Current Progress and Prospects in Rabbit Cloning

Abstract

Somatic cell nuclear transfer (SCNT) shows great value in the generation of transgenic animals, protection of endangered animals, and stem cell therapy. The combination of SCNT and gene editing has produced a variety of genetically modified animals for life science and medical research. Rabbits have unique advantages as transgenic bioreactors and human disease models; however, the low SCNT efficiency severely impedes the application of this technology. The difficulty in SCNT may be attributable to the abnormal reprogramming of somatic cells in rabbits. This review focuses on the abnormal reprogramming of cloned mammalian embryos and evaluates the progress and prospects of rabbit somatic cell cloning.

Introduction

Somatic cell nuclear transfer (SCNT) is the process by which a nucleus from a differentiated somatic cell is transferred into an enucleated oocyte, resulting in the development of a cloned individual through remodeling, activation, culture, and embryo transfer (Wilmut et al., 2015). In the research of first successful cloned animal, mammary epithelial cells of sheep were transferred into enucleated oocytes by electrofusion to obtain reconstructed embryos, and embryos at morulae or blastocyst stage were implanted into the uteruses of surrogate sheep, and then the first cloned mammal, Dolly, was born successfully (Wilmut et al., 1997).

Since then, more than 20 species of mammals have been successfully cloned, including cattle, mice, pigs, rabbits, rats, dogs, monkeys, and so on (Betthauser et al., 2000; Challah-Jacques et al., 2003; Chesné et al., 2002; Heyman et al., 1998; Lee et al., 2005; Liu et al., 2018; Onishi et al., 2000; Polejaeva et al., 2000; Wakayama et al. 1998; Wilmut et al., 2015; Zhou et al., 2003). SCNT is of great value in the construction of animal models of diseases, the rescue of endangered species, and therapeutic cloning (Matoba and Zhang, 2018; Mrowiec et al., 2021).

However, the cloning efficiency is very low in some species including rabbits, which hinders its application, and the main reason for this was owing to the abnormal reprogramming of SCNT embryos (Keefer, 2015). In fact, many pioneering achievements in early reproductive biology came from rabbits, which are considered as an ideal model for studying embryonic development (Fischer et al., 2012). In this study, we mainly review the progress and prospects of rabbit somatic cell cloning in detail.

Reprogramming of SCNT Embryo

During the process of SCNT, after the somatic cell nucleus is transferred into the enucleated oocyte, the nuclear membrane breaks down to form a pseudo-nucleus (Challah-Jacques et al., 2003). Under the control of reprogramming factors from the oocyte, the original epigenetic modifications of the somatic cell nucleus could not be completely erased and reprogrammed, resulting in abnormal gene expression and dysfunctional fetal and placental development (Sepulveda-Rincon et al., 2016).

Histone modification

Histone modification plays an important role in nuclear reprogramming. Previous studies reported that some regions enriched histone modification in donor cell DNA were resistant to reprogramming (Matoba et al., 2014). The abnormal histone H3 lysine 9 trimethylation (H3K9me3) modification of SCNT embryos, which impeded the expression of genes, caused failure of zygotic gene activation in SCNT embryos (Matoba et al., 2014). Researchers improved the cloning efficiency by injecting histone lysine demethylase 4D mRNA into the SCNT embryos, which resulted in the expression of the demethylase and removal of the methylation modification at H3K9me3, and improved cloning efficiency (Feng et al., 2021). Abnormal H3K4me3 levels in SCNT embryos can lead to the four-cell block (Zhang et al., 2016).

Maternal H3K27me3 can regulate DNA methylation, whereas abnormal H3K27me3 levels in SCNT embryos lead to disordered expression of imprinted genes (Matoba et al., 2018). The acetylation of H3 and H4 causes the chromatin to assume a relatively open state, and studies have shown that targeting histone acetylation can improve the efficiency of SCNT (Sawai et al., 2012; Yamanaka et al., 2009). For rabbit cloning, treatment of rabbit donor cells with NaBU (histone deacetylase inhibitor [HDACi]) could increase the level of acH3K9/14 in early embryos and increase blastocyst development rate (Yang et al., 2007). The acetylation levels of H3K14/12/5 in SCNT embryos can be adjusted to be more similar to that of fertilized embryos by treatment with the HDACi, trichostatin A (TSA), which benefits for SCNT embryo developmental competence (Chen et al., 2013; Meng et al., 2009; Shi et al., 2008; Sugimoto et al., 2015).

