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.

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.

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.

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 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.

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.

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.

Somatic Cell Nuclear Transfer in Pigs.

Somatic cell nuclear transfer (SCNT) has been successfully applied to clone animals of several species. Pigs are one of the main livestock species for food production and are also important for biomedical research due to their physiopathological similarities with humans. In the past 20 years, clones of several swine breeds have been produced for a variety of purposes, including biomedical and agricultural applications. In this chapter, we describe a protocol to produce cloned pigs by SCNT.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

Production of Water Buffalo SCNT Embryos by Handmade Cloning.

Cloning by somatic cell nuclear transfer (SCNT) involves the transfer of a somatic nucleus into an enucleated oocyte followed by chemical activation and embryo culture. Further, handmade cloning (HMC) is a simple and efficient SCNT method for large-scale embryo production. HMC does not require micromanipulators for oocyte enucleation and reconstruction since these steps are carried out using a sharp blade controlled by hand under a stereomicroscope. In this chapter, we review the status of HMC in the water buffalo (Bubalus bubalis) and further describe a protocol for the production of buffalo-cloned embryos by HMC and assays to estimate their quality.

Horse Somatic Cell Nuclear Transfer Using Zona Pellucida-Enclosed and Zona-Free Oocytes.

Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.

Effects of epigenetic modifier on the developmental competence and quantitative expression of genes in male and female buffalo (Bubalus bubalis) cloned embryos.

Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2'-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was higher (P < 0.05) and that of PGK, G6PD, OCT 4, IFN-tau and CASPASE3 was significantly lower (P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1, IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher (P < 0.05), and that of XIST and CASPASE3 was significantly lower (P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT, PGK, G6PD, DNMT1, IFN-tau, IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.

Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.