Cloned ferrets produced by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost.

Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse

It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70–80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase, and the short-term treatment of nuclear donor cells with these two methods could improve the efficiency of SCNT.

Survival to term following embryo transfer is usually low after cloning by somatic cell nuclear transfer (SCNT). The aims of this study were to evaluate the effects of the zona pellucida (presence or absence) and the cell nucleus transfer method (cell fusion or cell injection) on the in vitro survival and pregnancy outcome of Day-1 goat cloned embryos transferred into the oviduct of recipient females. In vitro-matured goat oocytes from slaughterhouse ovaries were polar body selected, with a group of oocytes subjected to enzymatic zona pellucida removal. Zona-free (ZF) and zona-intact (ZI) oocytes were enucleated by micromanipulation procedures (Oback and Wells 2003 Cloning Stem Cells 5, 3–12; Keefer et al. 2000 Biol. Reprod. 66, 199–203). Somatic nucleus donor cells from 3 transgenic females for mammary gland expressing human lysozyme (Maga et al. 2003 Trans. Res. 12, 485–496) were either fused (CF) with enucleated ZI and ZF oocytes or injected (CI) into enucleated ZI oocytes, with the assessment of fusion or injection survival rates performed after 60 min. Two direct-current (DC) pulses were used to induce fusion in the ZI group (2 kV cm–1 each for 10 µs) and in the ZF group (1.0 kV cm–1 for 20 µs). Embryo reconstruction using ZI oocytes was done by micromanipulation. Zona-free (CF) or ZI (CF or CI) reconstructed cloned embryos were chemically activated in ionomycin/DMAP, followed by in vitro culture for 12 h prior to the surgical embryo transfer into the oviducts of synchronous female recipients (Day 1). Pregnancy diagnosis was carried out on Day 30 of gestation by ultrasonography. Survival after cell fusion, cell injection, and embryo transfer were compared by the χ2 test, for P < 0.05. After 16 replications, 1047 in vitro-matured oocytes obtained from 208 does were used for embryo reconstruction (Table 1). In vitro survival was higher in ZI oocytes that were injected with somatic cells than in ZI oocytes or ZF subjected to cell fusion. Pregnancy rates were similar between groups, irrespective of the cell nucleus transfer method or the presence or not of the zona pellucida, but the overall efficiency (fetal survival/recipients) was higher in the ZI-CF group. Currently, 2 ongoing pregnancies carrying 3 cloned concepti from ZI oocytes fused to somatic cells are in late gestation (>110 days). In conclusion, the cell injection method promoted higher survival and, consequently, better efficiency than cell fusion for the reconstruction of goat cloned embryos. However, the zona removal did not affect subsequent in vivo embryo development, as the transfer of zona-free embryos into the oviducts of synchronous recipients resulted in similar pregnancy rates than with zona-intact embryos. Table 1.In vitro survival and pregnancy outcome of goat cloned embryos after embryo reconstruction and transfer to female recipients on Day-1 of development

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.

In order to improve somatic cell nuclear transfer (SCNT) efficiency and to understand cellular changes in SCNT, the dynamic changes in microtubules/DNA and early development of SCNT embryos with single or multiple pronuclei were investigated, along with activation timing on efficiency of SCNT, were studied in the Cynomolgus monkey. The confocal images showed that microtubules assembled around condensed DNA at 1h after cell injection; normal or abnormal reconstructed spindle formed at 2 h after cell injection; and reconstructed spindle separated at 2 h after activation. The results of nuclear formation showed that 61.3% of the reconstructed embryos did not form pronuclei; 19.3% formed a single nucleus, and 11.9% and 7.5% formed two and more than two reconstructed pronuclei, respectively. The cleavage and 8-cell development rates of SCNT embryos with pronuclei were significantly higher than those without pronuclei, but there was no difference in development rates among NT embryos with single, two and more then two pronuclei. Activation at 2 h after cell injection yielded more embryos with pronuclei and yielded 8-cell NT embryos more reliably than did activation at 3-4 h. In conclusion, microtubules assembled around condensed DNA at 1-2 h after cell injection, and formed a spindle at 2 h after SCNT, which separated at 2 h after activation; early development was affected by activation time, but no different between single and multiple pronuclei.

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2-60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

