Nuclear Transplantation

nuclear transplantation refers to moving of a cell nucleus and its genetic material from one cell to another cell. Nuclear transfer is a form of cloning. If the cloned cells are placed in the uterus of a female mammal, a cloned organism develops to term in rare instances. This is how Dolly the Sheep and many other species were cloned. Cows are commonly cloned to select those that have the best milk production. Nuclear transfer is a two-part process: first, scientists remove the nucleus from an egg, and second, they replace it with the nucleus of an older donor cell. A new clonea genetic copy of the donorforms when the egg starts to divide. Nuclear transplantation experiment with frog:

Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is articially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extra embryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments.

Process:
The actual techniques in SCNT are usually involves the nucleus of a somatic cell (eg. a normal body cell such as a blood cell, heart cell/cardiocyte, skin cell/fibroblast; the sperm and egg are germ cells not somatic cells) being physically transferred into an unfertilised egg cell that has had its own nucleus removed (referred to as 'enucleation'). There Are Generally These steps are involved: Step 1: Preparation of the somatic cell The somatic cell, as stated, can be any type of normal cell in the body apart from the sperm or egg. Most researchers appear to favour skin fibroblasts, because the skin is easy to access, non-invasive and fairly painless. However, cells from the breast/mammary gland[20] and cumulus cells[21] have also been used. A tiny amount of skin is cut and placed in a trypsin enzyme-buffer solution that frees the target fibroblasts from the extracellular matrix. The mixture is placed on a serum medium and incubated for three weeks, in order to obtain a single layer of fibroblasts without any other cell types. Step 2: Preparation of the egg/oocyte Typically, researchers will select the target egg that is in the antral stage and exhibits the 1st polar body. When the researchers can see follicles at least 18mm wide, human chorionic gonadotropin (hCG) is injected into the female donor. hCG is used since it is a strong inducer of ovulation, and allows a more 'comfortable' way of obtaining the egg without any invasive and direct surgical procedure to the ovaries and the donors themselves. Consequently, the ovulated egg is collected by ultrasound-guided transvaginal needle aspiration in a procedure similar to in-vitro fertilisation. Once the egg is extracted, it is placed in liquid human serum. Fluorescent tags are bound to the oocyte's DNA, allowing the researchers to check all the oocyte's DNA/nucleus has been removed when exposing the egg to UV light.

The egg's nucleus is removed using an inverted microscope, UV light and a glass needle. This setup minimises damage to the delicate egg as it can cut through the thick zona pellucida shell, and is fairly easy to manipulate. At this point with its nucleus removed, the oocyte is called a cytoplast. Step 3: Nuclear Transfer Both fibroblast and egg are placed in a thin human serum solution with cytochalasin B. Once the donor fibroblast's nucleus is extracted from the fibroblast with a pipette, it is called a karyoplast. Subsequently, this karyoplast is injected into the egg/cytoplast past the zona pellucid. At this point, the karyoplast and cytoplast are still functionally separate , therefore a few electric pulses are given to the entire solution causing fusion between the two entities (see Figure 7 at 10 minutes). Step 4: Post Nuclear Transfer Procedures The complete process of nuclear transfer is completed approximately 35-45 hours after the original hCG was administered to the female donor. However, it takes an additional three hours before cleavage can be seen if the transfer and activation has been successful. Finally, the egg is incubated in a culture medium at 37C in highly humidified conditions. This could be both an artificial attempt and natural requirement that replicates the uterine conditions, which are conducive to embryonic development. After this activation, in approximately four days for human donors, cleavage of the egg can be clearly seen. Step 5: Embryogenesis Once the hybrid egg has developed into a blastocyst, what happens to it from this point depends on its application: For reproductive cloning (creating an entire organism): the blastocyst is implanted into a surrogate mother who carries the developing embryo like a normal pregnancy.

For applications in regenerative medicine (obtaining specific cell/tissue types that can be surgically grafted for a patient): the ICM is harvested from the blastocyst onto mice-derived feeder cells for nutrients and differentiated into the required tissue/cell types, using certain growth and differentiation factors over two days.

The actual differentiation factors required for specific somatic cells has been determined over the years by many different researchers, for example, stem cells exposed to dimethyl sulfoxide would diffentiate into different proportions of muscle cells, while stem cells exposed to retinoic acid would become neurons.

