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Somatic cell cloning always produces female offspring?

Somatic cell cloning always produces female offspring?



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I was looking at the somatic cell cloning of goats.

One of the advantages of the process said was

  • All offspring produced are female.

Now I'm confused. How is this the case?

I know that the embryo after electrofusion is injected into a surrogate mother, but I don't understand how the advantage is that they are "all… female."


For most species of livestock only the females are of economic value. And if you clone a female, you will only get female offspring.

I can also imagine that you would want to use an oocyte and somatic nucleus from the same animal. This way, you also preserve the mitochondrial DNA from your golden goat. And males just don't make eggcells. It could also potentially avoid any incompatibilities between host and donor, somewhat comparable to donor organs. But I don't know if this is actually an issue.


Somatic cell

A somatic cell (from Ancient Greek σῶμα sôma, meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. [1]

The cell which takes part in composition of the body of an organism and divides through the process of binary fission and mitotic division is called somatic cell.

In contrast, gametes are cells that fuse during sexual reproduction, germ cells are cells that give rise to gametes, and stem cells are cells that can divide through mitosis and differentiate into diverse specialized cell types. For example, in mammals, somatic cells make up all the internal organs, skin, bones, blood and connective tissue, while mammalian germ cells give rise to spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, which divides and differentiates into the cells of an embryo. There are approximately 220 types of somatic cell in the human body. [1]

Theoretically, these cells are not germ cells (the source of gametes) they transmit their mutations, to their cellular descendants (if they have any), but not to the organism's descendants. However, in sponges, non-differentiated somatic cells form the germ line and, in Cnidaria, differentiated somatic cells are the source of the germline. Mitotic cell division is only seen in diploid somatic cells. Only some cells like germ cells take part in reproduction. [2]


Nuclear Transfer for Stem Cells

Somatic cell nuclear transfer for pluripotent stem cells is established for animal species, in particular in the mouse and non-human primates. However, there have been major difficulties in deriving pluripotent stem cells by nuclear transfer in the human. Despite this difficulty, there are strong interests in establishing somatic cell nuclear transfer (SCNT) in the human for study of early human development, production of gametes for infertile/sterile patients, comparisons of developmental and differentiation potential with induced pluripotential stem cells (iPSCs), and research in stem cell biology. The combination of oocyte derived factors and transcription factors for efficient and effective reprogramming of somatic nuclei for pluripotent stem cells is a strong inducement for further research in SCNT.


Nuclear Transfer☆

C. Lorthongpanich , . D. Solter , in Reference Module in Life Sciences , 2017

Abstract

Nuclear transfer techniques have been and are being used for a variety of purposes. In the broadest terms, nuclear transfer encompasses the transfer of the entire genetic material from one cell to another cell whose nucleus has been removed or inactivated in order to study nuclear-cytoplasmic interaction. The gene expression pattern of the donor nucleus will be reprogrammed after transfer into recipient cytoplasm under the influence of the cytoplasmic factors present. This reprogramming and the analysis of the mechanisms involved is the basic scientific paradigm of nuclear transfer. The transfer of the nuclei of somatic cells into enucleated egg cytoplasm for the purpose of creating a novel organism – cloning – represents one specific but very important aspect of nuclear transfer.


Contents

Somatic cell nuclear transfer is a technique for cloning in which the nucleus of a somatic cell is transferred to the cytoplasm of an enucleated egg. After the somatic cell transfers, the cytoplasmic factors affect the nucleus to become a zygote. The blastocyst stage is developed by the egg to help create embryonic stem cells from the inner cell mass of the blastocyst. [3] The first animal to be developed by this technique was Dolly, the sheep, in 1996. [4]

The process of somatic cell nuclear transplant involves two different cells. The first being a female gamete, known as the ovum (egg/oocyte). In human SCNT experiments, these eggs are obtained through consenting donors, utilizing ovarian stimulation. The second being a somatic cell, referring to the cells of the human body. Skin cells, fat cells, and liver cells are only a few examples. The genetic material of the donor egg cell is removed and discarded, leaving it 'deprogrammed.' What is left is a somatic cell and an enucleated egg cell. These are then fused by inserting the somatic cell into the 'empty' ovum. [5] After being inserted into the egg, the somatic cell nucleus is reprogrammed by its host egg cell. The ovum, now containing the somatic cell's nucleus, is stimulated with a shock and will begin to divide. The egg is now viable and capable of producing an adult organism containing all necessary genetic information from just one parent. Development will ensue normally and after many mitotic divisions, the single cell forms a blastocyst (an early stage embryo with about 100 cells) with an identical genome to the original organism (i.e. a clone). [6] Stem cells can then be obtained by the destruction of this clone embryo for use in therapeutic cloning or in the case of reproductive cloning the clone embryo is implanted into a host mother for further development and brought to term.

Stem cell research Edit

Somatic cell nuclear transplantation has become a focus of study in stem cell research. The aim of carrying out this procedure is to obtain pluripotent cells from a cloned embryo. These cells genetically matched the donor organism from which they came. This gives them the ability to create patient specific pluripotent cells, which could then be used in therapies or disease research. [7]

Embryonic stem cells are undifferentiated cells of an embryo. These cells are deemed to have a pluripotent potential because they have the ability to give rise to all of the tissues found in an adult organism. This ability allows stem cells to create any cell type, which could then be transplanted to replace damaged or destroyed cells. Controversy surrounds human ESC work due to the destruction of viable human embryos. Leading scientists to seek an alternative method of obtaining stem cells, SCNT is one such method.

A potential use of stem cells genetically matched to a patient would be to create cell lines that have genes linked to a patient's particular disease. By doing so, an in vitro model could be created, would be useful for studying that particular disease, potentially discovering its pathophysiology, and discovering therapies. [8] For example, if a person with Parkinson's disease donated his or her somatic cells, the stem cells resulting from SCNT would have genes that contribute to Parkinson's disease. The disease specific stem cell lines could then be studied in order to better understand the condition. [9]

Another application of SCNT stem cell research is using the patient specific stem cell lines to generate tissues or even organs for transplant into the specific patient. [10] The resulting cells would be genetically identical to the somatic cell donor, thus avoiding any complications from immune system rejection. [9] [11]

Only a handful of the labs in the world are currently using SCNT techniques in human stem cell research. In the United States, scientists at the Harvard Stem Cell Institute, the University of California San Francisco, the Oregon Health & Science University, [12] Stemagen (La Jolla, CA) and possibly Advanced Cell Technology are currently researching a technique to use somatic cell nuclear transfer to produce embryonic stem cells. [13] In the United Kingdom, the Human Fertilisation and Embryology Authority has granted permission to research groups at the Roslin Institute and the Newcastle Centre for Life. [14] SCNT may also be occurring in China. [15]

In 2005, a South Korean research team led by Professor Hwang Woo-suk, published claims to have derived stem cell lines via SCNT, [16] but supported those claims with fabricated data. [17] Recent evidence has proved that he in fact created a stem cell line from a parthenote. [18] [19]

Though there has been numerous successes with cloning animals, questions remain concerning the mechanisms of reprogramming in the ovum. Despite many attempts, success in creating human nuclear transfer embryonic stem cells has been limited. There lies a problem in the human cell's ability to form a blastocyst the cells fail to progress past the eight cell stage of development. This is thought to be a result from the somatic cell nucleus being unable to turn on embryonic genes crucial for proper development. These earlier experiments used procedures developed in non-primate animals with little success.

A research group from the Oregon Health & Science University demonstrated SCNT procedures developed for primates successfully using skin cells. The key to their success was utilizing oocytes in metaphase II (MII) of the cell cycle. Egg cells in MII contain special factors in the cytoplasm that have a special ability in reprogramming implanted somatic cell nuclei into cells with pluripotent states. When the ovum's nucleus is removed, the cell loses its genetic information. This has been blamed for why enucleated eggs are hampered in their reprogramming ability. It is theorized the critical embryonic genes are physically linked to oocyte chromosomes, enucleation negatively affects these factors. Another possibility is removing the egg nucleus or inserting the somatic nucleus causes damage to the cytoplast, affecting reprogramming ability.

Taking this into account the research group applied their new technique in an attempt to produce human SCNT stem cells. In May 2013, the Oregon group reported the successful derivation of human embryonic stem cell lines derived through SCNT, using fetal and infant donor cells. Using MII oocytes from volunteers and their improved SCNT procedure, human clone embryos were successfully produced. These embryos were of poor quality, lacking a substantial inner cell mass and poorly constructed trophectoderm. The imperfect embryos prevented the acquisition of human ESC. The addition of caffeine during the removal of the ovum's nucleus and fusion of the somatic cell and the egg improved blastocyst formation and ESC isolation. The ESC obtain were found to be capable of producing teratomas, expressed pluripotent transcription factors, and expressed a normal 46XX karyotype, indicating these SCNT were in fact ESC-like. [12] This was the first instance of successfully using SCNT to reprogram human somatic cells. This study used fetal and infantile somatic cells to produce their ESC.

In April 2014, an international research team expanded on this break through. There remained the question of whether the same success could be accomplished using adult somatic cells. Epigenetic and age related changes were thought to possibly hinder an adult somatic cells ability to be reprogrammed. Implementing the procedure pioneered by the Oregon research group they indeed were able to grow stem cells generated by SCNT using adult cells from two donors aged 35 and 75, indicating that age does not impede a cell's ability to be reprogrammed. [20] [21]

Late April 2014, the New York Stem Cell Foundation was successful in creating SCNT stem cells derived from adult somatic cells. One of these lines of stem cells was derived from the donor cells of a type 1 diabetic. The group was then able to successfully culture these stem cells and induce differentiation. When injected into mice, cells of all three of the germ layers successfully formed. The most significant of these cells, were those who expressed insulin and were capable of secreting the hormone. [22] These insulin producing cells could be used for replacement therapy in diabetics, demonstrating real SCNT stem cell therapeutic potential.