Study question Is it possible to generate genotyped oocytes by utilizing accessible somatic cells in a somatic cell nuclear transfer model? Summary answer Genotyped oocytes generated through haploidization of endometrial somatic cells exhibited reliably completed embryo development and yielded healthy offspring. What is known already The generation of genotyped oocytes through neogametogensis or somatic cell nuclear transfer (SCNT) would be the ultimate solution to treat female infertility due to primary ovarian insufficiency or agonadism. Among the various approaches, somatic cell haploidization appears more feasible (Lee, et al 2022). Unfortunately, this approach has only been successful in a murine model using cumulus cells. However, this donor cell type is not ideal when translated into clinical setting, due to the absence of cumulus cells in patients who are void of oocytes, which arguably is the patient population that would potentially benefit the most from this technique. Study design, size, duration In a mouse SCNT model, endometrial stromal cells (EMSCs) were injected into enucleated oocytes. Cumulus cells (CCs) were used as another source of nuclear donor. Intact MII oocytes were used as control. Post-SCNT oocytes and controls were inseminated by spermatozoa from B6-EGFP mice to monitor paternal genome presence through embryo development and offspring. Embryos were cultured in a time-lapse microscope. To prove gamete competence, blastocysts were transferred into pseudo-pregnant recipients and offspring health was assessed. Participants/materials, setting, methods Endometrial stromal cells were cultured for 4 days and exposed to aphidicolin overnight to synchronize at G1 phase of cell cycle. Cumulus cells were collected after denudation. Oocytes from B6D2F1 mice were enucleated under polarized light microscope. SCNT was performed with CCs or EMSCs by lipofectamine-mediate fusion. Oocytes were inseminated with spermatozoa from GFP-positive transgenic mice. Embryos morphokinesis were assessed by a time-lapse system. Blastocysts were transferred into 2.5-dpc surrogate CD1 mice. Main results and the role of chance Control oocytes(n = 75) fertilized at 93.3%(70/75) and progressed to 2-cell stage at 93.3%(70/75), 8-cell at 93.3%(70/75) and blastocyst at 89.3%(67/75). The control embryos started syngamy at 14.4±1h, reaching the 4-cell stage at 37.3±3h and blastulation at 80.6±8h. A total of 484 oocytes were enucleated, 470(97.1%) survived and underwent SCNT using CCs(n = 85) or EMSCs(n = 385). Fusion rate was 95.5% for CC with a haploidization rate of 70.9%(56/79) that led to a fertilization rate of 64.3%(36/56). The CC cohort developed to 2-cell, 8-cell, and morula at 47.4%, 35.4%, and 22.8%, with a delay in development at syngamy at 19.0±8h(P&amp;lt;0.0001). In the EMSC cohort, fusion rate was 96.0%(361/376). Haploidization rate was 65.4%(236/361), comparable to the CC cohort(P = 0.35). Fertilization was lower in the EMSC cohort at 47.0%(P&amp;lt;0.01) compared to the CC cohort. Subsequently, EMSC cohort yielded similar cleavage rates to 2-cell stage at 30.7%, 8-cell stage at 29.4%, and morula stage at 28.5%. The blastocyst rate was higher in the EMSC cohort at 28.0%(P&amp;lt;0.05). The EMSC cohort yielded similar developmental timing comparing to controls from pronuclear appearance to 4-cell cleavage but a delay manifested afterward at 37.3±3h(P&amp;lt;0.0001). In the EMSC cohort, 42 blastocysts were transferred, yielding 3 healthy pups(2 male,1 female). Limitations, reasons for caution While we generated consistent haploidization and satisfactory fertilization, full pre-implantation development remained low. Even more limited was the live birth rate, which probably related to subtle factors such as an imprinting issue due to insufficient reprogramming of the somatic cell donor. Wider implications of the findings The SCNT model described to generate genotyped oocytes, although still with limited efficiency, represents a feasible reproductive option for women with agonadism and compromised ovarian reserve. This model is still a better approach to complete neogametogenesis. Trial registration number not applicable

Abstract: Cloning by somatic cell nuclear transfer (SCNT) can be used for the conservation of endangered and extinct animals and elite livestock, and their somatic cells have often been cryopreserved as genetic resources. However, viable cells are not always available when individuals are already dead or when their species have extinct. If the intact genome could be retrieved from such animals, cloned animals could be produced by SCNT technology. Recently, we have demonstrated that viable somatic cells can be obtained from certain bovine organs frozen without cryoprotectant for a decade and that viable cloned animals can be produced from the retrieved cells by SCNT technology. In this mini-review, we discuss recent attempts to rescue animal genetic resources from tissues, organs or bodies frozen without cryoprotectant for a long time.

Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n=16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n=69/95 clones v. 96%, n=96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n=14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n=7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.

We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.

The use of somatic cell nuclear transfer (SCNT) to create agricultural and biomedical models is extremely useful for understanding how we can improve the efficiency of production agriculture, as well as how we can better understand the progression of human diseases. Pigs are especially useful for these types of models because of their importance for meat production and their anatomical similarity to humans for the study of disease. Although SCNT is a reliable way to create these models, the efficiency of the process is very low. Since the first pigs created by SCNT were born almost 20 years ago, many attempts have been made in order to increase the efficiency of porcine SCNT with only marginal improvements. Particular focus has been placed on understanding the way in which the somatic nucleus used for the SCNT process can be better primed for remodeling and reprogramming. Metabolism has been shown in many studies to affect the pluripotency of cells, with a switch from anaerobic to aerobic metabolism coinciding with remodeling of the cellular genome and differentiation. The purpose of this literature review is to: 1) understand the previous attempts that have been made to improve SCNT efficiency, 2) highlight research that has allowed better understanding of stem cell qualities by assessing the similarities between cancer cells and embryonic cells, 3) evaluate the role that metabolism plays in the reprogramming of cells to a multipotent state, and 4) discover the capacity that the transcription factor, hypoxia inducible factor (HIF), has to modulate cellular metabolism.

The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri- and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT.