appreciation of these biological constraints, warranting further\nresearch, and reaffirm…\nThey mention in the discussion section possible beneficial mutations, but have they\nanalyzed the sequencing data to identify which ones?\nWe thank the reviewer for this important question. We did not identify gene mutations\npreviously reported to improve the efficiency of SCNT, including those related to\nimprinting or epigenetic regulation.\nAlthough the list of variants shown in Supplementary Table 8 may include previously\nunrecognized candidate mutations that could influence cloning efficiency, we were not\nable to distinguish potentially beneficial mutations from neutral variants based on the\navailable data. Therefore, the mention of beneficial mutations in the Discussion was\nintended as a conceptual possibility rather than a conclusion supported by the\nidentification of specific variants.\nThe Discussion was revised to clarify this point, as follows:\nLine287\nDuring this period, although we were not able to detect adaptive mutations by whole-\ngenome sequencing (WGS) analysis, some mutations may have transiently enhanced\ncloning performance or somatic cells that were more easily reprogrammable and\ncapable of generating viable re-clones may have been preferentially selected, resulting\nin a positive selection effect.\nOn the production of offspring from cloned mice, G50 and G55 had litter sizes of 2.8\nand 2.2, respectively. Authors should clarify whether the reproductive function of these\nfemales was examined beyond oocyte quality. Specifically, they should address ovarian\nreserve and reproductive hormone levels.\nThank you for your valuable comments. As the mating experiments were conducted\nmore than two years ago, those mice have since died. Therefore, unfortunately, we were\nunable to examine their ovarian function or reproductive hormones. However, despite\nthe significant reduction in litter size, there was no change in the number of oocytes\novulated, and pregnancy was possible. Based on these outcomes, we believe the\nindividual re-cloned mice were almost normal. The following text has been added.\nLine 166\nThe number of oocytes recovered from each generation was recorded when collecting\ncumulus cells as donor cells (Supplementary Table 7). Although superovulation was\ntriggered by hormonal treatment, the number of oocytes did not decrease even with\nfurther generations of re-cloned mice. Therefore, the decrease in litter size was not due\nto a reduced number of oocytes ovulated from the re-cloned mice.\nAuthors should avoid stating in the discussion section that ‘re-cloned mice retained\nnormal fertility,’ as their results show that only G20 re-cloned mice had normal fertility\nin terms of litter size.\nThank you for this important point. Although the re-cloned mice retained reproductive\ncapacity, the number of offspring decreased despite the high number of oocytes\novulated, thus preventing the conclusion that all were completely normal. Therefore, the\nword “normal” was deleted from the sentence.\nLine 326\n“…re-cloned mice retained normal fertility…”\nThe discussion about the Red Queen Hypothesis doesn’t add to the manuscript;\nhowever, Muller’s ratchet theory is appropriate.\nWe agree that a discussion about Müller’s ratchet theory is appropriate in this paper. As\nyou pointed out, it is clear that the experiments in this study were insufficient to\ndisprove the Red Queen Hypothesis in mammals. However, we believe that a\ncomparison of the Red Queen Hypothesis would be beneficial to emphasize the\nimportance of Müller’s ratchet theory. However, since the Red Queen Hypothesis is not\nabsolutely necessary to enhance the value of this study, we will defer to the Editor’s\njudgment.\nThe authors should also mention that Trichostatin A could contribute to mutation rates.\nLiterature shows that TSA can reactivate endogenous retroviruses and transposable\nelements, and it may also interfere with DNA repair mechanisms. The reviewer is not\noverly concerned about this due to the short exposure time; however, the authors should\ninclude this point in the discussion section.\nThank you for this very important suggestion. The following text was added\naccordingly.