Optimization of handmade cloning technique in sheep and preliminary investigation of umbilical cord mesenchymal stem cells as donor nuclei.

Handmade cloning (HMC) has a higher yield and is relatively less difficult to operate compared to traditional micromanipulation cloning. Yet, there are few reports on handmade cloning in sheep. Therefore, this study investigates the key nodes such as AC and DC voltage, denucleation method and fusion method in sheep handmade cloning. In addition, it compares the effects of fibroblasts (FC) and umbilical cord mesenchymal stem cells (UC-MSCs) of different states as donors on the development of HMC embryos. Furthermore, the effect of different freezing solutions on the survival rate of frozen blastocysts without zona pellucida was also investigated. The results indicate that an AC voltage of 150 V/cm and a DC voltage of 1800 V/cm significantly enhanced the fusion and blastocyst rates (p < .01). The blastocyst rate achieved with umbilical cord MSCs as nucleus donors was significantly higher (40.3%) than that achieved with fibroblasts and differentiated umbilical cord MSCs (21.5%, 22.5%) (p < .01). The highest survival rate was achieved using 20% DMSO + 20% EG for freezing without zona pellucida. In conclusion, the most efficient and pregnant ovine HMC cloning method using 150 V/cm AC, 1800 V/cm DC, knife-cut denucleation, two-step fusion and the use of UC-MSCs as nucleus donors resulted in the highest overall efficiency and pregnancy after transplantation.

Improved surrogate eggshell incubation system for assisted reproductive technologies such as cloning, genome modification, and de-extinction efforts.

A germinal disc located on the egg yolk surface drives genetic modification. Windowed and surrogate eggshell incubation methods have been developed, but these exhibit limited abilities to generate transgenic chickens. In the present study, we investigated the frequency of observing the germinal disc according to the preincubation positioning direction and time and found that it depended on those conditions, but only a few chicks (2.8-5.6%) hatched using the windowed method. Then, we attempted to improve surrogate method via one- or two-step procedures. All eggs, including surrogates that were 10 g heavier than the donor eggs, were obtained from a poultry flock of the same age. With the one-step surrogate method, where the donor egg was transferred only once through a 3.5 cm hole on the point end, at the first day of preincubation, into the surrogate egg, the survival rate at day 4 was 30.8%, and the hatching rate was 11.8%. With the two-step surrogate method (transfer was on the 1st and 4th day of incubation), the survival rate at day 4 was improved to 90.7%, and a hatching rate of 70.0% was achieved. Therefore, this method can be effective for in ovo artificial incubation.

Involvement of METTL3-mediated m6A methylation in the early development of porcine cloned embryos.

METTL3-mediated N6-methyladenosine (m6A) modification is critical for gametogenesis and early embryonic development. However, the function of METTL3-mediated m6A modification in the early development of somatic nuclear transfer embryos (SCNT) remains unclear. Here, we found that METTL3 mRNA and protein levels exhibit dynamic changes during the early development of porcine SCNT embryos. The levels of METTL3 mRNA and protein in SCNT embryos at specific developmental stages differ from those in parthenogenetic activation (PA) counterparts. SiRNA injection effectively reduced the levels of METTL3 mRNA and protein in 4-cell embryos and blastocysts. METTL3 knockdown significantly reduced the cleavage and blastocyst rates of SCNT embryos. METTL3 knockdown significantly reduced the number of total cells and trophectoderm (TE) cells in the resulting blastocysts and perturbed cell lineage allocation. In addition, METTL3 knockdown reduced the levels of m6A modification in 4-cell embryos and blastocysts. Importantly, METTL3 knockdown decreased the expression levels of CDX2, GATA3, NANOG and YAP, and increased the expression levels of SOX2 and OCT4. Taken together, these results demonstrate that METTL3-mediated m6A modification regulates early development and lineage differentiation of porcine SCNT embryos.

A retrospective analysis of sheep generated by somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24-26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15-30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

LEUTX regulates porcine embryonic genome activation in somatic cell nuclear transfer embryos.

Emerging evidence highlights the regulatory role of paired-like (PRD-like) homeobox transcription factors (TFs) in embryonic genome activation (EGA). However, the majority of PRD-like genes are lost in rodents, thus prompting an investigation into PRD-like TFs in other mammals. Here, we showed that PRD-like TFs were transiently expressed during EGA in human, monkey, and porcine fertilized embryos, yet they exhibited inadequate expression in their cloned embryos. This study, using pig as the research model, identified LEUTX as a key PRD-like activator of porcine EGA through genomic profiling and found that LEUTX overexpression restored EGA failure and improved preimplantation development and cloning efficiency in porcine cloned embryos. Mechanistically, LEUTX opened EGA-related genomic regions and established histone acetylation via recruiting acetyltransferases p300 and KAT2A. These findings reveal the regulatory mechanism of LEUTX to govern EGA in pigs, which may provide valuable insights into the study of early embryo development for other non-rodent mammals.

Reduction of H3K9 methylation by G9a inhibitors improves the development of mouse SCNT embryos.

Removal of somatic histone H3 lysine 9 trimethylation (H3K9me3) from the embryonic genome can improve the efficiency of mammalian cloning using somatic cell nuclear transfer (SCNT). However, this strategy involves the injection of histone demethylase mRNA into embryos, which is limiting because of its invasive and labor-consuming nature. Here, we report that treatment with an inhibitor of G9a (G9ai), the major histone methyltransferase that introduces H3K9me1/2 in mammals, greatly improved the development of mouse SCNT embryos. Intriguingly, G9ai caused an immediate reduction of H3K9me1/2, a secondary loss of H3K9me3 in SCNT embryos, and increased the birth rate of cloned pups about 5-fold (up to 3.9%). G9ai combined with the histone deacetylase inhibitor trichostatin A further improved this rate to 14.5%. Mechanistically, G9ai and TSA synergistically enhanced H3K9me3 demethylation and boosted zygotic genome activation. Thus, we established an easy, highly effective SCNT protocol that would enhance future cloning research and applications.