DNA methylation

Compared with fertilized embryos, the overall DNA methylation level of SCNT embryos is relatively higher. The DNA methylation level of cloned embryos at the one-cell stage is almost the same as that of the donor cell (Gao et al., 2018). Targeting this abnormal DNA methylation is a reliable way to improve the efficiency of SCNT (Gao et al., 2018). For example, the ten-eleven translocation protein 3 (TET3) can induce DNA demethylation and gene regulation at the epigenetic level by converting 5-methylcytosine to 5-hydroxymethylcytosine. Overexpressing TET3 in donor cells could improve the efficiency of SCNT (Zhang et al., 2020). Introduction of siRNAs against DNA methyltransferases prevents remethylation defects and improves the developmental competence of early embryos (Cao et al., 2019; Liang et al., 2018; Yamanaka et al., 2010). DNA methylation patterns of SCNT embryos have been well described in mice, cattle, and pigs, but less in rabbits.

Transcriptional memory

Transcriptional memory acts as a reprogramming barrier for SCNT embryo development, and transcriptional profiles of cloned embryos retain some features of those in the donor cell, which demonstrates the existence of active and silent memory genes inherited from donor cells in early SCNT embryos (Eilertsen et al., 2007). As the previous study reported, a nucleus of an endodermal cell expressing the gene edd (an endoderm-specific marker) was transferred into an enucleated egg, and then edd was stably expressed in nonendoderm cells and the edd gene expression remained after the donor cell was treated with 5-aza-deoxycytidine, a powerful demethylating agent (Ng and Gurdon, 2005).

Specific transcription profiles of transcriptional memory genes of mice, bovine, and porcine SCNT reprogramming have been revealed and pointed that transcriptional memory was responsible for the low efficiency of nuclear reprogramming (Liu et al., 2020; Zhou et al., 2020). Although studies on the effects of transcriptional memory on clone reprogramming in rabbits are not reported, studies of other animals seem to indicate that repairing abnormal transcriptional reprogramming of SCNT embryos might be a new strategy for promoting nuclear reprogramming efficiency of rabbits.

Role of sperm in reprogramming

Sperm-derived regulators play an important role in cytoskeletal remodeling and nuclear reprogramming (Qu et al., 2020a). The lack of sperm-derived regulators may be a key reason for the low efficiency of SCNT. Studies have proved that SCNT embryos had abnormal and earlier first cleavage times compared with IVF embryos. Injecting miR-202, a specifically and highly expressed miRNA in bovine sperm, into SCNT embryos can delay the first cleavage and enhance the developmental potential of embryos by downregulating the expression of the SEPT7 gene (Wang et al., 2021). In addition, miR-34c and miR-449b specifically expressed in sperm could improve the developmental competence of SCNT embryos by regulating the levels of α-tubulin K40 acetylation and H3K9me3 (Qu et al., 2019).

The importance of sperm source regulatory factors has also been partially proven in rabbit SCNT embryos. Injecting miRNAs derived from sperm into SCNT embryos can significantly decrease the level of H3K9me3 and increase the total number of cells in the rabbit SCNT blastocysts (Qin et al., 2021).

Spermatogenesis is largely conserved from flies to mammals. During spermatogenesis, protamine replaces the vast majority of the nuclear histone content and forms a highly concentrated DNA protamine complex, which maintains DNA in a stable nontranscriptional state until fertilization. The level of H3K9me3 can be reduced by transient transfection with protamine, resulting in a sperm-like nuclear shape (Czernik et al., 2016; Iuso et al., 2015). Sperm-specific and crucial RNA, proteins, or other epigenetic factors should be screened, and a regulatory network of these sperm factors for embryo development needs to be constructed to further improve the efficiency of SCNT.