\nLine 309\nConversely, in this study, TSA treatment was conducted during nuclear transfer for\nproduction of all generations of re-cloned mice. TSA is a standard class I/II histone\ndeacetylase (HDAC) inhibitor known to not only activate genes involved in\ntranscription and promote nuclear reprogramming (27,39), but also reactivate\nendogenous retroviruses (40,41). However, since a very low concentration of TSA (5\nnM) was used during nuclear transfer and lasted only about 8 h, it is unlikely that TSA\ntreatment caused the high mutation rate of the re-cloned mice.\nTwo issues in the methods section\n1- The authors mention the isolation of fibroblasts. What were they used for?\nWe apologize for the lack of an explanation. Fibroblasts were isolated from the tail tips\nof re-cloned mice to examine the karyotype.\nLine 394\nFibroblasts were collected from the tail tips of G54 re-cloned mice for karyotype\nanalysis as described previously\n2- Explain what ‘CB’ means, cytochalasin B?\nThank you for pointing out the failure to define this abbreviation. This oversight has\nbeen corrected in the revised article.\nReviewer #2 (Remarks to the Author):\nOverall Comments:\nFirst of all, I admire the work by the authors very much. It’s an extraordinary work,\nlasting for 20 years, cloning/recloning the animals for 58 generations. Simply amazing!\nAs the authors state in their Discussion part, this work is the first empirical\ndemonstration that mutational meltdown, as predicted by Muller’s ratchet, can indeed\noccur in mammals. In my opinion, this alone makes this work deserving to be\npublication in Nat Comm or any other journal.\nWe thank the reviewer for the comprehensive analysis of the presented evidence and\nhelping to improve the quality of this report. We made appropriate corrections\nthroughout the manuscript in accordance with the reviewer’s suggestions.\nI have some minor comments:\n1. Figure 1 b and d, please mark the actual year, e.g., 2005, 2025 below each generation.\nIn particular, I am interested to see if COVID years have any impacts?\n2. Any changes in technician throughout years? Any major changes in the cloning\nprotocols?\nThank you for your useful comments. The implementation years were added to Figure\n1b. There was no discernable impact from the COVID-19 pandemic. The following text\nhas been added:\nLine 80\n…and thus continued this experiment. To maintain consistent experimental conditions\nas much as possible, TW and SW always participated in the production of next\ngeneration re-cloned mice. Approximately 3–4 generations of re-cloned mice were\nproduced each year.\nLine 301\nThis study was conducted over a period of approximately 20 years. Therefore,\nconsidering variations to the quality of culture medium or equipment, it was impossible\nto maintain completely identical experimental conditions throughout the entire period.\nHowever, two researchers (TW and SW) consistently participated in the nuclear transfer\nexperiments across all generations. Furthermore, during the nuclear transfer\nexperiments conducted during this period using BDF1 cumulus cells, there was no\nchange to the birth rate of cloned mice and no restriction due to the COVID-19\npandemic (Supplemental Table 13).\n3. Any insights onto the roles of enucleated oocytes? Mitochondria?\nThank you for your question. While some studies have reported the effects of\nenucleation of MII spindle on oocyte development (Simerly et al., Science. 2003 Apr\n11;300(5617):297; Van Thuan, et al., Biol Reprod. 2006 May;74(5):777-87), there was\nno clear impact on mouse cloning. Regarding mitochondria, the oocytes used in this\nstudy were collected from BDF1 mice, and the donors were BD129F1 mice/cloned\nmice. Since both the oocytes and somatic cells possessed mitochondria derived from\nC57BL/6N mice, we believe there was no impact on development. Furthermore, this\nmethod has remained unchanged since the first generation of clones. Therefore, we\nbelieve there was no effect on the results.\n4. Line 310, why does this finding provide “a plausible explanation for why sexual\nreproduction has been maintained through evolution”? I suggest delete this.\nThank you for your suggestion. We apologize for the unclear explanation. The first part\nof the text has been significantly revised and the sentence you mentioned was removed.\nLine 325\nSince sexual system function is not required for the survival of individual clones, it is\nimprobable that only cloned embryos with normal reproductive system were selected\nand born. Instead, these results imply that the sexual reproductive system is unusually\nrobust against mutational damage, even under conditions that foster accumulation of\ndeleterious variants.