Production of cleavage-resistant phytase transgenic pigs by handmade cloning.

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry.

High Expression of ZFP42 Improves Early Development of Pig Embryos Produced by Handmade Cloning.

Handmade Cloning (HMC) is a pivotal technique for cloning pig embryos. Despite its significance, the low efficiency of this method hampers its widespread application. Although numerous factors and signaling pathways influencing embryo development have been studied, the mechanisms underlying low developmental capacity and insufficient reprogramming of cloned embryos remain elusive. In the present study, we sought to elucidate key regulatory factors involved in the development of pig HMC embryos by comparing and analyzing the gene expression profiles of HMC embryos with those of naturally fertilized (NF) embryos at the 4-cell, 8-cell, and 16-cell stages. The results showed that ZFP42 expression is markedly higher in NF embryos than in cloned counterparts. Subsequent experiments involving the injection of ZFP42 messenger RNA (mRNA) into HMC embryos showed that ZFP42 could enhance the blastocyst formation rate, upregulate pluripotent genes and metabolic pathways. This highlights the potential of ZFP42 as a critical factor in improving the development of pig HMC embryos.

Efficient generation of cloned cats with altered coat colour by editing of the KIT gene.

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.

The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and somatic cloned embryo development.

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial ß-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm.

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.

Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Astaxanthin enhances the development of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes.

Low cloning efficiency limits the wide application of somatic cell nuclear transfer technology. Apoptosis and incomplete DNA methylation reprogramming of pluripotency genes are considered as the main causes for low cloning efficiency. Astaxanthin (AST), a powerfully antioxidative and antiapoptotic carotenoid, is recently shown to improve the development of early embryos, however, the potential role of AST during the development of cloned embryos remains unclear. This study displayed that treating cloned embryos with AST significantly increased the blastocyst rate and total blastocyst cell number in a concentration dependent manner, and also alleviated the damage of H2O2 to the development of cloned embryos. In addition, compared with the control group, AST significantly reduced the apoptotic cell number and rate in cloned blastocysts, and the significantly upregulated expression of anti-apoptotic gene Bcl2l1 and antioxidative genes (Sod1 and Gpx4) and downregulated transcription of pro-apoptotic genes (Bax, P53 and Caspase3) were observed in the AST group. Moreover, AST treatment facilitated DNA demethylation of pluripotency genes (Pou5f1, Nanog and Sox2), in accompany with the improved transcription levels of DNA methylation reprogramming genes (Tet1, Tet3, Dnmt1, Dnmt3a and Dnmt3b) in cloned embryos, and then, the significantly upregulated expression levels of embryo development related genes including Pou5f1, Nanog, Sox2 and Cdx2 were observed in comparison with the control group. In conclusion, these results revealed that astaxanthin enhanced the developmental potential of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes, and provided a promising approach to improve cloning efficiency.

Blastocyst Cell Number and Allocation Affect the Developmental Potential and Transcriptome of Bovine Somatic Cell Nuclear Transfer Embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. Although the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117 ± 6 total cells, whereas embryos with an average of 128 ± 5 cells did not establish pregnancy (P < 0.05). A lesser average number of 41 ± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (<0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then subjected to RNA sequencing for transcriptome analysis. Using weighted gene coexpression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology analysis of these clusters revealed enriched biological processes in coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total and ICM cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling.

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

Cloning in action: can embryo splitting, induced pluripotency and somatic cell nuclear transfer contribute to endangered species conservation?

The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.

Mouse Cloning Using Outbred Oocyte Donors and Nontoxic Reagents.

Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprogramming research. However, the standard mouse SCNT protocol remains expensive, labor-intensive, and requires hard work for many hours. Therefore, we have been trying to reduce the cost and simplify the mouse SCNT protocol. This chapter describes the methods to use low-cost mouse strains and steps from the mouse cloning procedure. Although this modified SCNT protocol will not improve the success rate of mouse cloning, it is a cheaper, simpler, and less tiring method that allows us to perform more experiments and obtain more offspring with the same working time as the standard SCNT protocol.

Somatic Cell Nuclear Transfer in Rabbits.

Somatic cell nuclear transfer (SCNT) is a technology that enables differentiated somatic cells to acquire a totipotent state, thus making it of great value in developmental biology, biomedical research, and agricultural applications. Rabbit cloning associated with transgenesis has the potential to improve the applicability of this species for disease modeling, drug testing, and production of human recombinant proteins. In this chapter, we introduce our SCNT protocol for the production of live cloned rabbits.

Production of Cloned Pigs by Handmade Cloning.

Somatic cell nuclear transfer (SCNT) in pigs is a promising technology in biomedical research by association with transgenesis for xenotransplantation and disease modeling technologies. Handmade cloning (HMC) is a simplified SCNT method that does not require micromanipulators and facilitates the generation of cloned embryos in large quantities. As a result of HMC fine-tuning for porcine-specific requirements of both oocytes and embryos, HMC has become uniquely efficient (>40% blastocyst rate, 80-90% pregnancy rates, 6-7 healthy offspring per farrowing, and with negligible losses and malformations). Therefore, this chapter describes our HMC protocol to obtain cloned pigs.