The impetus for SCNT-based stem cell research has been decreased by the development and improvement of alternative methods of generating stem cells. Methods to reprogram normal body cells into pluripotent stem cells were developed in humans in 2007. The following year, this method achieved a key goal of SCNT-based stem cell research: the derivation of pluripotent stem cell lines that have all genes linked to various diseases. [23] Some scientists working on SCNT-based stem cell research have recently moved to the new methods of induced pluripotent stem cells. Though recent studies have put in question how similar iPS cells are to embryonic stem cells. Epigenetic memory in iPS affects the cell lineage it can differentiate into. For instance, an iPS cell derived from a blood cell will be more efficient at differentiating into blood cells, while it will be less efficient at creating a neuron. [24] This raises the question of how well iPS cells can mimic the gold standard ESC in experiments, as stem cells are defined as having the ability to differentiate into any cell type. SCNT stem cells do not pose such a problem and continue to remain relevant in stem cell studies.

Reproductive cloning Edit

This technique is currently the basis for cloning animals (such as the famous Dolly the sheep), [25] and has been proposed as a possible way to clone humans. Using SCNT in reproductive cloning has proven difficult with limited success. High fetal and neonatal death make the process very inefficient. Resulting cloned offspring are also plagued with development and imprinting disorders in non-human species. For these reasons, along with moral and ethical objections, reproductive cloning in humans is proscribed in more than 30 countries. [26] Most researchers believe that in the foreseeable future it will not be possible to use the current cloning technique to produce a human clone that will develop to term. It remains a possibility, though critical adjustments will be required to overcome current limitations during early embryonic development in human SCNT. [27] [28]

There is also the potential for treating diseases associated with mutations in mitochondrial DNA. Recent studies show SCNT of the nucleus of a body cell afflicted with one of these diseases into a healthy oocyte prevents the inheritance of the mitochondrial disease. This treatment does not involve cloning but would produce a child with three genetic parents. A father providing a sperm cell, one mother providing the egg nucleus, and another mother providing the enucleated egg cell. [10]

In 2018, the first successful cloning of primates using somatic cell nuclear transfer, the same method as Dolly the sheep, with the birth of two live female clones (crab-eating macaques named Zhong Zhong and Hua Hua) was reported. [2] [29] [30] [31] [32]

Interspecies nuclear transfer Edit

Interspecies nuclear transfer (iSCNT) is a means of somatic cell nuclear transfer being used to facilitate the rescue of endangered species, or even to restore species after their extinction. The technique is similar to SCNT cloning which typically is between domestic animals and rodents, or where there is a ready supply of oocytes and surrogate animals. However, the cloning of highly endangered or extinct species requires the use of an alternative method of cloning. Interspecies nuclear transfer utilizes a host and a donor of two different organisms that are closely related species and within the same genus. In 2000, Robert Lanza was able to produce a cloned fetus of a gaur, Bos gaurus, combining it successfully with a domestic cow, Bos taurus. [33]

Interspecies nuclear transfer provides evidence of the universality of the triggering mechanism of the cell nucleus reprogramming. For example, Gupta et al., [34] explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Furthermore, ovine oocyte cytoplast may be used for remodeling and reprogramming of human somatic cells back to the embryonic stage. [35]

Somatic cell nuclear transfer (SCNT) can be inefficient due to stresses placed on both the egg cell and the introduced nucleus in early research were enormous. This can result in a low percentage of successfully reprogrammed cells. For example, in 1996 Dolly the sheep was born after 277 eggs were used for SCNT, which created 29 viable embryos, giving it just a measly 0.3% efficiency. [36] Only three of these embryos survived until birth, and only one survived to adulthood. [25] Millie, the offspring that survived, took 95 attempts to produce, [36] because the procedure was not automated, but had to be performed manually under a microscope, SCNT was very resource intensive. Another reason why there is such high mortality rate with the cloned offspring is due to the fetus being larger than even other large offspring, resulting in death soon after birth. [36] The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg was also far from understood. Another limitation is trying to use one-cell embryos during the SCNT. When using using just one-cell cloned embryos, the experiment has a 65% chance to fail in the process of making morula or blastocyst. The biochemistry also has to be extremely precise, as most late term cloned fetus death are caused as the result of inadequate placentation. [36] However, by 2014, researchers were reporting success rates of 70-80% with cloning pigs [37] and in 2016 a Korean company, Sooam Biotech, was reported to be producing 500 cloned embryos a day. [38]

In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus. This fact may also hamper the potential benefits of SCNT-derived tissues and organs for therapy, as there may be an immuno-response to the non-self mtDNA after transplant.

Proposals to use nucleus transfer techniques in human stem cell research raise a set of concerns beyond the moral status of any created embryo. These have led to some individuals and organizations who are not opposed to human embryonic stem cell research to be concerned about, or opposed to, SCNT research. [39] [40] [41]

One concern is that blastula creation in SCNT-based human stem cell research will lead to the reproductive cloning of humans. Both processes use the same first step: the creation of a nuclear transferred embryo, most likely via SCNT. Those who hold this concern often advocate for strong regulation of SCNT to preclude implantation of any derived products for the intention of human reproduction, [42] or its prohibition. [39]

A second important concern is the appropriate source of the eggs that are needed. SCNT requires human egg cells, which can only be obtained from women. The most common source of these eggs today are eggs that are produced and in excess of the clinical need during IVF treatment. This is a minimally invasive procedure, but it does carry some health risks, such as ovarian hyperstimulation syndrome.

One vision for successful stem cell therapies is to create custom stem cell lines for patients. Each custom stem cell line would consist of a collection of identical stem cells each carrying the patient's own DNA, thus reducing or eliminating any problems with rejection when the stem cells were transplanted for treatment. For example, to treat a man with Parkinson's disease, a cell nucleus from one of his cells would be transplanted by SCNT into an egg cell from an egg donor, creating a unique lineage of stem cells almost identical to the patient's own cells. (There would be differences. For example, the mitochondrial DNA would be the same as that of the egg donor. In comparison, his own cells would carry the mitochondrial DNA of his mother.)

Potentially millions of patients could benefit from stem cell therapy, and each patient would require a large number of donated eggs in order to successfully create a single custom therapeutic stem cell line. Such large numbers of donated eggs would exceed the number of eggs currently left over and available from couples trying to have children through assisted reproductive technology. Therefore, healthy young women would need to be induced to sell eggs to be used in the creation of custom stem cell lines that could then be purchased by the medical industry and sold to patients. It is so far unclear where all these eggs would come from.

Stem cell experts consider it unlikely that such large numbers of human egg donations would occur in a developed country because of the unknown long-term public health effects of treating large numbers of healthy young women with heavy doses of hormones in order to induce hyper-ovulation (ovulating several eggs at once). Although such treatments have been performed for several decades now, the long-term effects have not been studied or declared safe to use on a large scale on otherwise healthy women. Longer-term treatments with much lower doses of hormones are known to increase the rate of cancer decades later. Whether hormone treatments to induce hyper-ovulation could have similar effects is unknown. There are also ethical questions surrounding paying for eggs. In general, marketing body parts is considered unethical and is banned in most countries. Human eggs have been a notable exception to this rule for some time.

To address the problem of creating a human egg market, some stem cell researchers are investigating the possibility of creating artificial eggs. If successful, human egg donations would not be needed to create custom stem cell lines. However, this technology may be a long way off.

SCNT involving human cells is currently legal for research purposes in the United Kingdom, having been incorporated into the Human Fertilisation and Embryology Act 1990. [43] [5] Permission must be obtained from the Human Fertilisation and Embryology Authority in order to perform or attempt SCNT.

In the United States, the practice remains legal, as it has not been addressed by federal law. [44] However, in 2002, a moratorium on United States federal funding for SCNT prohibits funding the practice for the purposes of research. Thus, though legal, SCNT cannot be federally funded. [45] American scholars have recently argued that because the product of SCNT is a clone embryo, rather than a human embryo, these policies are morally wrong and should be revised. [46]

In 2003, the United Nations adopted a proposal submitted by Costa Rica, calling on member states to "prohibit all forms of human cloning in as much as they are incompatible with human dignity and the protection of human life." [47] This phrase may include SCNT, depending on interpretation.

The Council of Europe's Convention on Human Rights and Biomedicine and its Additional Protocol to the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine, on the Prohibition of Cloning Human Being appear to ban SCNT of human beings. Of the Council's 45 member states, the Convention has been signed by 31 and ratified by 18. The Additional Protocol has been signed by 29 member nations and ratified by 14. [48]


Cloning competence of various somatic cell types

Many somatic cell types, including mammary epithelial cells, ovarian cumulus cells, fibroblast cells from skin and internal organs, various internal organ cells, Sertoli cells [38, 56], macrophage [56] and blood leukocytes [34, 35] have been successfully utilized for nuclear transfer. A clear consensus, however, has not yet been reached as to the superior somatic cell type for nuclear transfer. This is due in part to the fact that different laboratories employ diverse procedures and cell culture, nuclear transfer, and micromanipulation all require critical technical skills. In order to make these comparisons valid, the procedures and techniques used, as well as the skill of lab personnel, must be identical for each donor animal and cell type. To compare the competence of different cell types for reprogramming by cloning, we avoided animal variation by looking at the cloning competence of three cell types: ovarian cumulus, mammary epithelial and skin fibroblast cells, all from the same donor animal, a 13-year-old elite diary cow.

The ability of donor cells to be reprogrammed was assessed by the development of cloned embryos in vitro and by the birth of cloned calves following embryo transfer. As shown in Tables 2 and 3, although no differences were detected in the cleavage rates of embryos from three different cell types, cumulus cells produced the highest rate of blastocyst development in this study and resulted in 6 full-term cloned calves. Furthermore, four out of the six calves derived from cumulus cells survived and were still healthy at nearly 4 years of age (Table 3). In contrast, the poorest in vitro development, and no full-term survival, was obtained with mammary epithelial cells. Skin fibroblast cells resulted in an intermediate rate of in vitro development and gave rise to 4 full-term cloned calves.