\n5. Line 333, find another example other than “pet loss syndrome.”\nThank you for your observation. This text was deleted.\n6. Line 225, in Supplementary Table 9, the telomere lengths are labeled around 5 kb,\nwhich is much shorter than the typical 10 to 50 kb length in mice. Please check if the\nvalues are correct.\nThe data provided in Supplementary Table 9 were estimated using Telomerecat from\nshort-read WGS analysis and represent relative rather than absolute telomere lengths. To\navoid confusion, the following clarification was added to the footnote of Supplementary\nTable 9:\nSupplementary Table 9\nThe data were estimated from short-read WGS data using Telomerecat (version 4.0.0)\nand, although reported in base pairs, represent relative estimates rather than absolute\ntelomere lengths.\n7. More regarding telomere lengths. I know authors published findings in earlier\ngenerations of these recloned animals that telomeres do not get shortened with increased\ngenerations. I wonder if the “shortest” telomeres may have contributed to the\ncollapse of cloning success. In other words, do authors have any telomere length data at\nindividual chromosome level?\nWe agree that nanopore long-read WGS can, in principle, provide telomere length\ninformation at the level of individual chromosomes by directly measuring telomeric\nrepeat tracts anchored to subtelomeric regions.\nHowever, in the present study, sequencing conditions differed across generations, which\nprecluded accurate quantitative comparisons of chromosome-specific telomere lengths\nbetween generations. Nevertheless, in sample G57-2, we were able to confirm telomere\nlengths of at least 10 kb at all examined chromosome ends, supporting the conclusion\nthat extreme telomere shortening did not occur, even in later generations.\nLine228\nNanopore long-read WGS data also revealed no evidence of extreme telomere\nshortening at individual chromosome ends (≥10 kb in G57-2).\n8. It’s interesting that late generation cloned embryos lose their “responsiveness” to\nepigenetic remodeler TSA. Does this suggest mutational changes related to histone\ndeacetylase?\nThank you for your feedback. We believe this confusion was due to our inability to\nprovide an adequate explanation in English, which is not our native language.\nNonetheless, the responsiveness to TSA was not lost even after 50 re-cloning cycles.\nThe results of this experiment are described in the text.\nLine 122–123．\nThus, even after more than 50 nuclear transfer cycles, the somatic cell nuclei of G50 re-\ncloned mice retained sensitivity to TSA.\n9. Line 106, It will be good if an example could be presented to show how to measure\nthe labyrinth zone and spongiotrophoblast layer.\nWe sincerely apologize for the incomplete documentation. For clarification, the\nfollowing text was added the Methods section.\nSince the spongiotrophoblast samples were poorly preserved, accurate measurement\nwas impossible. We kindly ask for your understanding.\nLine 457\nTrophoblasts in the labyrinth layers were detected by AP staining, and the area of AP\nstaining-positive labyrinth layers were compared between natural, re-cloned mice and\ngrandchild mice.\n10. Line 111, The “lifespan-related abnormalities”. Not very clear. Figure 1D shows that\nthe lifespan of re-cloning mice is similar to those of control cloned mice.\nWe sincerely apologize for the confusing text and made the following corrections.\nLine 110\nThe average lifespan of the re-cloned mice was approximately two years (Fig. 1d,\nSupplementary Table 2), similar to that of the control cloned mice, suggesting no\nabnormality.\n11. Line 122, in the epigenetic staining experiment, Figure 2B and 2C do not clearly\ndemonstrate the “abnormality” of epigenetics in nuclear transferred embryos. On the\nother hand, the Extended data Figure 2 has higher quality results and better\ndemonstrates the statement in the text. I recommend switching the figures or\nreorganizing the way to demonstrate the results.\nThank you for your suggestion. Accordingly, the figures were switched. See Figure 2b.\n12. Line 143, In Figure 2e and f, specify the markers that are used for staining, in\naddition to labeling them as ICM or TE.\nThank you for your suggestion. The legends of Figure 2e and f were revised as\nsuggested.\n13. Line 157, I did not see any discussion of “fusion transcripts” abnormality in\nrecloned embryos in the main text. Please check if the statement needs to be clarified or\nremoved.