Somatic Cell Nuclear Transfer Technology Research Articles

Generation of RAG2 Knockout Immune-Deficient Miniature Pigs.

Recombination-activating genes (RAGs) play a crucial role in the V(D)J recombination process and the development of immune cells. The development of the immune system and its mechanisms in pigs exhibit greater similarity to those of humans compared to other animals, thus rendering pigs a valuable tool for biomedical research. In this study, we utilized CRISPR/Cas9 gene editing and somatic cell nuclear transfer technology to generate RAG2 knockout (KO) pigs. Furthermore, we evaluated the impact of RAG2 KO on the immune organs and immune cell development through morphological observations, blood analysis and flow cytometry technology. RAG2 KO cell lines were used as donors for cloning. The reconstructed embryos were transplanted into 4 surrogate sows, and after 116 days of gestation, 2 sows gave birth to 12 live piglets, all of which were confirmed to be RAG2 KO. The thymus and spleen sizes of RAG2 KO pigs were significantly smaller than those of wild-type (WT) pigs. Hematoxylin-eosin staining results revealed that the thymus and spleen tissue structures of RAG2 KO pigs were disorganized and lacked the characteristic structures, indicating that RAG2 KO leads to dysplasia of the thymus and spleen. Hematological analysis demonstrated that the total number of white blood cells and lymphocytes in the circulation of RAG2 KO pigs was significantly lower, while the number of eosinophils was higher. Flow cytometry results indicated that the proportions of mature T and B lymphocytes were significantly reduced compared to WT pigs. These findings successfully verified the immunodeficiency phenotype of RAG2 KO pigs. This study may provide experimental animals for the development of tumor models and humanized animals.

Integrating Transcriptomics, Proteomics, and Metabolomics to Investigate the Mechanism of Fetal Placental Overgrowth in Somatic Cell Nuclear Transfer Cattle.

A major factor limiting the development of somatic cell nuclear transfer (SCNT) technology is the low success rate of pregnancy, mainly due to placental abnormalities disrupting the maternal-fetal balance during pregnancy. Although there has been some progress in research on the abnormal enlargement of cloned bovine placenta, there are still few reports on the direct regulatory mechanisms of enlarged cloned bovine placenta tissue. In this study, we conducted sequencing and analysis of transcriptomics, proteomics, and metabolomics of placental tissues from SCNT cattle (n = 3) and control (CON) cattle (n = 3). The omics analysis results indicate abnormalities in biological functions such as protein digestion and absorption, glycolysis/gluconeogenesis, the regulation of lipid breakdown, as well as glycerolipid metabolism, and arginine and proline metabolism in the placenta of SCNT cattle. Integrating these analyses highlights critical metabolic pathways affecting SCNT cattle placenta, including choline metabolism and unsaturated fatty acid biosynthesis. These findings suggest that aberrant expressions of genes, proteins, and metabolites in SCNT placentas affect key pathways in protein digestion, growth hormone function, and energy metabolism. Our results suggest that abnormal protein synthesis, growth hormone function, and energy metabolism in SCNT bovine placental tissues contribute to placental hypertrophy. These findings offer valuable insights for further investigation into the mechanisms underlying SCNT bovine placental abnormalities.

Treatment of Donor Cells with Oxidative Phosphorylation Inhibitor CPI Enhances Porcine Cloned Embryo Development.

Somatic cell nuclear transfer (SCNT) technology holds great promise for livestock industry, life science and human biomedicine. However, the development and application of this technology is limited by the low developmental potential of SCNT embryos. The developmental competence of cloned embryos is influenced by the energy metabolic status of donor cells. The purpose of this study was to investigate the effects of CPI, an oxidative phosphorylation inhibitor, on the energy metabolism pathways of pig fibroblasts and the development of subsequent SCNT embryos. The results showed that treatment of porcine fibroblasts with CPI changed the cellular energy metabolic pathways from oxidative phosphorylation to glycolysis and enhanced the developmental ability of subsequent SCNT embryos. The present study establishes a simple, new way to improve pig cloning efficiency, helping to promote the development and application of pig SCNT technology.

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

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12iMax.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.

Genome editing pig models with elements for controllable gene expression

AbstractConditional gene regulation systems can control gene expression in predefined tissues or organs at a desired time. Site‐specific recombinase systems and chemically induced gene expression systems are the two most widely used approaches for creating genetically modified (GM) animals with conditional regulation of gene expression. Generation of GM pigs with controllable elements, usually involving multiple gene editing, used to be a major challenge due to a lack of germ line‐competent pluripotent stem cells. With the emergence of artificial endonuclease‐mediated gene editors, a variety of GM pigs with recombinase‐specific recognition elements or chemically induced elements for conditional regulation of gene expression have been generated by the combination of site‐directed knock‐in of somatic cells and somatic cell nuclear transfer technology, allowing conditional deletion of endogenous genes or overexpression of exogenous genes in pigs. These inducible tool pig models will greatly facilitate the production of GM pigs and broaden the applications of transgenic pigs in biomedicine and agriculture fields. In this paper, we review the progress in the construction and application of pigs with controllable elements using gene editing techniques.