SCNT Protocol Optimization

Much researches related to abnormal reprogramming have been carried out with beneficial effects on SCNT, but in fact, few of the findings have been widely used in commercial applications, and the SCNT protocols for most species are similar. From the first cloned animal until now, many improvements in SCNT protocol have been made; but the procedure still consists of enucleation of the oocyte, injection of donor cell nucleus, reconstruction of enucleated oocyte and donor cell, activation, in vitro culture of embryo, and embryo transplantation.

Donor cell type and cell cycle synchrony

The effect of various factors about donor cells on the developmental potential of SCNT embryos has received attention. There is still little agreement on what constitutes the most suitable cell type. As donors, cumulus cells, fibroblasts, ovarian mesenchymal cells, breast epithelial cells, fallopian tube epithelial cells, nerve cells, lymphocytes, stem cells, and other cells all can produce cloned individuals successfully (Al-Mashhadi et al., 2013; Du et al., 2009; Li et al., 2007, 2009; Liu et al., 2015; Vajta and Callesen, 2012; Wakayama et al., 1999; Zhai et al., 2018).

The donor cell used to clone the sheep, Dolly, was in G0 phase, and some researchers hold the point that donor cells in G0/G1 phase are more suitable for SCNT (Shufaro and Reubinoff, 2011; Wilmut et al., 2007). Methods to keep donor cells in G0/G1 phase include serum starvation, contact inhibition, and chemical induction (Ma et al., 2015). Subjecting rabbit fibroblasts to serum starvation and contact inhibition significantly increased the proportion of G0/G1 phase cells (Yang et al., 2007). In rabbit cloning with fibroblasts as donor cells, the rate of blastocysts with serum starvation treatment was significantly higher than that with contact inhibition group (45% vs. 21%) (Li et al., 2006). It is also possible to obtain cloned animals by using cells in G2 and M phases as nuclear donors (Shufaro and Reubinoff, 2011).

Few reports state that donor cells in a poor state could still support full-term development of SCNT embryos, for instance, cells from cadavers that have been frozen for many years had been used as donor cells to obtain cloned embryos that developed into individuals (Miranda Mdos et al., 2009; Wakayama et al., 2008). In addition, the sex of donor cells results in some differences in terms of epigenetic modification, but their influence on the early development of SCNT embryos and the efficiency of SCNT is still controversial. More in-depth studies about the effects of donor cells on SCNT embryos are needed.

Fusion and activation

The donor nucleus need to enter the oocyte cytoplasm before activation can occur and fusion is mainly accomplished by chemical fusion, inactivated Sendai virus, or electrofusion (Song et al., 2011). The use of polyethylene glycol for chemical fusion was one of the earliest methods, but chemical reagents are rarely used now because of toxicity to the embryo (D'Souza and Shegokar, 2016). The insertion of inactivated Sendai virus into the oocyte membrane to form a channel can be used but this method is limited by safety and low fusion efficiency (Song et al., 2011). Electrofusion is the most widely used fusion method at present. Based on the fluid mosaic model of cells, when the plasma membrane of the recipient cells is in contact with the donor cell, an electrical pulse is administered that rearranges the phospholipid molecules in the cell membrane, resulting in fusion (Willadsen, 1986).

In natural fertilization, a sperm enters an oocyte and activates it by triggering the cytosolic Ca²⁺ oscillations and reducing M-phase-promoting factor and cytostatic factor (Castillo et al., 2018; Svoboda, 2018). Compared with fertilized embryos, SCNT embryos lack the activation of sperm factors, so artificial intervention, which mimics the activation of fertilized embryos, is required for activation of SCNT embryos.