Our results showed that the donor cell type can significantly affect embryo development in vitro as well as in vivo. Cumulus cells proved to be the most effective cell type for somatic cloning according to both the in vitro development test as well as full-term survival. These results suggest that DNA from cumulus cells is more effectively reprogrammed following nuclear transfer. Our results agreed with those obtained in mice [57] where they compared the nuclear transfer efficiency of neuronal, Sertoli and cumulus cells, and obtained the best live birth rate from cumulus cell-derived cloned embryos. Furthermore, it was reported that cumulus cell-derived cloned mice do not have widespread dysregulation of imprinting [23]. Kato et al. [15, 36] compared cells from the liver, testis, skin, ear, along with cumulus and oviductal cells and concluded that cumulus and oviduct epithelial cells are the most suitable for nuclear donors. Evidence supporting the superiority of cumulus cells for nuclear transfer also comes from the study of Forsberg et al. [58] who conducted large numbers of embryo transfer in cattle. It was shown that cumulus cells gave an overall 15.2% calving rate, while fetal genital ridge cells, and fibroblast cells produced a 9% calving rate. Adult fibroblast cells, in this study, gave the lowest calving rate of only 5%.

In summary, among the somatic cell types tested, the consensus from numerous laboratories is that cumulus cells give the highest cloning efficiency and result in the least number of abnormalities in cloned animals.


Recent advancements in cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

1. Introduction

Somatic cell nuclear transfer (SCNT) in mammals is an assisted reproductive technique used to produce an animal from a single cell nucleus using an enucleated oocyte as a recipient. As somatic cells can be proliferated and gene-modified in vitro, this technique has been expected to contribute extensively to the farm animal production industry, drug production, regenerative medicine and conservation of invaluable genetic resources [1,2]. Besides its broad practical applications, SCNT can provide unique and interesting experimental systems for genomic research, especially in epigenetics, to learn how the somatic cell genome is reprogrammed into a state equivalent to that of the fertilized oocyte: the so-called totipotent state [3]. Although the somatic cell genome can be reprogrammed (or dedifferentiated) by other methods, including the introduction of transcription factors [4], incubation of permeabilized cells with cell extracts [5] and cell–cell fusion [6], the resultant reprogrammed state of this genome is ultimately a pluripotent state equivalent to that of embryonic stem (ES) cells (figure 1). Therefore, SCNT is the sole technique to date that can endow the somatic cell genome with totipotency.

Figure 1. Two types of reprogramming of the somatic cell genome. The genome of differentiated somatic cells can be reprogrammed to the pluripotent state by introduction of the so-called Yamanaka factors [4], incubation of permeabilized cells with cell extracts [5] or cell fusion [6]. However, the resultant genomic state even with the best reprogramming is that of embryonic stem (ES) cells, which are epigenetically distinct from the inner cell mass (ICM) cells of blastocysts (see the text). It can be assumed that there is a somatic cell–embryo epigenetic barrier between preimplantation embryos and postimplantation embryonic tissues. Only somatic cell nuclear transfer (SCNT) using enucleated oocytes can overcome this barrier to endow the somatic genome with totipotency, the epigenetic state equivalent to fertilized oocytes (zygotes). Experimentally, SCNT bypasses the normal path of germ cell development, during which somatic epigenetic marks are erased and germ cell epigenetic marks are imposed. Conceivably, this might cause reprogramming errors in the somatic cell genome and the associated abnormal phenotypes following SCNT. By contrast, nuclear transfer (NT) using embryonic blastomeres is more efficient. The epigenetic barrier proposed here might be more stringent in mice and humans than in ungulates because the latter species have a non-attached preimplantation phase before the placenta is established in the uterus, after embryonic differentiation. iPS, induced pluripotent stem cell.

ES cells, especially those of mice and rats at their most ground state, have full pluripotency and can differentiate into all cell lineages comprising the body [7,8] and even into placental cells under specific culture conditions [9]. Their open hyperdynamic chromatin structure [10] and global transcriptional hyperactivity [11] sets them apart from differentiated somatic cells [12]. However, in a broad sense, ES cells seem to be a kind of somatic cell because they share some important characteristics common to other somatic cells or to postimplantation embryos, such as their DNA methylation patterns in promoter and centric/pericentric regions and complete loss of the germline-specific X-chromosome inactivation memory [9,13–16]. It has also been demonstrated that the process of ES cell derivation causes robust changes in methylation of histone H3 at lysines 4 (H3K4me3) and 27 (H3K27me3) [17]. Similarly, many differences in transcriptional programmes between ES cells and inner cell mass (ICM) cell outgrowths have been detected by RNA-seq profiling [18,19]. Taken together, during the derivation of ES cells, they accumulate somatic cell-type epigenetic characters, faithfully reflecting the phenomena occurring during implantation in vivo. In accordance with this, it is known that cloned mice derived from ES cells show SCNT-specific hyperplastic placentas, which are about twice as large as those cloned from ICM cells [20,21]. By contrast, nuclear transfer (NT) from blastomeres of preimplantation mouse embryos is more efficient in terms of the birth rate per number of embryos transferred and causes very few abnormalities in the resulting clones [21,22]. As far as has been tested in mammals, there might be a gap between blastomere NT cloning and SCNT in terms of the birth rates and the normality of cloned offspring [20–23]. Therefore, it is reasonable to assume that there is an ‘epigenetic barrier’ between preimplantation embryos and postimplantation somatic cells. This barrier might be more stringent in mice and humans than in ungulates because the latter species have a non-attached preimplantation phase before the placenta is established in the uterus, after embryonic differentiation. Some epigenetic marks are probably imposed on the somatic cell genome at the time of implantation, and remain in the genome throughout the subsequent life cycle. These somatic cell marks are erased only through gonadal germ cell development in a step-by-step manner, although the mechanism is still unclear [24]. SCNT forces the somatic cell genome to be reprogrammed directly to a totipotent state by bypassing these erasing steps, and this might make the technique prone to epigenetic errors and cause frequent death and loss of embryos. In addition, SCNT should also erase the cell-type-specific memory that had been imposed during differentiation. This differentiation-associated memory might be easier to reprogramme than the former putative somatic cell marks because during induced pluripotent stem (iPS) cell generation, most if not all of the epigenetic characters of original donor cells are erased successfully [25].

In sum, the SCNT technique should somehow overcome these two epigenetic hurdles: somatic cell marking and cell-type-specific differentiation memory. Each hurdle might cause specific reprogramming errors and clone-associated abnormalities. We expect that while we seek to improve the efficiency of SCNT, such relationships between the epigenetic hurdles and their specific reprogramming errors will become clearer, leading to more precise understanding of epigenetic control mechanisms functioning during implantation and cell differentiation. The mouse probably provides the best experimental model for this purpose.

2. What determines genomic reprogrammability? Lessons from mice

Since the birth of Dolly the sheep in 1996, many attempts have been made to clone animals of different species using different somatic cell types [26]. Based on the accumulated information on the various levels of efficiency in those cloning experiments, it is broadly accepted that the efficiency of cloning in terms of the birth rates of offspring can be affected by a number of biological and technical factors. However, in cloning farm animals, there are inevitably considerable individual differences in the quality of recipient oocytes, donor cells and recipient females, so it is usually very difficult to determine the decisive factors statistically [2]. Therefore, attempts to determine the best experimental conditions for cloning farm animals can be compromised, and sometimes become issues of controversy. By contrast, laboratory mice offer more reproducible experimental systems because of the availability of defined genetic backgrounds [27] and well-established protocols for superovulation, embryo culture and embryo transfer. Since the first report of successful mouse cloning in 1998 [28], cumulus cells with a B6D2F1 genetic background have been the standard nuclear donor source and have been used as controls for the assessment of other donor cells for their ‘clonability’ [29]. A large-scale mouse cloning experiment using two different cell types and six different genotypes of donor cells revealed that the birth rates of clones were determined by the combination of these two factors [30]. In this analysis, the birth rate using B6D2F1 cumulus cells was 2.2 per cent while that of (B6 × 129) F1 neonatal Sertoli cell clones was 10.8 per cent. Overall, SCNT with neonatal Sertoli cells resulted in better efficiencies than with cumulus cells. This has been true in more recent studies [31,32] and is in accordance with the results of global gene expression analysis of blastocysts cloned from cumulus or Sertoli cells [33]. ES cells have also been used in cloning studies in mice, especially in the second step of a serial cloning protocol where NT-derived ES cells were generated as an intervening step between the first and second rounds of SCNT [34] (figure 2). The efficiency of cloning mice using ES cells is generally very high, provided their cell cycle is synchronized successfully with that of the ooplasm and genetic/epigenetic errors are avoided during in vitro culture. The birth rates per number of embryos transferred ranged from 12 per cent in the original report by Wakayama et al. [20] to 33 per cent in an experiment using F1 hybrid ES cells [37]. The high reprogrammability (the ability of the genome to be reprogrammed) of the ES cell genome might be attributed to the consecutive expression of Oct3/4, a key upstream factor required for normal early embryonic development [38]. Better cell cycle synchrony between the ES cell nucleus and the recipient ooplasm can be achieved by confluent culture [39] or through cell cycle arrest at the M-phase with nocodazole treatment [40].

Figure 2. Two-step SCNT in mice. NT-derived embryonic stem (ntES) cells are generated by the first round of SCNT. These are then used to generate cloned mice by a second SCNT procedure or via an intervening stage using chimeric embryo production. This protocol has been used successfully for mouse cloning studies where conventional one-step SCNT is unsuccessful or when it is very difficult to generate mice for example, when cloning lymphocytes [35] and when using somatic cells retrieved from frozen cadavers [36].