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.

Interspecific Nuclear Transfer Blastocysts Reconstructed from Arabian Oryx Somatic Cells and Domestic Cow Ooplasm.

Cloning, commonly referred to as somatic cell nuclear transfer (SCNT), is the technique of enucleating an oocyte and injecting a somatic cell into it. This study was carried out with interspecific SCNT technology to clone the Arabian Oryx utilizing the oryx's fibroblast cells and transfer it to the enucleated oocytes of a domestic cow. The recipient oocytes were extracted from the cows that had been butchered. Oryx somatic nuclei were introduced into cow oocytes to produce embryonic cells. The study was conducted on three groups, Oryx interspecific somatic cell nuclear transfer into enucleated oocytes of domestic cows, cow SCNT "the same bovine family species", used as a control group, and in vitro fertilized (IVF) cows to verify all media used in this work. The rates of different embryo developmental stages varied slightly (from 1- cell to morula stage). Additionally, the oryx interspecies Somatic cell nuclear transfer blastocyst developmental rate (9.23%) was comparable to that of cow SCNT (8.33%). While the blastula stage rate of the (IVF) cow embryos exhibited a higher cleavage rate (42%) in the embryo development stage. The results of this study enhanced domestic cow oocytes' ability to support interspecific SCNT cloned oryx, and generate a viable embryo that can advance to the blastula stage.

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

Emerging technologies and ethics—exogenic chimeric humanized organs

Organ transplantation is limited due to the scarcity of donor organs. In order to expand the supply of organs for transplantation, interspecies chimeras have been examined as a potential future source of humanized organs. Recent studies using gene editing technologies in combination with somatic cell nuclear transfer technology and hiPSCs successfully engineered humanized skeletal muscle in the porcine embryo. As these technologies progress, there are ethical issues that warrant consideration and dialogue.

Effects of Crotonylation on Reprogramming of Cashmere Goat Somatic Cells with Different Differentiation Degrees.

Simple SummaryCurrently, not enough is known about the effect of histone modification on the epigenetic reprogramming of somatic cells, and the lack of basic study limits the development of somatic cell nuclear transfer technology. The aim of this study was to explore the influence of lysine crotonylation, a newly discovered histone post-translational modification, on the reprogramming of somatic cells from Cashmere goats. The results showed that the crotonylation level was increased in somatic cells with sodium crotonate treatment. At the same time, the treatment of somatic cells improved the cloned embryo cleavage rate. In conclusion, an increasing crotonylation level could promote the reprogramming of somatic cells and cloned embryo development. This finding provides an important reference for future improvements in the efficiency of in vitro Cashmere goat somatic cell nuclear transfer embryo production.Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr.

Dogs lacking Apolipoprotein E show advanced atherosclerosis leading to apparent clinical complications.

Atherosclerotic cardiovascular disease resulting from dysregulated lipid metabolism is the leading cause of morbidity and mortality worldwide. Apolipoprotein E (ApoE) plays a critical role in cholesterol metabolism. Knockouts in lipid-metabolizing proteins including ApoE in multiple model organisms such as mice and rats exhibiting elevated levels of cholesterol have been widely used for dissecting the pathology of atherosclerosis, but few of these animal models exhibit advanced atherosclerotic plaques leading to ischemia-induced clinical symptoms, limiting their use for translational studies. Here we report hypercholesterolemia and severe atherosclerosis characterized by stenosis and occlusion of arteries, together with clinical manifestations of stroke and gangrene, in ApoE knockout dogs generated by CRISPR/Cas9 and cloned by somatic cell nuclear transfer technologies. Importantly, the hypercholesterolemia and atherosclerotic complications in F0 mutants are recapitulated in their offspring. As the ApoE-associated atherosclerosis and clinical manifestations in mutant dogs are more similar to that in human patients compared with those in other animal models, these mutant dogs will be invaluable in developing and evaluating new therapies, including endovascular procedures, against atherosclerosis and related disorders.

Antioxidant Activity and Anti-Apoptotic Effect of the Small Molecule Procyanidin B1 in Early Mouse Embryonic Development Produced by Somatic Cell Nuclear Transfer.

As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H2O2) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H2O) and oxygen (O2) in order to reduce the harm caused by H2O2. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H2O2 accumulates excessively, it produces radical-(HO-) through Fe2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H2O2 by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

Nuclear Transfer Arrest Embryos Show Massive Dysregulation of Genes Involved in Transcription Pathways.

