Somatic Cell Nuclear Transfer Research Articles

Testosterone Supplementation Promotes Estrogen Synthesis of Buffalo Cumulus Cells Surrounding In Vitro-Matured Oocytes.

Cumulus cells (CCs) synthesize estrogens that are essential for follicular development. However, the effects of androgen on estrogen production in buffalo CCs remain unknown. In the present study, the impacts of testosterone on estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes were investigated. The results showed that testosterone supplementation improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 17β-HSD) and the secretion levels of estradiol in buffalo CCs surrounding in vitro-matured oocytes. Furthermore, testosterone treatment enhanced the sensitivity of buffalo CCs surrounding in vitro-matured oocytes to follicle-stimulating hormone (FSH). This study indicated that testosterone supplementation promoted the estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes mainly through strengthening the responsiveness of CCs to FSH. The present study serves as a foundation of acquiring high-quality recipient oocytes for buffalo somatic cell nuclear transfer.

Current status of interspecies somatic cell nuclear transfer and meta‐analysis of the effects of phylogenetic distance on embryonic and fetal development

Abstract Interspecies somatic cell nuclear transfer (iSCNT) has the capacity to rescue endangered species, ethically produce human embryonic stem cells, and allow for more specific analysis of pre‐implantation embryos. However, several obstacles negatively affect iSCNT efficiency. Over 100 iSCNT studies have been published between 1999 and the spring of 2023. This review details the possible benefits of iSCNT, describes the hypothesized issues that prevented the development of some embryos and/or live offspring from developing; and analyzes the cleavage, blastocyst, pregnancy, live birth, and iSCNT efficiency rates based on published data. Embryos produced by iSCNT may help with the rescue of endangered species and the production of human embryonic stem cells with the use of oocytes from domestic species, as well as in‐depth analyses of embryonic genome activation (EGA) and mRNA transcripts. But the combination of the cells of two species in a single embryo presents several challenges: lack of EGA synchronisation, presence of mitochondrial DNA (mtDNA) from both species in the embryo, requirement of cell and embryo culture optimization for each species combination, lack of embryo transfer data, and an extremely low overall efficiency in terms of producing live offspring. Additional research on the aforementioned topics is warranted to increase iSCNT efficiency.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200µm and a spacing of 150µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

Bta-miR-301a targets ACVR1 to influence cleavage time and blastocyst formation rate of early embryos in cattle.

Accumulating evidence indicates that paternally-derived miRNAs play a crucial role in the development of early embryos and are regarded as the key factor in the successful development of somatic cell cloned embryos. In our previous study, bta-miR-301a was found to be highly expressed in bovine sperm, and was delivered into oocytes during fertilization. In this study, bioinformatics, dual luciferase reporter assays, rescue experiments and gain- and loss-of-function experiments indicated that ACVR1 is the target gene of bta-miR-301a in early bovine embryos. By microinjecting bta-miR-301a mimic into embryos of parthenogenetic or somatic cell nuclear transfer, we observed that bta-miR-301a prolonged the first cleavage time of the embryos and increased the blastocyst formation rate. Thus, this study provides preliminary evidence that bta-miR-301a influences remodeling of the microfilament skeleton, prolongs the first cleavage time, and improves the developmental competence of embryos by negatively regulating ACVR1 translation.

Monitoring swine virus transmission in embryos derived from commercial abattoir oocytes.

Pigs are pivotal in agriculture and biomedical research and hold promise for xenotransplantation. Specific-pathogen-free (SPF) herds are essential for commercial swine production and xenotransplantation research facilities. Commercial herds aim to safeguard animal health, welfare, and productivity, and research facilities require SPF status to protect immunocompromised patients. Somatic cell nuclear transfer (SCNT) embryos are the norm for producing cloned and genetically edited animals. Oocytes for embryo reconstruction are most conveniently sourced from commercial abattoirs with unclear disease statuses. However, research on viral clearance from donor oocytes during embryo reconstruction remains limited. SCNT has previously been shown to reduce the transmission of Porcine reproductive and respiratory syndrome virus, Bovine viral diarrhea virus, Porcine Circovirus type 2, and Porcine parvovirus. Still, it is lacking for other pathogens, including endogenous viruses. This project contains two preliminary studies investigating the polymerase chain reaction (PCR) assay detection of common swine viruses through the phases of producing parthenogenic and SCNT embryos. Exogenous pathogens detected in oocyte donor tissue or the oocyte maturation media were not detected in the produced embryos. Porcine endogenous retrovirus type C (PERVC) was not removed by parthenogenic embryo activation and was detected in 1 of the 2 tested SCNT embryos reconstructed using a PERVC-negative cell line. SCNT and parthenogenic embryo construction similarly reduced exogenous virus detection. SCNT embryo construction helped reduce endogenous virus detection. This project demonstrates the importance of screening embryos for endogenous viruses and shows the usefulness of parthenogenic embryos in future exogenous virus clearance studies.

Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.

Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24–48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24–48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

Cloned Foal Born from Postmortem-Obtained Ear Sample Refrigerated for 5 Days Before Fibroblast Isolation and Decontamination of the Infected Monolayer Culture.

A 6-year-old mare, a valuable polo horse, died of complications following postcolic surgery. To preserve its genetics, ear skin samples were collected immediately after death and stored in an equine embryo transfer medium at 4°C for 5 days. After trypsin digestion, monolayer fibroblast cultures were established, but signs of massive bacterial infection were found in all of them. As an ultimate attempt for rescue, rigorously and repeatedly washed cells were individually cultured in all wells of four 96-well dishes. New monolayers were established from the few wells without contamination and used for somatic cell nuclear transfer. Four of the six Day 7 blastocysts derived from 14 reconstructed zygotes were transferred in four naturally cycling mares on Day 5 after ovulation. The embryo transfers resulted in 2 pregnancies, one from a fresh and one from a vitrified blastocyst. The vitrified embryo transfer resulted in a healthy offspring, now 21 months old, genetically and phenotypically identical to the somatic cell donor animal.

Reprogramming mechanism dissection and trophoblast replacement application in monkey somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) successfully clones cynomolgus monkeys, but the efficiency remains low due to a limited understanding of the reprogramming mechanism. Notably, no rhesus monkey has been cloned through SCNT so far. Our study conducts a comparative analysis of multi-omics datasets, comparing embryos resulting from intracytoplasmic sperm injection (ICSI) with those from SCNT. Our findings reveal a widespread decrease in DNA methylation and the loss of imprinting in maternally imprinted genes within SCNT monkey blastocysts. This loss of imprinting persists in SCNT embryos cultured in-vitro until E17 and in full-term SCNT placentas. Additionally, histological examination of SCNT placentas shows noticeable hyperplasia and calcification. To address these defects, we develop a trophoblast replacement method, ultimately leading to the successful cloning of a healthy male rhesus monkey. These discoveries provide valuable insights into the reprogramming mechanism of monkey SCNT and introduce a promising strategy for primate cloning.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

Determination of the Copy Number of Porcine Endogenous Retroviruses (PERV) in Auckland Island Pigs Repeatedly Used for Clinical Xenotransplantation and Elimination of PERV-C.

Auckland Island pigs represent an inbred population of feral pigs isolated on the sub-Antarctic island for over 100 years. The animals have been maintained under pathogen-free conditions in New Zealand; they are well characterized virologically and have been used as donor sources in first clinical trials of porcine neonatal islet cell transplantation for the treatment of human diabetes patients. The animals do not carry any of the xenotransplantation-relevant viruses, and in the first clinical trials, no porcine viruses, including porcine endogenous retroviruses (PERVs) were transmitted to the human recipients. PERVs pose a special risk in xenotransplantation, since they are part of the pig genome. When the copy number of PERVs in these animals was analyzed using droplet digital PCR and primers binding to a conserved region of the polymerase gene (PERVpol), a copy number typical for Western pigs was found. This confirms previous phylogenetic analyses of microsatellites as well as mitochondrial analyses showing a closer relationship to European pigs than to Chinese pigs. When kidney cells from very young piglets were analyzed, only around 20 PERVpol copies were detected. Using these cells as donors in somatic cell nuclear transfer (SCNT), animals were born showing PERVpol copy numbers between 35 and 56. These data indicate that Auckland Island pigs have a similar copy number in comparison with other Western pig breeds and that the copy number is higher in adult animals compared with cells from young piglets. Most importantly, PERV-C-free animals were selected and the absence of an additional eight porcine viruses was demonstrated.

Incomplete reprogramming of DNA replication timing in induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are the foundation of cell therapy. Differences in gene expression, DNA methylation, and chromatin conformation, which could affect differentiation capacity, have been identified between iPSCs and embryonic stem cells (ESCs). Less is known about whether DNA replication timing, a process linked to both genome regulation and genome stability, is efficiently reprogrammed to the embryonic state. To answer this, we compare genome-wide replication timing between ESCs, iPSCs, and cells reprogrammed by somatic cell nuclear transfer (NT-ESCs). While NT-ESCs replicate their DNA in a manner indistinguishable from ESCs, a subset of iPSCs exhibits delayed replication at heterochromatic regions containing genes downregulated in iPSCs with incompletely reprogrammed DNA methylation. DNA replication delays are not the result of gene expression or DNA methylation aberrations and persist after cells differentiate to neuronal precursors. Thus, DNA replication timing can be resistant to reprogramming and influence the quality of iPSCs.

Reproductive ability of minipigs as surrogates for somatic cell nuclear transfer.