\nThank you for your observation. Since there was no abnormality, the description is not\nneeded and, therefore, deleted.\nAgain, I admire this work very much. Regardless of authors’ answers to my above\nquestions, their work showcased that somewhere along the asexual reproduction course,\ndisastrous events would happen, which provides an explanation why sexual\nreproduction is dominant in mammals.\nThank you for your high regard for our research. All of your comments were\nconstructive and aimed at improving our paper. Therefore, all of your suggestion were\nincorporated into the main text. Thank you very much.\nReviewer #3 (Remarks to the Author):\nThe paper by Wakayama et al. not only highlights the limitations of cloning technology\nbut also offers numerous biological insights into areas such as evolutionary biology and\nmutational genomics. The detailed genomic analyses, including whole-genome\nsequencing (WGS), serve as extremely valuable records. It is also intriguing that\ncloning efficiency began to decline around the G25 milestone. Throughout the paper,\nthe logic is sound, and the experimental results support the conclusions. The\nexperimental methods are also generally accepted methods.This paper is descriptive in\nnature and simply consists of a compilation of clone's data, but it represents the only\nlong-term serial clone data available.\nWe thank the reviewer for the comprehensive analysis of the presented evidence and\nhelping to improve the quality of this report. We have also made appropriate corrections\nas suggested throughout the manuscript.\nThe major weakness of this study is that its reproducibility cannot be evaluated by other\nlaboratories. Therefore, it is essential to demonstrate that clone experiments using BDF1\ncumulus cells as standard donors in the authors’ laboratory have maintained consistent\ncloning efficiency. While it is not necessary to provide data for every generation, please\ninclude a summary of the efficiency of these standard clones at several time points over\nthe past 20 years. Such stability of cloning technology ensures the reliability of this\nresearch.\nThank you for your very important feedback. To demonstrate that our technical changes\nhave not affected the cloning success rate in this study, we have created a new table\nsummarizing our laboratory’s cloning success rates. See Supplementary Table 13.\nThe following text has been added:\nLine 305\nFurthermore, nuclear transfer experiments conducted during this period using BDF1\ncumulus cells demonstrated no change to the birth rate of cloned mice, including the\nduration of the COVID-19 pandemic (Supplemental Table 13).\nThere are several areas in which clarity and impact could be improved in the\nmanuscript. Nevertheless, most of the points raised below are minor and are intended to\nstrengthen the paper rather than to challenge its main conclusions.\nWe are very grateful for your many suggestions to improve our paper. The paper was\nrevised in accordance with your suggestions.\nLine 106: Please show data of measurement of the spongiotrophoblast layer, which\ndepicts the cloned placental phenotype.\nThank you for your useful comment. We had also planned detailed measurements of the\nplacenta, but due to poor sample preservation, we were only able to obtain accurate\nmeasurements of the labyrinth zone. Therefore, we had to abandon measurements of the\nspongiotrophoblasts. We hope that you understand. Instead, we have provided a more\ndetailed description of the labyrinth measurement method.\nLine 457\nTrophoblasts in the labyrinth layers were detected by AP staining, and the area of AP\nstaining-positive labyrinth layers were compared between natural, re-cloned mice and\ngrandchild mice.\nLine 197: It is interesting that G56 oocytes exhibited damage to both the nucleus and\ncytoplasm. Given the genes involved in the mutation, is it possible to speculate what\naspect of the cytoplasm is affected?\nWe thank the reviewer for this insightful question. Our MII replacement experiments\nindicate that the reduced developmental competence of G56 oocytes cannot be\nexplained solely by nuclear defects, as impaired cytoplasmic oocyte competence was\nalso involved.