Somatic cell nuclear transfer (SCNT) technology can reprogram terminally differentiated cell nuclei into a totipotent state. However, the underlying molecular barriers of SCNT embryo development remain incompletely elucidated. Here, we observed that transcription-related pathways were incompletely activated in nuclear transfer arrest (NTA) embryos compared to normal SCNT embryos and in vivo fertilized (WT) embryos, which hinders the development of SCNT embryos. We further revealed the transcription pathway associated gene regulatory networks (GRNs) and found the aberrant transcription pathways can lead to the massive dysregulation of genes in NTA embryos. The predicted target genes of transcription pathways contain a series of crucial factors in WT embryos, which play an important role in catabolic process, pluripotency regulation, epigenetic modification and signal transduction. In NTA embryos, however, these genes were varying degrees of inhibition and show a defect in synergy. Overall, our research found that the incomplete activation of transcription pathways is another potential molecular barrier for SCNT embryos besides the incomplete reprogramming of epigenetic modifications, broadening the understanding of molecular mechanism of SCNT embryonic development.

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine

As a robust antagonist of myostatin (MSTN), follistatin (FST) is an important regulator of skeletal muscle development, and the delivery of FST to muscle tissue represents a potential therapeutic strategy for muscular dystrophies. The N terminus and FSI domain of FST are the functional domains for MSTN binding. Here, we aimed to achieve site-specific integration of FSI-I-I, including the signal peptide, N terminus, and three FSI domains, into the last codon of the porcine MSTN gene using a homology-mediated end joining (HMEJ)-based strategy mediated by CRISPR-Cas9. Based on somatic cell nuclear transfer (SCNT) technology, we successfully obtained FSI-I-I knockin pigs. H&E staining of longissimus dorsi and gastrocnemius cross-sections showed larger myofiber sizes in FSI-I-I knockin pigs than in controls. Moreover, the Smad and Erk pathways were inhibited, whereas the PI3k/Akt pathway was activated in FSI-I-I knockin pigs. In addition, the levels of MyoD, Myf5, and MyoG transcription were upregulated while that of MRF4 was downregulated in FSI-I-I knockin pigs. These results indicate that the FSI-I-I gene mediates skeletal muscle hypertrophy through an MSTN-related signaling pathway and the expression of myogenic regulatory factors. Overall, FSI-I-I knockin pigs with hypertrophic muscle tissue hold great promise as a therapeutic model for human muscular dystrophies.

Functions and Regulation of Endogenous Retrovirus Elements during Zygotic Genome Activation: Implications for Improving Somatic Cell Nuclear Transfer Efficiency.

Endogenous retroviruses (ERVs), previously viewed as deleterious relics of ancestral retrovirus infections, are silenced in the vast majority of cells to minimize the risk of retrotransposition. Counterintuitively, bursts of ERV transcription usually occur during maternal-to-zygotic transition (MZT) in preimplantation embryos; this is regarded as a major landmark event in the zygotic genome activation (ZGA) process, indicating that ERVs play an active part in ZGA. Evolutionarily, the interaction between ERVs and hosts is mutually beneficial. The endogenization of retrovirus sequences rewires the gene regulatory network during ZGA, and ERV repression may lower germline fitness. Unfortunately, owing to various limitations of somatic cell nuclear transfer (SCNT) technology, both developmental arrest and ZGA abnormalities occur in a high percentage of cloned embryos, accompanied by ERV silencing, which may be caused by the activation failure of upstream ERV inducers. In this review, we discuss the functions and regulation of ERVs during the ZGA process and the feasibility of temporal control over ERVs in cloned embryos via exogenous double homeobox (DUX). We hypothesize that further accurate characterization of the ERV-rewired gene regulatory network during ZGA may provide a novel perspective on the development of preimplantation embryos.

25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer.

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985-1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996-2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

Recent advances in critical nodes of embryo engineering technology.

The normal development and maturation of oocytes and sperm, the formation of fertilized ova, the implantation of early embryos, and the growth and development of foetuses are the biological basis of mammalian reproduction. Therefore, research on oocytes has always occupied a very important position in the life sciences and reproductive medicine fields. Various embryo engineering technologies for oocytes, early embryo formation and subsequent developmental stages and different target sites, such as gene editing, intracytoplasmic sperm injection (ICSI), preimplantation genetic diagnosis (PGD), and somatic cell nuclear transfer (SCNT) technologies, have all been established and widely used in industrialization. However, as research continues to deepen and target species become more advanced, embryo engineering technology has also been developing in a more complex and sophisticated direction. At the same time, the success rate also shows a declining trend, resulting in an extension of the research and development cycle and rising costs. By studying the existing embryo engineering technology process, we discovered three critical nodes that have the greatest impact on the development of oocytes and early embryos, namely, oocyte micromanipulation, oocyte electrical activation/reconstructed embryo electrofusion, and the in vitro culture of early embryos. This article mainly demonstrates the efforts made by researchers in the relevant technologies of these three critical nodes from an engineering perspective, analyses the shortcomings of the current technology, and proposes a plan and prospects for the development of embryo engineering technology in the future.