Given that the best donor cell types in terms of cloning efficiency are ES cells, followed by neonatal Sertoli cells and adult cumulus cells [28,30,34,41], this led us to hypothesize that the degree of differentiation might be correlated inversely with cloning efficiency. One of the strategies to test this assumption was to clone differentiated cells within the same cell lineage. So far as has been examined to date, neuronal lineage cells seem to confirm this assumption. When neural cells were collected from foetuses at 15.5–17.5 days postcoitus (dpc), normal cloned offspring were born at a relatively high rate (5.5%) [42]. By contrast, no live offspring were born from the neural cells of neonatal mice (days 0–4 after birth) because of embryonic death at around 10.5 dpc [43]. Adult neuronal cells also contributed to the reconstruction of cloned embryos, but supported their development only up to 6–7 dpc [28]. Neural stem cells derived from foetal or neonatal brains could be cloned to produce normal offspring by SCNT [31,44]. These results suggest that neural lineage cells lose their reprogrammability as they differentiate. However, another line of evidence using haematopoietic lineage cells suggested that this scenario is not always the case. Cloning T or B cells was extremely difficult while cloning natural killer T (NKT) cells was relatively easy, even though they are all terminally differentiated lymphocytes carrying rearranged DNA [35,45]. So far as has been tested to date, the generation of mice from T- or B-lymphocytes by direct SCNT has been unsuccessful and, therefore, a two-step round of NT—a technique involving ES cell generation and tetraploid complementation—is necessary [35] (figure 2). In this, the ES cell stage allows for extra reprogramming time, and tetraploid cell lines might contribute to most extra-embryonic tissues, which are commonly the more adversely affected components in cloned animals [46]. By contrast, we found that NKT cells with specific differentiated markers were suitable donors for the generation of cloned offspring and NT-derived embryonic stem (ntES) cell lines by direct SCNT [45]. We then further examined whether haematopoietic stem cells (HSCs) with innate differential plasticity could be cloned by SCNT. Unexpectedly, only 6 per cent of reconstructed embryos reached the morula or blastocyst stage in vitro (versus 46% for cumulus cell-derived clones) and at best only 0.7 per cent per embryo transferred reached full term [47]. No offspring were obtained when a standard B6D2F1 genetic background was used [47]. Sung et al. [48] also confirmed the unsuitability of HSCs for SCNT by comparing the birth rate of clones with that of granulocyte clones. The poor development of HSC-derived cloned embryos was consistent with their gene expression pattern at the 2-cell stage when major zygotic gene activation (ZGA) occurs in the mouse [49]. The HSC clones failed to activate five out of six important ZGA genes examined, including Hdac1 encoding histone deacetylase 1, a key regulator of ZGA [50,51]. As a result, only 34 per cent of the HSC clones reached the 4-cell stage (p < 0.05 versus 65–78% of other cloned embryos). This finding seems to be contradictory to the finding that HDAC inhibitors actually improve the development of cloned embryos (see below), but treatment with these drugs is usually restricted to the very early stage of development (less than 10 h after oocyte activation) to avoid their inhibitory effects on ZGA at a later stage [50]. We also confirmed that the expression of Hdac1 mRNA in HSCs was lower than in other somatic cells, probably reflecting an open chromatin structure that enables the easy access of transcriptional factors [52,53]. Taken together, we postulate that genomic reprogrammability is biologically distinct from the degree of genomic plasticity based on its differentiation status (or its ‘stemness’, in reverse). Rather, it might have a close correlation with the gene expression pattern or chromatin structure specific to each donor cell type. Oback [54] reviewed the relationships between the genomic reprogrammability of donors and their differentiation status in mice and other species in detail in 2009, and postulated that the differentiation status of the donor genome and its reprogrammability to totipotency might be unrelated.

According to Eminli et al. [55], HSCs and progenitor haematopoietic cells provided better efficiencies of iPS cell generation than did B or T cells. This is not surprising because genomic reprogramming to the pluripotent state does not need ZGA or activation of genes for early embryonic development. For example, the suppression of HDAC1 in HSCs might facilitate iPS generation, but might also hamper ZGA and subsequent embryonic development. Thus, the prerequisites for acquisition of pluripotency and totipotency are epigenetically different, although there might be a common machinery to reprogramme the chromatin structure and nuclear architecture.

After a series of SCNT experiments using different donor cell types with a common male (B6 × 129) F1 genotype, we found a high correlation (r = 0.92, p = 9.1 × 10 –5 ) between the rates of embryos that developed beyond the 2-cell stage and the rates of birth after embryo transfer (figure 3). This finding suggests that the degree of ZGA has a strong effect on embryonic development to term. The data also clearly indicate that there was no relationship between genomic reprogrammability and the undifferentiated status of the genome (figure 3).

Figure 3. Correlation between the rates of development beyond the 2-cell stage and full-term development in cloned embryos. There is a close relationship between these parameters while the degree of ‘stemness’ or undifferentiated status of the donor cells has no association with these cloning efficiencies. All experiments were undertaken using male donor cells with a (B6×129) F1 genetic background, except for female primordial germ cells (PGCs). This is based on data both published [30,44,45,47,56] and unpublished (A. Ogura, K. Inoue, unpublished data). The birth rates were calculated, including placenta-only conceptuses. Results using donor cells with incomplete genomic imprinting such as early PGCs [57] or with abnormal chromosomal constitutions such as long-cultured mesenchymal stem cells [44] or cancer cells [58] have been omitted here. TSA, trichostatin A.

As mentioned above, the genotype of donor cells can also affect cloning efficiency. In one study, Japanese Black calves were born following SCNT at a rate as high as 80 per cent (8/10 per embryos transferred) using cumulus and oviductal epithelial cells [59]. This efficiency was much higher than that of bovine cloning performed during the same period (about 10%) [60]. Since then, high pregnancy rates have been reported for bovine SCNT using the Japanese Black breed [61,62]. Sheep also showed breed-specific variability in terms of the success of cloned embryo development [63]. However, there are no directly comparable data for cattle and sheep clones that can allow precise evaluation of the effects of genetic backgrounds. By contrast, mouse cloning experiments have allowed us to assess the effect of genotype on the development of clones in a more precise manner thanks to the availability of genetically defined strains of mice [27]. According to Van Thuan et al. [64], cloned mice could be obtained from all the inbred strains so far tested [64]. The best birth rate was obtained with the 129 strain, followed by the DBA/2 strain (figure 4). Based on these studies, the presence of the genome from the 129 strain is expected to increase the reprogramming efficiency of the donor genome following SCNT [30]. Historically, the plasticity of this mouse genome has been demonstrated by the relatively easy establishment of ES cells from this strain [65] and by the high incidence of testicular carcinoma [66]. Interestingly, the placentas of clones derived from the 129 strain showed nearly normal morphology, unlike those from other strains, which showed placentomegaly [67]. Therefore, there might be certain factors within the 129 strain genome that ensure its high genomic plasticity. At present, we have no idea of the identity of these factors, but if we could identify them they would contribute greatly to the safe and efficient development of new technologies in regenerative medicine and pharmacy. SCNT experiments using a set of recombinant inbred strains based on crosses between the 129 strain and other strains should help in the execution of this strategy by the so-called ‘forward genetics’.

Figure 4. Effects of trichostatin A (TSA) and scriptaid treatment on inbred mouse cloning. Without such histone deacetylase inhibitor (HDACi) treatment (black bars), cloned mice could be obtained from hybrid and 129/Sv strains, but with a low success rate. Only one cloned mouse was obtained from the DBA/2 strain, but this animal never reproduced. When TSA (light grey bars) was used, the success rates for hybrid and outbred strains were increased but we have never succeeded in producing full-term cloned mice from inbred strains. However, when scriptaid (dark grey bars) was used, the overall success rate was increased even from inbred strains.

3. Technical improvements based on histone modifications

As mentioned above, the prevention of epigenetic errors during nuclear reprogramming is expected to improve the success rate of animal cloning. Enright et al. [68] have tried to alter the epigenetic status of donor nuclei before NT by using two chemicals: 5-azacytidine (an inhibitor of DNA methylation) and trichostatin A (TSA a histone deacetylase inhibitor (HDACi)). The in vitro developmental potential of bovine cloned embryos was improved slightly. However, these drugs that affect epigenetics are very toxic [69,70] and each drug must be tested pharmacologically for its appropriate exposure, timing, concentration and duration. Thus, Kishigami et al. [71] discovered by trial and error the optimum concentration, timing and period of TSA treatment for cloning mouse embryos. Eventually, this method led to a greater than fivefold increase in the success rate of mouse cloning, except for cloning ES cells (figure 5). Unlike the situation in mouse cloning, the effects of TSA on cloning efficiency are controversial for bovine [72,73], pig [74,75], rabbit [76,77] and rat [78] models. Moreover, some groups have reported that TSA treatment had detrimental effects on the in vitro and in vivo development of SCNT embryos [73,76]. To our knowledge, the effects of TSA treatment on full-term development have not been determined in any species other than the mouse.

Figure 5. Effects of HDACi treatment on mouse cloning using B6D2F1 cumulus cells as NT donors. Without HDACi treatment, cloned mice could be obtained but with a low success rate (Cont). When TSA, scriptaid, SAHA or oxamflatin were used, the success rates were increased significantly, but when APHA was used, no clones were obtained. All the HDACi agents listed here belong to the hydroxamic acid or hydroxamate chemical compound classes.

It must be emphasized that most cloned mice have only been generated from hybrid strains and have never been cloned from outbred or inbred strains [30,67]. However, when the drug scriptaid, which acts as an HDACi but is less toxic than TSA [79], was used for cloning, it could increase mouse cloning success rates not only in hybrid but also in supposedly ‘unclonable’ inbred strains of mice [64,80]. Similarly, when scriptaid was used instead of TSA, Zhao et al. [81] improved the success rate of pig cloning to full term. These results suggest that although the use of HDACi drugs can enhance reprogramming in cloned embryos, because of their toxicity the effects depend on the individual sensitivity of the donor strain or species. For this reason, we have tried to discover other useful HDACi drugs for mouse cloning, and we found that two other agents, suberoylanilide hydroxamic acid (SAHA) and oxamflatin, could also improve the full-term development of cloned mice significantly without leading to obvious abnormalities [82]. Another group found that m-carboxycinnamic acid bishydroxamide also improved the success rate of full-term mouse cloning [83]. However, although valproic acid was reported to increase the reprogramming efficiency of mouse fibroblasts by more than 100-fold to establish iPS cells [84], it had little [85] or no effect [82] on the success rate of mouse cloning. Another HDACi, aroyl pyrrolyl hydroxamide (APHA), also could not improve cloning efficiency [64]. Figure 4 summarizes the effect of HDACi agents on mouse cloning using BDF1 cumulus cells [64,71,82].