The combined electrical–chemical activation is a main method for activation of rabbit SCNT embryos. In majority of reported researches, rabbit SCNT embryos were usually treated with ionomycin (an activator of cytosolic Ca²⁺ oscillations) for 5 minutes, then treated for 1 hour with 2 mM 6-dimethylaminopurine (an inhibitor of protein phosphorylation) and 5 μg/mL cycloheximide (an inhibitor of protein synthesis), and combined with direct current pulses treatment during the electrical–chemical activation procedure (Daniel and Chesné, 2015; Li et al., 2006; Meng et al., 2009; Qin et al., 2021; Qu et al., 2020b; Zhang et al., 2019).

Although the combined electrical–chemical activation method can obtain a higher activation rate, it may induce oxidative stress, disturb epigenetic state, and cause endoplasmic reticulum (ER) stress of SCNT embryos (Qu et al., 2020b). A recent research reports that soluble sperm extract can properly modulate the activation of reconstructed eggs during SCNT, which might be a new strategy that is worth exploring to optimize activation methods for SCNT embryos of rabbits and other animals (Prukudom et al., 2019).

In vitro culture

Up to now, it seems that there is no commercial embryo culture medium specifically for animal cloned embryos. In most previous reports, the in vitro culture of cloned embryos used the culture medium of fertilized embryos, and some compounds were added into the culture medium to enhance the development of cloned embryos (Cordova et al., 2017). Adding tauroursodeoxycholic acid (an inhibitor of ER stress) into culture medium can enhance the developmental potential of SCNT embryos by attenuating ER stress and reducing apoptosis (Lin et al., 2016). Adding melatonin (an antioxidant and a free radical scavenger) into culture medium can reduce apoptosis and reactive oxygen species (ROS) in SCNT embryos and enhance cloning efficiency (Su et al., 2015). Adding TSA (a HDACi) into culture medium can enhance the early developmental competence of SCNT embryos through improvements in epigenetic status and protein expression (Cao et al., 2017).

For rabbit SCNT or fertilized embryos in vitro culture, the commonly used embryo culture mediums include modified F10, BSM II, TCM-199, Ham's F10, and synthetic oviductal fluid media, all of which are supplemented with fetal bovine serum or bovine serum albumin. The in vitro culture system of rabbit SCNT embryos was usually at 38.5°C, with 5% CO₂, 95% air, and 100% humidity (Challah-Jacques et al., 2003; Chesné et al., 2002; Lane and Gardner, 2007). Base on the current situation of animal SCNT, it is important to optimize a special culture medium for animal SCNT embryos, which is necessary for getting a stable cloning efficiency, and the research and development of special culture medium for rabbit SCNT embryos may provide a good paradigm for other animals.

Applications of SCNT in Rabbits

Many scientific achievements, such as development of the cholesterol theory, the arterial intimal injury theory, and the inflammation theory, were derived from experiments on rabbits (Cybulsky and Gimbrone, 1991; Finking and Hanke, 1997; Hansson et al., 1991). Studies elucidating the transport and movement of gametes in the reproductive tract, sperm capacitation, in vitro fertilization, implantation, and many other important studies were also carried out on rabbits (Fischer et al., 2012).

Bioreactor

Since the birth of the first transgenic mouse in 1980, one current research hot spot has focused on genetically modified organisms as bioreactors. Transgenic animals can produce large amounts of specific proteins, which have great value in treating disease. These desirable proteins can be produced by bacteria, mammalian cells, transgenic plants, and transgenic animals.

Compared with other technologies, transgenic animal bioreactors have significant advantages. Transgenic animals can produce more complex, biologically active proteins in an efficient and economic manner. The cost of constructing a single purification facility for breeding transgenic animals is much lower than the cost of a commercial cell-culture facility. Bacterial bioreactors are limited by their translation capacity, but transgenic animals can generate correctly folded and modified proteins for therapy and research (Dyck et al., 2003; Paleyanda et al., 1997).

Over the past 20 years, therapeutic monoclonal antibodies (mAbs) have become very successful both clinically and commercially. More than 60 antibody-based therapies have been approved by the FDA, and the total global revenue has exceeded 100 billion U.S. dollars (Carter and Lazar, 2018). Although currently all FDA-approved therapeutic mAbs have either murine, humanized murine, or human variable domain amino acid sequences, rabbit-based therapeutic mAbs have been tested in various clinical trials such as human ROR1, human HGF, human TNF-α, human CGRP, and so on (Mage et al., 2019).