Pigs are genetically, anatomically, and physiologically similar to humans. Recently, pigs are in the spotlight as a suitable source animal for xenotransplantation. However, to use pigs as source animals, pigs should be raised in designated pathogen-free facilities. There is abundant data from embryo transfer (ET) experiments using farm pigs as surrogates, but data on ET experiments using minipigs are scarce. Eighty minipigs were used for ET experiments and after transplantation, the implantation and delivery rates were investigated. It was also confirmed whether the pregnancy rate could be increased by changing the condition or surgical method of the surrogate. In the case of minipigs that gave birth, the size of the fetal sac on the 28th day of ET was also measured. The factors that can affect the pregnancy rate such as estrus synchronization program, ovulation status at the time of ET, the number of repeated ET surgeries, and the ET sites, were changed, and the differences on the pregnancy rate were observed. However there were no significant differences in pregnancy rate in minipigs. The diameter of the implanted fetal sac on the 28th day after ET in the minipigs whose delivery was confirmed was calculated to be 4.7 ± 0.5 cm. In conclusion, there were no significant differences in pregnancy rate of minipigs in the comparative experiment on various factors affecting the pregnancy rate. However, additional experiments and analyses are needed due to the large individual differences of the minipigs.

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

Piperine improves the quality of porcine oocytes by reducing oxidative stress

Oxidative stress caused by light and high temperature arises during in vitro maturation (IVM), resulting in low-quality embryos compared with those obtained in vivo. To overcome this problem, we investigated the influence of piperine (PIP) treatment during maturation of porcine oocytes on subsequent embryo development in vitro. Porcine oocytes were cultured in IVM medium supplemented with 0, 50, 100, 200, or 400 μM PIP. After parthenogenetic activation, the blastocyst (BL) formation was significantly higher and the apoptosis rate was significantly lower using 200 μM PIP-treated oocytes (200 PIP). In the 200 PIP group, the level of reactive oxygen species at the metaphase II stage was decreased, accompanied by an increased level of glutathione and increased expression of antioxidant processes (Nrf2, CAT, HO-1, SOD1, and SOD2). Consistently, chromosome misalignment and aberrant spindle organization were alleviated and phosphorylated p44/42 mitogen-activated protein kinase activity was increased in the 200 PIP group. Expression of development-related (CDX2, NANOG, POU5F1, and SOX2), anti-apoptotic (BCL2L1 and BIRC5), and pro-apoptotic (BAK, FAS, and CASP3) processes was altered in the 200 PIP group. Ultimately, embryo development was improved in the 200 PIP group following somatic cell nuclear transfer. These findings suggest that PIP improves the quality of porcine oocytes by reducing oxidative stress, which inevitably arises via IVM. In-depth mechanistic studies of porcine oocytes will improve the efficiencies of assisted reproductive technologies.

DNA methylation regulates RNA m6 A modification through transcription factor SP1 during the development of porcine somatic cell nuclear transfer embryos.

Epigenetic modifications play critical roles during somatic cell nuclear transfer (SCNT) embryo development. Whether RNA N6-methyladenosine (m6 A) affects the developmental competency of SCNT embryos remains unclear. Here, we showed that porcine bone marrow mesenchymal stem cells (pBMSCs) presented higher RNA m6 A levels than those of porcine embryonic fibroblasts (pEFs). SCNT embryos derived from pBMSCs had higher RNA m6 A levels, cleavage, and blastocyst rates than those from pEFs. Compared with pEFs, the promoter region of METTL14 presented a hypomethylation status in pBMSCs. Mechanistically, DNA methylation regulated METTL14 expression by affecting the accessibility of transcription factor SP1 binding, highlighting the role of the DNA methylation/SP1/METTL14 pathway in donor cells. Inhibiting the DNA methylation level in donor cells increased the RNA m6 A level and improved the development efficiency of SCNT embryos. Overexpression of METTL14 significantly increased the RNA m6 A level in donor cells and the development efficiency of SCNT embryos, whereas knockdown of METTL14 suggested the opposite result. Moreover, we revealed that RNA m6 A-regulated TOP2B mRNA stability, translation level, and DNA damage during SCNT embryo development. Collectively, our results highlight the crosstalk between RNA m6 A and DNA methylation, and the crucial role of RNA m6 A during nuclear reprogramming in SCNT embryo development.

Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.

Ellagic acid treatment during in vitro maturation of porcine oocytes improves development competence after parthenogenetic activation and somatic cell nuclear transfer

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 μM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

157 Production of β-lactoglobulin gene knockout blastocyst stage embryos of Indian water buffalo using CRISPR and somatic cell nuclear transfer technology

157 Production of β-lactoglobulin gene knockout blastocyst stage embryos of Indian water buffalo using CRISPR and somatic cell nuclear transfer technology

18 Development of interspecies somatic cell nuclear transfer (iSCNT) caprine-bovine embryos injected with demethylase mRNA and mitochondrial extract

18 Development of interspecies somatic cell nuclear transfer (iSCNT) caprine-bovine embryos injected with demethylase mRNA and mitochondrial extract