(a) How does HDACi treatment enhance reprogramming?

Although how HDACi treatment improves cloning efficiency remains unknown, it is thought that it can induce hyperacetylation of the core histones, resulting in structural changes in chromatin that permit transcription and enhanced DNA demethylation of the somatic cell-derived genome after SCNT [71]. This is a necessary part of genetic reprogramming [86]. In fact, several reports clearly showed that HDACi treatment during SCNT cloning improved histone acetylation [87], nascent mRNA production [64] and gene expression [88] in a manner similar to that in normally fertilized embryos. TSA treatment also improved the long-term consistency of genome-wide gene expression regulation: the total number of genes commonly exhibiting up- or downregulation in the TSA-treated clone pups decreased to half of the conventional SCNT pups and the total gene expression profile of the TSA clones came to resemble that of the intracytoplasmic sperm injection (ICSI) pups [89].

How histone methylation is modified in TSA-treated cloned embryos is not completely understood. Bui et al. [90] found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos, similar to embryos produced by ICSI [90]. This was associated with a more effective formation of DNA replication complexes in treated embryos. Those embryos could overcome a failure in the timely onset of embryonic gene transcription by the activation of rRNA genes and promotion of nucleolar protein allocation during the early phase of ZGA [91]. Those results suggest that HDACi can enhance the reprogramming of the somatic nuclei in terms of chromatin remodelling, histone modification, DNA replication and transcriptional activity.

(b) Why do cloned embryos require HDACi treatment for better genomic reprogramming?

In nature, the ooplasm contains reprogramming mechanisms, such as histone acetylation or DNA demethylation, that convert the sperm and oocyte nuclei to a totipotent state [87,92,93]. Given that SCNT can result in the birth of viable animals, the oocyte's reprogramming machinery is sufficient to reprogramme a somatic cell nucleus, at least in some cases. However, the potential reprogramming machinery of the oocyte cytoplasm is prepared for the receipt of a haploid sperm nucleus, not a somatic cell nucleus. In general, it is considered that the incomplete reprogramming of somatic cell nuclei following SCNT arises from poor genomic reprogramming in the oocyte. However, we now think that the oocyte cytoplasm might reprogramme the somatic cell nucleus too strongly, or that the somatic cell nucleus is more sensitive to oocyte reprogramming factors than are sperm cell nuclei. Therefore, by inhibiting a particular HDAC or one of the reprogramming factors in ooplasm during reprogramming, the donor nuclei in our studies were possibly reprogrammed more correctly, resulting in a higher success rate for cloning [64,71].

4. Technical improvements based on X-chromosome inactivation status

It is very probable that the effect of HDACi treatment on the chromatin remodelling of cloned embryos is genome wide rather than genome region-specific and leads to nearly normal reprogramming of the whole genome. However, the presence of many SCNT-specific phenotypes, such as placental abnormalities [21,94,95], obesity [96] and immunodeficiency [97], suggests that SCNT inevitably induces specific epigenetic errors into the donor genome. They might be non-random and definable characteristics, perhaps caused by the fundamental epigenetic nature imposed on somatic cell nuclei at the time of implantation described above. To examine this possibility, single cloned blastocysts were analysed for their global gene expression patterns by comparing them with genotype- and sex-matched controls produced by in vitro fertilization under the same environment. We noted that when the relative expression levels of all 41 233 genes in cloned embryos were plotted on the 20 chromosomes, many genes on the X chromosome were specifically downregulated [32]. This suppression of the X-linked gene was chromosome wide and was associated with elevated expression of the Xist gene, which is responsible for inactivation of one of the X chromosomes in female cells. RNA fluorescent in situ hybridization (FISH) analysis for Xist mRNA in blastomere nuclei revealed an excessive signal in male and female cloned embryos, indicating that Xist was expressed ectopically from the active X chromosome in both sexes. We then examined to what extent its ectopic expression was responsible for the low birth rates of clones, using knockout and knockdown strategies. As the X chromosome is only one of the 20 chromosomes in mice (except for the Y), we first anticipated that normalization of Xist expression would simply result in some slight improvements in the cloning efficiency. However, the results were much more remarkable than we expected. When donor cells containing an Xist-deficient X chromosome were used for NT, the birth rates increased 8- and 14-fold following cumulus and Sertoli cell cloning, respectively [32]. Similarly, knockdown of Xist by the injection of specific short interfering (si)RNA into reconstructed oocytes resulted in about a 10-fold increase in the birth rate [98]. Interestingly, normalization of the Xist expression level resulted in a decreased number of downregulated autosomal genes to 6 per cent and 25 per cent in female and male clones, respectively. These results indicate that the ectopic Xist expression in clones might have adversely affected the gene expression in cloned embryos in a genome-wide manner. Although this knockdown strategy is currently only applicable to male clones because of our inability to achieve precise quantitative control of Xist mRNA repression, this technology is more realistic for the future practical applications of SCNT because it is technically easy and does not alter the genomic constitution of the donor genome or of the cloned offspring. The XIST gene is upregulated in bovine SCNT embryos and has been implicated in the prenatal death of cloned pigs and calves [99–101]. In domestic animal species such as bovines and pigs, embryonic implantation takes place long after embryo transfer. Therefore, we should carefully examine whether the effect of XIST knockdown can persist through to the critical developmental period for the cloned embryos to survive.

Two discrete groups of genes remained suppressed in Xist-deleted mouse clone blastocysts. They were the Magea and Xlr gene clusters, localized in chromosomal regions XqF3 and XqA7.2–7.3, respectively [32]. These regions are within the blocks enriched with the dimethylation of histone H3 at lysine 9 (H3K9me2), which is responsible for gene silencing providing a constitutive heterochromatin status. They are called ‘large organized chromatin K9 modifications’ [102] and their pattern of distribution matched exactly between ES cells [102] and donor cumulus cells [32]. We postulated that the repressive state of the Magea and Xlr regions mediated by the somatic type-H3K9me2 modification in donor cells was resistant to reprogramming by the putative ooplasmic factor(s) and consequently transmitted to cloned embryos. The Magea and Xlr genes were actively transcribed in normal fertilization-derived blastocysts, but became shut down in ES cells [32], suggesting that the H3K9me2 blocks might serve as a ‘somatic signature’ imposed during implantation. These could be the next targets for technical improvements in SCNT.

5. Other factors that might affect the development of clones

(a) Placental abnormalities

Most SCNT-derived clone embryos arrest their development at some time during the early postimplantation period, depending on the species: before 6.5 days in mouse [103] and before 60 days in bovine embryos [104]. These periods are critical for early placentation. Indeed, SCNT-associated abnormal placental phenotypes have often been described in several of the species cloned to date [105]. Cloned mice are associated with hypoplastic placentas during early gestation [106,107] and with placental hyperplasia from mid-gestation to term [94,95,108]. Placentas of cloned calves have fewer but much larger placentomes, presumably to compensate for the reduced number of placental sites for maternal–foetal exchange [109]. Thus, it is possible that defects in the extra-embryonic cell lineage are one of the major causes of the low success rate of reproductive cloning [105]. At first, this assumption was thought to be consistent with the easy derivation of ntES cells from the inner mass cells of cloned blastocysts [110]. However, this scenario was found to be invalid because trophoblast stem (ntTS) cells were also efficiently established from SCNT embryos [111,112]. To determine how the extra-embryonic tissues are involved in poor development of clones and enlarged placenta phenotypes in mice, several laboratories have analysed chimeras generated by fusing cloned embryos and fertilized embryos. In addition, ntTS cells have also been characterized in vivo and in vitro for this purpose. As a result, some studies implied that disorganized interactions between embryonic and extra-embryonic tissues are responsible for inefficient cloning or for placental abnormalities [111,113,114], while others indicated the predominant involvement of the extra-embryonic tissues themselves [107,112]. Recently, Lin and co-workers [115] re-evaluated the developmental consequences of chimera embryos using isolated ICM cells, not whole blastocysts. By aggregating clone-derived ICM cells with tetraploid fertilized embryos, they successfully rescued abnormal placental phenotypes and increased the birth rate of clones up to 15.7 per cent, indicating that defects in the extra-embryonic lineage underlie the low success rate of SCNT cloning. The rescue of SCNT-derived embryos by Xist knockout or knockdown described above had no beneficial effect on the hyperplastic placental phenotype. It will be interesting to see whether the two strategies for rescuing SCNT embryos might have a synergistic effect.

(b) Maternal inheritance of the mitochondrial DNA

Since the early SCNT studies in the 1990s, whether the mitochondrial DNA (mtDNA) transmitted from the donor cells might cause poor development of cloned embryos has been an issue. This is a set of cytoplasmic genomes that encodes a subset of genes encoding oxidative phosphorylation and is transmitted to offspring by strict maternal inheritance [116,117]. Generally, a single oocyte contains more than 10 5 copies of mtDNA, whereas somatic cells contain only 10 2 –10 3 copies [117,118]. Theoretically, cloned animals possess two types of mtDNAs derived from oocytes and donor cells this condition is called heteroplasmy. Evans et al. [119] demonstrated that the first cloned mammal, Dolly, did not possess nuclear donor cell-derived mtDNA in its tissues, whereas Steinborn et al. [120] detected heteroplasmy in bovine embryos produced from three types of donor cells. Many attempts have been made to discriminate oocyte- from donor cell-derived mtDNA quantitatively. These have supported the presence of heteroplasmy in cloned animals, although its degree varied greatly among experiments [121–124]. In mice, 24 of 25 cloned offspring carried the donor cell-derived mtDNA up to a ratio of 13.1 per cent of the total [125]. However, as far as we know, there is no experimental evidence that heteroplasmy might compromise the development of cloned embryos or the health status of offspring, at least in conventional SCNT. By contrast, the situation of interspecies SCNT (iSCNT) is very different. Incompatibility between the mtDNA genes and the nuclear genes encoding mitochondrial proteins is thought to be one of the major causes of developmental arrest among iSCNT embryos [126]. In iSCNT among the Canidae and Felidae, this might not be a critical issue because wolves and wild cats have been born using oocytes from domestic dogs and cats, respectively [127,128].