\nCytoplasmic competence of mammalian MII oocytes is dependent on multiple\nprocesses, including mitochondrial bioenergetics, Ca²⁺ handling required for activation,\nmaternal mRNA regulation, cytoskeletal organization, and proteostasis. Within the\nheterozygous variant set identified in G56 oocytes (Supplementary Table 8), several\ngenes map to these pathways. However, because most variants are heterozygous and\ntheir functional consequences remain uncertain, we consider it premature to attribute the\ncytoplasmic phenotype to any single gene or pathway. We therefore speculate that\nmultiple heterozygous perturbations acting additively may underlie the observed\ncytoplasmic defects, consistent with the MII replacement results. The Discussion was\nrevised accordingly.\nLine 296\nThis transition suggests that the burden of accumulating mutations began to affect not\nonly nuclear genomic integrity but also oocyte-level functions essential for\ndevelopment. Consistent with this interpretation, our MII replacement experiments\nindicate that late-generation oocytes exhibited compromised cytoplasmic competence in\naddition to nuclear defects.\nLine 251: The author cites Csmd1 disruption as an example causing deleterious effects,\nbut knockout of this gene appears to manifest abnormalities after birth. Are there no\nother examples causing “deleterious effects” to embryonic/fetal development?\nWe thank the reviewer for this important comment. We agree that disruption of Csmd1\nis primarily associated with postnatal phenotypes rather than overt embryonic or fetal\nlethality. Our intention in citing Csmd1 was not to imply a specific role in embryonic or\nfetal development, but to highlight severe deleterious effects upon disruption.\nAs shown in Supplementary Table 8, Csmd1 was identified in a homozygous state,\nwhich occurred in the context of numerous additional heterozygous deleterious\nmutations present in the genome. In contrast, no other genes with homozygous\ndisruptions known to cause embryonic or fetal lethality were identified. Given this\ngenetic background, the acquisition of additional mutations could plausibly lead to\nlethality or severe developmental impairment. Therefore, the possibility of\nCsmd1disruption, together with the accumulation of other deleterious variants, may\nhave contributed to serious developmental effects.\nTo avoid overinterpretation, the text was revised to clarify this point and to describe the\ndeleterious effects in a broader context rather than attribution solely to the embryonic or\nfetal stages.\nLine243\nAcross generations, two large deletions were identified in an adjacent region, resulting\nin homozygous loss of Csmd1 gene function (Extended Data Fig. 7). Considering the\ncomplete mutation spectrum, approximately 30 loss-of-function mutations and\napproximately 50 missense mutations had accumulated in G57 mice, even when\nexcluding X chromosome loss (Table 1). In this context of an extensive background\nmutation load, the emergence of additional deleterious mutations near the final\ngeneration, including Csmd1 disruption, may have exceeded a tolerable threshold,\nleading to lethality or severe developmental impairment, ultimately contributing to the\ncollapse of cloning success (Extended Data Fig. 8). Considered together, these findings\nindicate that the progressive decline of serial cloning success is strongly associated with\nthe accumulation of de novo mutations—including large SVs and harmful SNVs/indels\nafter G23–G25— rather than the effect of any single mutation alone.\nP305: Please cite a recent paper that reported a DNA repair mechanism in oocytes\n( https://pubmed.ncbi.nlm.nih.gov/41241715/ ) and discuss the involvement of this\nability in the reproductive performance of the cloned mice.\nThank you for introducing such an important paper. The following passage is cited in\nthe revised text.\nLine 330\nThroughout this study, we consistently utilized oocytes collected from young\nindividuals, and it is known that the oocytes of young individuals can effectively\nsuppress age-related DNA damage (42), and this ability may have been particularly\neffective against mutations in the sexual reproductive system."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have properly addressed this reviewer's concerns.", "reviewer_2": "The authors have addressed all my questions.", "reviewer_3": "The author has responded appropriately to all my comments and revised the manuscript. It is regrettable that the\nspongiotrophoblast analysis was not performed. For future studies, I recommend that the author histologically identify\nspongiotrophoblast using PAS staining after standard aldehyde fixation, rather than frozen sections."}, "author_response": ""}]

Decision letter