Applications of SCNT
Conserving wild animals for next generations: Another area where SCNT can be useful is conservation. This use can be effective to preserve and propagate endangered species that are being produced poorly in the zoos. With effective reproduction, these species can be reintroduced to the wild again, allowing maintenance of genetic diversity of species by introducing new genes. The use of SCNT can also be helpful to even create the extinct species, if any tissues or cells are available. The idea of producing mammoth is being considered as an intact animal was discovered frozen in the tundra. The close relative of the mammoth, the elephant, could be used both as a surrogate mother and an oocyte donor. Mass production of animals: As farm animals are being used for human use, SCNT can be used to produce high quality farm animals in infinite number. Cloning technology can be applied, without compromising animal welfare, if integrated in breeding programs and these transgenic clones will be delivering the expected products. Researches show that, somatic cell cloned cattle reportedly were physiologically, immunologically, and behaviorally normal and this makes use of SCNT useful for mass production.

*****The Cloning of Dolly (example):

Disease resistant animal production: By using SCNT, genes causing diseases can be manipulated in order to have healthier farm animals that live a lot longer. Xenotransplantation: Shortage of organs is a big problem considering the amount of patients needing them. Transplant organs can be a solution for this. Genetically modified animals such as pigs are being developed as a solution but so far the modifications are limited to adding genes. By using SCNT, deleting genes that are responsible for rejection from pigs is possible and this way it is aimed to avoid rejection of an organ transplanted from a normal pig to a human patient. Events during fertilization and pre-implantation embryos: knowledge of the complex mechanisms and the various controlling factors during embryonic development will be understood better with SCNT. Cell Therapy using dedifferentiated stem cells: This use is being developed for a range of diseases including heart attack, stroke and diabetes etc. Patients own cell can be used as transplanted cells are likely to be rejected. Cloning of adult animals shows that egg and the embryo have the capability of reprogramming. This use may make it possible to reprogramming patients own cells without creating and destroying embryos. The differentiating stem cells can then be grown into several hundred or thousand cells and surgically transported into the patient where they will produce the required tissue. To give an example, a child's problem of severe immnunodefficiency due to chemotheraphy and whole body radiation because of having Hodgkin's Lymphoma, can be corrected as child's skin fibroblast can be obtained and embryonic stem cells obtained as per the procedures mentioned above. Transcription factors would differentiate the stem cells into bone marrow cells, which can be transplanted into the marrow cavities of the child, and they would gradually rebuild the child's haemopoietic system and also, their immune system.

Advantages of SCNT
Retains genetic code of the donor nucleus: Resultant tissue that is transplanted into the donor patient will not be exposed to potentially fatal immune rejection. Common Procedures and Methods: Relatively similar and uniform between different researchers - the main difference appears to be the choice of culture mediums which do not seem to affect the actual outcome. Easy Access: Easily obtainable equipment and growth substrates via purchases from biotechnology and biological supply companies.

Disadvantages of SCNT
Adverse effects of somatic nucleus choice: Somatic nucleus adversely determines cloned offspring's post-birth growth, eg. cumulus cell lead to morbidly obese adults, and Sertoli cells lead to premature deaths of offspring. Inefficient steps: ie. maximum 4% non-human embryoes become live offspring, while others display fatal abnormalities resulting in spontaneous loss with current techniques and technology. Difficulty in inducing the re-expression of differentiated genes: This is especially the case where the donor nuclei have been obtained from adults as opposed to fetal or newborn stage. This phenomenon appears to be in common with in vitro fertilisation in human and non-human species, where higher rates of spontaneous pregnancy terminations occur with adult-age nuclei, as opposed to higher term births with fetal/embryonic nuclei. Electric Fusion damage: Eiges and colleagues have suggested electroporation, which has been demonstrated to be fatal to stem cell survival in other non-SCNT techniques, may disrupt the delicate interaction between the fusing oocyte and somatic nucleus, thereby preventing them from communicating properly. Other methods for cytoplast-karyoplast fusion in SCNT have not been assessed yet. Cross-species incompatibilities: Gurdon et al. suggests irreconcilable genetic differences between egg and nucleus species leads to low success rate; that is, mice eggs should not be mixed with human nuclei. Though amphibian and mammal sources were used by Gurdon and colleagues, there has been no other evidence to suggest why this incompatibility could not affect human-only procedures as well.