6. Future prospects

(a) The possibility of resurrecting an extinct animal

Cloning animals by SCNT provides an opportunity to preserve endangered mammalian species, provided viable cells can be collected. However, the ‘resurrection’ of extinct species from permafrost (such as the woolly mammoth) is thought to be impractical, because no live cells will be available. On the other hand, it is known that ‘dead’ spermatozoa from freeze-drying treatments [129] or from a whole frozen cadaver [130] still possess a complete haploid genome. When such spermatozoa were injected into oocytes, the resulting embryos could develop to full-term healthy offspring. Surprisingly, the toughness of DNA was demonstrated not only in the sperm head but also in somatic cells. We attempted to produce cloned mice from cadavers kept frozen at –20°C for up to 16 years without any cryoprotection. These conditions are similar to those of a frozen body recovered from permafrost, and the cells from all organs of the cadavers were completely disrupted. When we injected those cell nuclei into enucleated mouse oocytes, some of the embryos could develop to blastocysts. Although we could not produce cloned offspring from the somatic cells directly, several ES cell lines were established from the cloned embryos. Finally, healthy cloned mice were produced from these ES cells by a second round of NT (figure 2) [36,131]. Thus, these techniques could be used to resurrect animals or to maintain genome stocks from tissues that have been frozen for prolonged periods or even when no live cells are available. In such cases, all the anticipated clones would be the same gender as the donor and they could never propagate by natural breeding. In a recent cloning experiment, we obtained a female mouse from an immature Sertoli cell. This ‘male-derived female’ clone grew into a normal adult and produced offspring by natural mating [132]. Although this was an accidental phenomenon arising from a sex chromosomal error, the result unequivocally suggests the possibility of producing females from male donor animals if the techniques of sex chromosome manipulation are sufficiently well developed.

(b) The possibility of selecting high-quality embryos before transfer

In addition to epigenetic alterations, genetic abnormalities arising during early cleavage stages, such as chromosomal abnormalities [105,133,134], might also be reasons for the low success rate of cloning. If so, it is probable that the SCNT-derived embryos could develop to term if such epigenetic reprogramming were to occur correctly leading to normal chromosomal segregation. Unfortunately, the morphology and/or rate of development to the blastocyst stage are not significant predictive markers for the full-term development of cloned embryos. However, we have recently succeeded in developing a ‘less-damage’ live-cell fluorescent imaging system optimized for preimplantation mouse embryos [135]. Using this system, we succeeded in selecting chromosomally normal cloned embryos and could improve the cloning success rate after embryo transfer [136]. If these selection and epigenetic modification methods could be combined, exploration of the mechanisms involved in genomic reprogramming would be accelerated and questions about the differences between embryonic cell-derived clones and SCNT-derived clones might be solvable. The cloning success rate per transferred embryo would be sufficient for applications in commercial animal breeding.

(c) Nuclear transfer techniques for analysing germ cell epigenetics

Pre-meiotic germ cells can also be used for constructing diploid embryos by NT, and this can work as a powerful tool to determine the dynamics of epigenetic changes during germ cell development. One important study in this category is the analysis of the genomic imprinting status of germ cells, especially primordial germ cells (PGCs) and gonocytes. Genomic imprinting involves an epigenetic ‘memory’ for parental allele-specific expression in about 100 genes in eutherian mammals [137,138]. Based on the principle that reprogramming in the mature ooplasm does not alter the genomic imprinting, cloned foetuses generated from germ cells are expected to reflect the donor's genomic imprinting status faithfully [139]. Therefore, analysis of foetuses and placentas reconstructed from germ cells might give invaluable information on their genomic imprinting status based on DNA methylation status and gene expression pattern. It is usually difficult to determine the gene expression profile of imprinted genes by conventional direct analysis of germ cells because most imprinted genes are primarily expressed in developing foetuses and placentas [137]. Furthermore, such direct analysis gives us only an averaged picture of a mixed population of germ cells. NT cloning of germ cells can overcome these biological and technical disadvantages. By employing the NT technique, we could analyse the dynamics of the imprinting erasure process in PGCs at 11.5 dpc and found the coordinated order of erasure specific for each imprinted gene [57]. This is consistent with the results from a comprehensive genomic analysis using PGCs at each developmental stage [140]. We identified the ‘default status’ of each imprinted gene either as a biallelic expression or as no expression, which were achieved in PGCs by 12.5 dpc. Similarly, this experimental system can also be applied to pre-meiotic male germ cells such as gonocytes and spermatogonia. According to the gene expression pattern of foetuses generated from these cells, the paternal genomic imprinting was thought to be imposed by 16.5 dpc for both the H19-DMR (differentially methylated region) and IG-DMR (A. Ogura, K. Inoue, unpublished data). Furthermore, by using germ cells at different developmental stages (PGCs, gonocytes, germline stem cells, round spermatids and parthenotes), we are now examining which genome ensures the correct Xist expression after NT. The results from RNA FISH and quantitative mRNA expression analysis imply that, for the Xist gene, transcription from the 4-cell stage onwards is the default pattern, and some unknown imprinting machinery, probably imposed during oogenesis, represses this transcription (A. Ogura, K. Inoue, unpublished data). This is consistent with the previous finding that the X chromosome derived from non-growing oocytes was inactivated whereas that from fully grown oocytes remained active [141]. Interestingly, the establishment of this Xist-repressing mechanism was independent of de novo DNA methylation [142].

We also expect that NT using germ cells will provide important clues in understanding the factors that ensure the normal process of genomic reprogramming for producing totipotency. Our recent findings on the phenotypes of cloned embryos and offspring derived from PGCs at 10.5 dpc suggested a somatic cell nature of their genome they showed stage-specific developmental arrest and hyperplastic placentas as those derived from SCNT [56]. Therefore, at some time during germ cell development between 10.5 dpc and the mature gamete stage, the germ cell genome acquires the ability to be reprogrammed fully for the beginning of new life. Recently developed high-resolution and genome-wide epigenetic analysis of germ cells could be combined effectively to achieve this research goal [143].


Somatic cell cloning always produces female offspring? - Biology

PART III. MOLECULAR BIOLOGY, CELL DIVISION, AND GENETICS

11. Applications of Biotechnology

11.3. The Genetic Modification of Organisms

For thousands of years, civilizations have attempted to improve the quality of their livestock and crops. Cows that produce more milk or more tender meat were valued over those that produced little milk or had tough meat. Initial attempts to develop improved agricultural stocks were limited to selective breeding programs, in which only the organisms with the desired characteristics were allowed to breed. As scientists asked more sophisticated questions about genetic systems, they developed ways to create and study mutations.

Although this approach was a very informative way to learn about the genetics of an organism, it lacked the ability to create a specific desired change. Creating mutations is a very haphazard process. However, today the results are achieved in a much more directed manner using biotechnology’s ability to transfer DNA from one organism to another. Transformation takes place when a cell gains new genetic information from its environment. Once new DNA sequences are transferred into a host cell, the cell is genetically altered and begins to read the new DNA and produce new cell products, such as enzymes. The resulting new form of DNA is called recombinant DNA.

A clone is an exact copy of biological entities, such as genes, organisms, or cells. The term refers to the outcome, not the way the results are achieved. Many whole organisms “clone” themselves simply by how they reproduce bacteria divide by cell division and produce two genetically identical cells. Strawberry plants clone themselves by sending out runners and establishing new plants. Many varieties of fruit trees and other plants are cloned by making cuttings of the plant and rooting the cuttings. With the development of advanced biotechnology techniques, it is now possible to clone specific genes from an organism. It is possible to put that cloned gene into the cell of an entirely different species.

Genetically Modified Organisms

Genetically modified (GM) organisms contain recombinant DNA. Viruses, bacteria, fungi, plants, and animals are examples of organisms that have been engineered so that they contain genes from at least one unrelated organism.

As this highly sophisticated procedure has been refined, it has become possible to splice genes quickly and accurately from a variety of species into host bacteria or other host cells by a process called gene cloning (How Science Works 11.4). Genetically modified organisms are capable of expressing the protein-coding regions found on recombinant DNA. Thus, the organisms with the recombinant DNA can make products they were previously unable to make. Since they can rapidly reproduce to large numbers, industrial-sized cultures of bacteria can synthesize large quantities of proteins. For example, recombinant DNA procedures are responsible for the production of:

• Human insulin, used in the control of diabetes (figure 11.6)

• Nutritionally enriched “golden rice,” capable of supplying poor people in less developed nations with beta-carotene, which is missing from normal rice

• Interferon, used as an antiviral agent

• Human growth hormone, used to stimulate growth in children lacking this hormone

• Somatostatin, a brain hormone implicated in growth.

The primary application of GM technology is to put herbicide-resistance or pest-resistance genes into crop plants. Edible GM crops are used mainly for animal feed. In agricultural practice, two kinds of genetically modified organisms have received particular attention. One involves the insertion of genes from a specific kind of bacterium called Bacillus thuringiensis israeliensis (Bti). Bti produces a protein that causes the destruction of the lining of the gut of insects that eat it. It is a natural insecticide. To date, the gene has been inserted into the genetic makeup of several crop plants, including corn. In field tests, the genetically engineered corn was protected against some of its insect pests, but there was some concern that pollen grains from the corn might be blown to neighboring areas and affect nontarget insect populations. In particular, a study of monarch butterflies indicated that populations of butterflies adjacent to fields of this genetically engineered corn were negatively affected. One could argue that since the use of Bti corn results in less spraying of insecticides in cornfields, this is just a trade-off.