Compared with cattle, sheep, dogs, cats, pigs, and other mammals, rabbits have many advantages in production of therapeutic antibodies in term with short reproductive cycle, low feeding costs, a known genetic background, lack of serious human diseases, and ease of biological purification (Fan et al., 2015; Wang et al., 2013). Moreover, rabbits have a strong innate immune response and the antibodies have high affinity and specificity, and also the spleen is large enough for substantial fusion experiments. Although bioreactors are still limited by genetic safety controversies and bioethics, the continued progress of researches will undoubtedly mean that rabbit bioreactors will occupy an important position in the future of medicine.

Human disease model

Rabbits have a history of more than 100 years as a human disease model, and the rabbit models play an important role in atherosclerosis, heart disease, infectious diseases, orthopedic surgery, cardiovascular surgery, and cancer research (Chentoufi et al., 2010; Fan et al., 2021). In recent years, researchers have obtained a variety of genetically modified rabbits, including apoE, apoC-III, liver esterase, phospholipid transfer protein, C-reactive protein, and others (Masson et al., 2011; Niimi et al., 2016; Torzewski et al., 2014; Yan et al., 2020). These genetically modified rabbits have far-reaching significance in the development of new drugs and understanding the mechanisms of pathogenesis. Rabbit models have also shown advantages in human noroviruses, hepatic fulminant diseases, tuberculosis, and other diseases (Le Pendu et al., 2014; Nyström et al., 2011; Okumura et al., 2018).

Rabbits have a high degree of histopathological similarity with humans after being infected with tuberculosis (Esteves et al., 2018). The similarity between rabbit and human in some diseases will provide great benefits to our research. By utilizing the CRISPR/Cas9 technique to achieve site-specific insertion of human genes at the donor cell, and then obtaining humanized rabbits through SCNT, this approach affords a good solution for overcoming the low success rate of large gene insertion.

Stem cell therapy

Treatment of most diseases has traditionally relied on drugs and interventional procedures, but, in the past two decades, biological therapy has been recognized as a safe and highly effective new method. SCNT reprograms donor cells into pluripotent embryonic stem cells (ESCs) and is conceived to produce patient-matched nuclear transfer-ESCs for understanding disease mechanisms and developing specific therapies. Stem cell biology provides new hope for treatment of diseases and disorders that currently cannot be effectively treated. Rabbit stem cells may have the potential to be a powerful tool for exploring lung disease and stem cell therapy (Kamaruzaman et al., 2013).

Mesenchymal stem cells engraftment had a protective effect on rabbit smoke inhalation injury through regulation of the local and systematic vascular endothelial cell growth factor and reduction of lung water content (Zhu et al., 2010). Using autologous bone marrow mononuclear cells engraftment can mitigate elastase-induced pulmonary emphysema in rabbits (Yuhgetsu et al., 2006). Studies using rabbit models have shown the feasibility of autologous and allogeneic cell therapy. Relevant researches are in the early stage, and still need further studies about how to enhance production, survival, and integration of transplanted cells.

Future Perspectives

Undoubtedly, rabbits have an important position in the field of medical research, and the cloning of rabbits offers great potential in disease model construction, stem cell therapy, and bioreactors. From the theoretical and epigenetic perspective, many breakthroughs have been made in SCNT, but few could be widely used in commercial applications. Continued research and protocol optimization on nuclear reprogramming strategies should be encouraged to improve cloning efficiency. Erasing somatic memory or acquiring sperm-derived regulators may provide new strategies for improved cloning efficiency. With all these advantages and proven effectiveness, rabbit SCNT has great future potential.

Authors' Contribution

All authors have read and agreed to the published version of the article.

Footnotes

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

This research was funded by the Project funded by China Postdoctoral Science Foundation under Grant No. 2020TQ0240 and the Natural Science Foundation of Shaanxi Province under Grant No. 2020PT-001, No. 2020PT-004 and No. 2021PT-039.

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