FIGURE 11.6. Human Insulin from Bacteria

The gene-cloning process is used to place a copy of the human insulin gene into a bacterial cell. As the bacterial cell reproduces, the human DNA it contains is replicated along with the bacterial DNA. The insulin gene is expressed along with the bacterial genes and the colony of bacteria produces insulin. This bacteria-produced human insulin is both more effective and cheaper than previous therapies, which involved obtaining insulin from the pancreas of slaughtered animals.

A second kind of genetically engineered plant involves inserting a gene for herbicide resistance into the genome of certain crop plants (figure 11.7a). The value of this to farmers is significant. For example, a farmer could plant cotton with very little preparation of the field to rid it of weeds. When both the cotton and the weeds begin to grow, the field could be sprayed with a specific herbicide that would kill the weeds but not harm the herbicide-resistant cotton. This has been field-tested and it works. Critics have warned that the genes possibly could escape from the crop plants and become part of the genome of the weeds that we are trying to control, thus creating “super-weeds.”

Many more products have been manufactured using these methods. Genetically modified cells are not only used as factories to produce chemicals but also for their ability to break down many toxic chemicals. Bioremediation is the use of living organisms to remove toxic agents from the environment. There has been great success in using genetically modified bacteria to clean up oil spills and toxic waste dumps.

FIGURE 11.7. Application of Genetically Modified Organisms

Soybeans, corn, cotton, Hawaiian papaya, tomatoes, rapeseed, sugarcane, sugar beets, sweet corn, and rice are a short list of GM crops being grown and sold. (a) One of the most important applications of this technology involves the insertion of genes that make a crop plant resistant to herbicides. Therefore, the field can be sprayed with an herbicide and kill the weeds without harming the crop plant. (b) Normal rice does not produce significant amounts of beta-carotene. Beta-carotene is a yellow-orange compound needed in the diet to produce vitamin A. (c) Genetically modified “golden rice” can provide beta-carotene to populations that have no other sources of this nutrient.

Cloning a specific gene begins with cutting the source DNA into smaller, manageable pieces with restriction enzymes. Next, there are several basic steps that occur in the transfer of DNA from one organism to another:

1. The source DNA is cut into a usable size by using restriction enzymes.

The source DNA is usually isolated from a large number of cells. Therefore, It consists of many copies of an organism's genome. The source DNA is cut into many small fragments with restriction enzymes. Isolating the small portion of DNA that contains the gene of interest can be difficult because the gene of interest is found on only a few of these fragments. To identify the desired fragments, scientists must search the entire collection. The search involves several steps.

2. The DNA fragments are attached to a carrier DNA molecule.

The first step is to attach every fragment of source DNA to a carrier DNA molecule. A vector is the term scientists use to describe a carrier DNA molecule. Vectors usually contain special DNA sequences that facilitate attachment to the fragments of source DNA. Vectors also contain sequences that promote DNA replication and gene expression.

A plasmid is one example of a vector that is used to carry DNA into bacterial cells. A plasmid is a circular piece of DNA that is found free in the cytoplasm of some bacteria. Therefore, the plasmid must be cut with a restriction enzyme, so that the plasmid DNA will have sticky ends, which can attach to the source DNA. The enzyme ligase creates the covalent bonds between the plasmid DNA and the source DNA, so that a new plasmid ring is formed with the source DNA inserted into the ring. The plasmid and its inserted source DNA is recombinant DNA. Because there are many different source DNA fragments, this process results in many different plasmids, each with a different piece of source DNA. All of these recombinant DNA plasmids constitute a DNA library for the entire source genome.

3. The carrier DNA molecule, with its attached source DNA, is moved into an appropriate cell for the carrier DNA. In the cell, the new DNA is replicated or expressed.

The first step in cloning a specific gene is to cut the source DNA into smaller, manageable pieces with restriction enzymes.

The source DNA is cut with restriction enzymes to create sticky ends. The vector DNA (orange) has compatible sticky ends, because it was cut with the same restriction enzyme. The enzyme ligase is used to bond the source DNA to the vector DNA.

The second step in the cloning process is to mix the DNA library with bacterial cells that will take up the DNA molecules. Transformation occurs when a cell gains genetic information from its environment. Each transformed bacterial cell carries a different portion of the source DNA from the DNA library. These cells can be grown and isolated from one another.

Bacterial cells pick up the plasmids with recombinant DNA and are transformed. Different cells pick up plasmids with different genomic DNA inserts.

The third step is to screen the DNA library contained within the many different transformed bacterial cells to find those that contain the DNA fragment of interest. Once the bacterial cells with the desired recombinant DNA are identified, the selected cells can be reproduced and, in the process, the desired DNA is cloned.

Screening the DNA Library

A number of techniques are used to eliminate cells that do not carry plasmids with attached source DNA. Once these cells are eliminated from consideration, the remaining cells are screened to find those that contain the genes of interest.

Genetically Modified Foods

Although some chemicals have been produced in small amounts from genetically engineered microorganisms, crops such as turnips, rice, soybeans, potatoes, cotton, corn, and tobacco can generate tens or hundreds of kilograms of specialty chemicals per year. Such crops have the potential of supplying the essential amino acids, fatty acids, and other nutrients now lacking in the diets of people in underdeveloped and developing nations. Researchers have also shown, for example, that turnips can produce interferon (an antiviral agent), tobacco can create antibodies to fight human disease, oilseed rape plants can serve as a source of human brain hormones, and potatoes can synthesize human serum albumin that is indistinguishable from the genuine human blood protein (figure 11.7b and c).

Many GM crops also have increased nutritional value yet can be cultivated using traditional methods. There are many concerns regarding the development, growth, and use of GM foods. Although genetically modified foods are made of the same building blocks as any other type of food, the public is generally wary. Countries have refused entire shipments of GM foods that were targeted for hunger relief. However, we may eventually come to a point where we can no longer choose to avoid GM foods. As the world human population continues to grow, GM foods may be an important part of meeting the human population’s need for food. The following are some of the questions being raised about genetically modified food:

• Is tampering with the genetic information of an organism ethical?

• Is someone or an agency monitoring these crops to determine if they are moving beyond their controlled ranges?

• What safety precautions should be exercised to avoid damaging the ecosystems in which GM crops are grown?

• What type of approval should these products require before they are sold to the public?

• Is it necessary to label these foods as genetically modified?

The field of biotechnology allows scientists and medical doctors to work together and potentially cure genetic disorders. Unlike contagious diseases, genetic diseases cannot be transmitted, because they are caused by a genetic predisposition for a particular disorder—not separate, disease-causing organisms, such as bacteria and viruses. Gene therapy involves inserting genes, deleting genes, and manipulating the action of genes in order to cure or lessen the effect of genetic diseases. These therapies are very new and experimental. While these lines of investigation create hope, many problems must be addressed before gene therapy becomes a reliable treatment for many disorders.

The strategy for treating someone with gene therapy varies, depending on the disorder. When designing a gene therapy treatment, scientists have to ask exactly what the problem is. Is the mutant gene not working at all? Is it working normally but there is too little activity? Is there too much protein being made? Or is the gene acting in a unique, new manner? If there is no gene activity or too little gene activity, the scientists need to introduce a more active version of the gene. If there is too much activity or if the gene is engaging in a new activity, this excess activity must first be stopped and then the normal activity restored.

To stop a mutant gene from working, scientists must change it. This typically involves inserting a mutation into the protein-coding region of the gene or the region that is necessary to activate the gene. Scientists have used some types of viruses to do this in organisms other than humans. The difficulty in this technique is to mutate only that one gene without disturbing the other genes and creating more mutations in other genes. Developing reliable methods to accomplish this is a major focus of gene therapy. Once the mutant gene is silenced, the scientists begin the work of introducing a “good” copy of the gene. Again, there are many difficulties in this process:

• Scientists must find a way of returning the corrected DNA to the cell.

• The corrected DNA must be made a part of the cell’s DNA, so that it is passed on with each cell division, it doesn’t interfere with other genes, and it can be transcribed by the cell as needed (figure 11.8).

• Cells containing the corrected DNA must be reintroduced to the patient.

One method of introducing the correct genetic information to a cell is to use a virus as a vector. Here, a dog is treated for a degenerative disorder of the retina. The normal gene is spliced into the viral genome. The virus is then used to infect the defective retinal cells. When the virus infects the retinal cells, it carries the functional gene into the cell.

Cloning does not always refer to exchanging just a gene. Another type of cloning is the cloning of an entire organism. In this case, the goal is to create a new organism that is genetically identical to the previous organism. Cloning of multicellular organisms, such as Protists, plants, fungi and many kinds of invertebrate animals, often occur naturally during asexual reproduction and is duplicated easily in laboratories. The technique used to accomplish cloning in vertebrates is called somatic cell nuclear transfer. Somatic cell nuclear transfer removes a nucleus from a cell of the organism that will be cloned. After chemical treatment, that nucleus is placed into an egg cell that has had it original nucleus removed. The egg cell will use the new nucleus as genetic information. In successful cloning experiments with mammals, an electrical shock is used to stimulate the egg to begin to divide as if it were a normal embryo. After transferring the egg with its new nucleus into a uterus, the embryo grows normally. The resulting organism is genetically identical to the organism that donated the nucleus.

In 1996, a team of scientists from Scotland successfully carried out somatic cell nuclear transfer for the first time in sheep. The nucleus was taken from the mammary cell of an adult sheep. The embryo was transplanted into a female sheep’s uterus, where it developed normally and was born (figure 11.9). This cloned offspring was named Dolly. This technique has been applied to many other animals, such as monkeys, goats, pigs, cows, mice, mules, and horses, and has been used successfully on humans. However, for ethical reasons, the human embryo was purposely created with a mutation that prevented the embryo from developing fully. The success rate of cloning animals is still very low for any animal, however only 3-5% of the transplanted eggs develop into adults (figure 11.10).

FIGURE 11.9. Cloning an Organism

The nucleus from the donor sheep is combined with an egg from another sheep. The egg’s nucleus had previously been removed. The egg, with its new nucleus, is stimulated to grow by an electrical shock. After several cell divisions, the embryo is artificially implanted in the uterus of a sheep, which will carry the developing embryo to term.

FIGURE 11.10. Success Rate in Cloning Cats

Out of 87 implanted cloned embryos, CC (Copy Cat) is the only one to survive. This is comparable to the success rate in sheep, mice, cows, goats, and pigs. (a) Notice that CC is completely unlike her tabby surrogate mother. (b) “Rainbow” is her genetic donor, and both are female calico domestic shorthair cats.

A cloning experiment has great scientific importance, because it represents an advance in scientists’ understanding of the processes of determination and differentiation. Recall that determination is the process a cell goes through to select which genes it will express. A differentiated cell has become a particular cell type because of the proteins that it expresses. Differentiation is more or less a permanent condition. The techniques that produced Dolly and other cloned animals use a differentiated cell and reverse the determination process, so that this cell is able to express all the genes necessary to create an entirely new organism. Until this point, scientists were not sure that this was possible.

9. A scientist can clone a gene. An organism can be a clone. How is the use of the word clone different in these instances? How is the use of the word clone the same in both uses?

10. What are some of the advantages of creating genetically modified (GM) foods? What are some of the concerns?

11. Describe how viruses are used in gene therapy.

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The Embryo Project Encyclopedia

In the second half of the twentieth century, scientists learned how to clone some species of mammals. Scientists have applied somatic cell nuclear transfer to clone human and mammalian embryos as a means to produce stem cells for laboratory and medical use. Somatic cell nuclear transfer (SCNT) is a technology applied in cloning, stem cell research, and regenerative medicine. Somatic cells are cells that have gone through the differentiation process and are not germ cells. Somatic cells donate their nuclei, which scientists transplant into eggs after removing their nucleuses (enucleated eggs). Therefore, in SCNT, scientists replace the nucleus in an egg cell with the nucleus from a somatic cell.

Although Karl Illmensee first cloned a mammal in 1981, other scientists had theorized and developed the techniques needed for SCNT in the form of nuclear transfer. Hans Spemann, who taught zoology at the University of Freiburg in Freiburg, Germany, theorized about SCNT in his 1938 book Embryonic Development and Induction. Spemann proposed to transplant a nucleus from already differentiated cell from an embryo into an egg after removing the egg's nucleus. However, the technology required for this kind of experiment was not available to Spemann at that time, so he could not test his theory of nuclear transfer or SCNT. Robert King and Thomas Briggs developed the necessary protocol to conduct preliminary nuclear transfer at the Institute for Cancer Research and Lankenau Hospital Research Institute in Philadelphia, Pennsylvania, in 1952. The same nuclear transfer techniques serve as the basis for SCNT.

While researching how embryos differentiate in 1952, Briggs and King transplanted the nucleus from an early embryonic blastula cell of a Rana pipiens frog embryo to an unfertilized egg after removing its nucleus. To enucleate the eggs, Briggs and King used a small glass needle to puncture the cell membrane, enter the cytoplasm, and suck out the nucleus of the egg cell. Briggs and King then transplanted the donor nucleus from a separate blastula cell to replace the nucleus that they removed from the egg cell. Briggs and King observed that the embryo developed normally.

Researchers struggled to clone mammals using the same procedure that Briggs and King used on frogs. In 1975, Derek Bromhall in Oxford, UK, conducted experiments using rabbit embryos and showed that, after a certain stage in development called the morula stage, embryos produced from nuclear transfer died. Bromhall hypothesized that they died as the result of complications from the punctures made in the cell membrane during the transfer.

Scientists performed nuclear transfer only on amphibians until 1981, when Illmensee in Geneva, Switzerland, claimed to have cloned mice using nuclear transfer technique. His work resulted in the birth of three live mice. Illmensee's experiments came under scrutiny and an investigation occurred concerning the veracity of his claims. Although the investigators never found conclusive evidence against Illmensee, the investigation cast doubts as to whether or not he had used nuclear transfer to clone the mice.

Scientists struggled to perform nuclear transfer on mammals larger than mice. Steen Willadsen at the Institute of Animal Physiology in Babraham Institute in Babraham, United Kingdom, was the first to clone a sheep embryo in 1984. Willadsen modified the technique of Briggs and King. After transferring the nucleus, Willadsen fused the embryo together using an electrofusion apparatus that has small electrodes that produce an electrical current. Willadsen coated the embryo with an agar jelly made from algae to reduce the damage caused by entry of the glass needle into the cell membrane. Once he had coated the embryos with agar jelly, Willadsen placed the embryos into the tied oviducts of a sheep, and he observed that the embryos were growing. From this experiment, Willadsen made viable mammalian embryos using his modified techniques, but they didn't grow into adult organisms.

In 1996, Keith Campbell, Jim McWhir, William Ritchie, and Ian Wilmut at the Roslin Institute in Edinburgh, UK, used nuclear transfer techniques to clone a sheep that was born and gre into an adult. The team manipulated a stage in the cell cycle called quiescence, when the cell undergoes a period of supposed hibernation and ceases to develop. Campbell induced quiescence in the donor blastocyst nuclei before transferring them to recipient egg cells by depriving the cells of proteins called growth factors. The change in the state of the donor nuclei before entering the receiving egg cells enabled embryos to develop to term in surrogate ewes.

According to Wilmut, the next experiment applied the same procedure to the nucleus of a fully differentiated adult cell as opposed to a blastocyst cell. The Roslin team hypothesized that the nuclear transfer procedure started by Briggs and King could be applied to somatic cells, thus becoming somatic cell nuclear transfer as opposed to just nuclear transfer. The Roslin Institute performed this step in 1997. The result of the experiment was Dolly the sheep.

Dolly was the first mammal cloned from a fully differentiated adult cell. The main difference in the techniques producing Dolly was that the scientists used adult cell nuclei as opposed to the embryonic cell nuclei used in previous sheep experiments. After Dolly was born, the scientists applied these techniques in genetically modified mammalian embryos. Quiescence enabled the scientists to perform genetic modifications on the nucleus of the cell because growth factors were not altering the inserted DNA. In 1997, a Roslin Institute team used similar techniques to genetically modify Polly the sheep to express a human protein. After the success of Dolly and Polly, some scientists worked to clone human embryos using SCNT, however there were social, ethical and legal controversies over the practice. Many disagreed with the claims that scientists could or should clone, or perhaps genetically modify, humans using SCNT.

Scientists sought ways to clone human embryos without causing controversy. In 2011, Scott Noggle and his team at the New York Stem Cell Foundation in New York, New York, used SCNT to retrieve human embryonic stem cells. Although, Noggle's team did not perform SCNT using the same methods that produced Dolly. In fact, Noggle and his colleagues aimed to avoid the social and ethical implications of working with human embryos. Instead of removing the nucleus of the receiving egg cell before transfer, the scientists kept the egg nucleus and inserted the donor nucleus into the egg cell. As a result, the embryo developed into the blastocyst stage where scientists could extract stem cells. The chromosome count, however, was sixty-nine as opposed to the normal forty-six, because it contained the chromosomes from the full nucleus as well as the egg nucleus, which only contains half, or twenty-three of the chromosomes in a zygote. This result meant that the blastocyst could not result in a pregnancy leading to birth because the cells would not progress to a further developmental state. Embryonic stem cells are derived from these blastocyst cells.

Scientists report that SCNT is a plausible technique for creating human embryonic stem cells without extra chromosomes. In 2013, scientists in Oregon succeeded in using SCNT to reprogram somatic cells to become embryonic stem cells. After examining Noggle's research, Masahito Tachibana and his team at the Oregon National Primate Research Center in Hillsboro, Oregon, used the same methods that Campbell and his team had used to clone Dolly, but they also added a few extra procedures. The key differences were that they removed the spindle apparatus, which is a responsible for movement of chromosomes in cellular mitosis and meiosis, from the donor egg cell before removing the egg cell nucleus. They reinserted the spindle apparatus into the cell when they inserted the donor nucleus. After removing the spindle apparatus, they also added caffeine, which inhibits the enzyme protein phosphatase, which activates proteins that begin cellular replication in the cytoplasm. Because the spindle apparatus was removed and the cytoplasm inactivated, the scientists could perform their procedures without risk of premature activation of the cell resulting in cellular damage and death. The results of the experiment showed that the cells altered with SCNT reached the blastocyst stage and produced viable embryonic stem cell lines of normal chromosome count. As of 2014, doctors use this version of SCNT for medical therapies and treatment, described as therapeutic cloning.

Controversies due to SCNT largely arise from the possibility of cloning humans. In 2003, a private company called Clonaid headquartered in Las Vegas, Nevada, claimed to have cloned the first human baby, called Eve, using SCNT. However, Clonaid did not allow scientists to perform a DNA test on Eve to confirm that she was indeed a clone, and therefore many in the scientific community doubted their claims. As of 2014, controversies arose over the possibility of human clones from SCNT. Some criticized the scientists who used SCNT to clone human stem cells in Oregon. The Oregon scientists justified their research by claiming that their only goal was to produce embryonic stem cells and not to produce a fully developed human being.


Related Biology Terms

  • Cell – The basic biological unit of living things.
  • Gamete – A sperm or egg cell.
  • Apoptosis – Programmed cell death in which a cell self-destructs.
  • Diploid – A cell with two copies of each chromosome somatic cells are diploid.

1. Which type of cell is NOT a somatic cell?
A. Leukocyte
B. Myocyte
C. Osteoblast
D. Gamete

2. What is the approximate lifespan of an erythrocyte?
A. 3-4 days
B. 5-9 days
C. 100-120 days
D. 365-395 days

3. What is the function of an osteoclast?
A. To form and help maintain bone
B. To attach to bone and allow it to move
C. To resorb old bone
D. To release neurotransmitters


Watch the video: Somatic Cell Nuclear Transfer Presentation (August 2022).