Gene Transfer | BiotechStudies

Gene Transfer by Nuclear Transfer

• Nuclear transfer, the introduction of the nucleus from a cell into an enucleated egg cell (an egg cell that has had its own nucleus removed). This can be accomplished through fusion of the cell to the egg or through the direct removal of the nucleus from the cell and the subsequent transplantation of that nucleus into the enucleated egg cell. The donor nucleus used for nuclear transfer may come from an undifferentiated adult cell (somatic cell); in the latter case, the technique is called somatic cell nuclear transfer (SCNT).
• SCNT involves taking any differentiated somatic cell’s nucleus, for example, skin and muscle cells, and placing that nucleus into an unfertilized egg cell. The egg’s own nucleus has already been removed. This initiates a process called nuclear reprogramming, which causes the donor nucleus to become pluripotent again by certain chemicals in the egg’s cytoplasm. The combined hybrid egg can develop into an embryo without normal fertilization, and the ES cells can be extracted.
• SCNT avoids or at least minimizes the potential ethical issues that other embryo sources, such as surplus in-vitro fertilization embryos, have encountered in many countries including Australia.
• The concept of nuclear transfer was first conceived in 1928 by German embryologist Hans Spemann, who initially experimented with transferring salamander embryonic cell nuclei into egg cells.
• In the 1990s British developmental biologist Ian Wilmut and his team of scientists at Roslin Institute in Scotland used nuclear transfer to produce sheep clones, the best known of which was the Finn Dorset sheep Dolly, born in 1996. Dolly was created through cell fusion and nuclear transfer of a differentiated cell and an enucleated egg cell.

Somatic body cell with desired genes

Stem Cells in Generals

ES cells are pluripotent, which means they are the first cells to form in embryos. Subsequently, they will undergo continual division to become all the cell type in our bodies. Stem cells found in two places in human:
(1) In a developing embryo called blastocyst. Only the inner cell mass contains stem cells. The surrounding shell of trophoblast cells do not- they give rise to the placenta, instead.
(2) Adult and child tissues such as bone marrow and skin contain stem cells, which produce new cells to regularly replace old/dead cells. However they are rarer and difficult to identify amongst the crowded somatic cells, and do not survive long enough outside the human body for potential therapeutic use.

Methodology

The actual techniques in SCNT are common but they differ by the materials and equipment used. Stated below is an outline of the steps taken in order to perform SCNT for humans.

Step 1: Preparation of the somatic cell

The somatic cell can be any type of normal cell in body apart from sperm or egg. A tiny amount of skin is cut and placed in trypsin enzyme-buffer solution that frees the target fibroblasts from the extracellular matrix. The mixture is placed in serum 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

Researchers select the target egg that is the antral stage and exhibits the 1st polar body. When follicles are seen at least 18mm wide, hCG is injected into the female donor. hCG is used since it is a strong inducer and allows more comfortable way of obtaining egg without any invasive and direct surgical procedure. The ovulated egg is collected by ultrasound guided transvaginal needle aspiration.
Once the egg is extracted, it is placed in liquid human serum. The egg’s nucleus is removed using an inverted microscope, UV light and a glass needle. This setup minimizes damage to the delicate egg as it can cut through the thick zona pellucid shell. At this point with its nucleus removed, the oocyte is called a cytoplast.

Generating cytoplasts by oocyte enucleation

 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. 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.

An adult cell nucleus us injected into a encucleated egg

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 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 37•C in highly humified conditions. This could be both artificial attempt and natural requirement that replicates the uterine conditions. After this activation, for approx four days cleavage of egg can be clearly seen.

Step 5: Embryogenesis 

Once the hybrid cell has developed into a blastocyst what happens to it from this point depends on its application. It can be used for reproductive cloning (creating an entire organism), for applications in regenerative medicine (obtaining specific cell/tissue types that can be surgically grafted for a patient).

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.
• The SCNT technique has the potential to be used to reproduce extinct species. This advantage of SCNT has been proved to work by experiment conducted on mice.

 Disadvantages of SCNT

• Adverse effects of somatic nucleus choice- Somatic nucleus adversely determine cloned offspring’s post birth growth.
• Inefficient steps- i.e. maximum 4% non-human embryos 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.
• Electric fusion damage- Electroporation becomes 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.
• Cross-species incompatibilities- Irreconcilable genetic differences between egg and nucleus species leads to low success rate; that is, mice eggs should not be mix with human nuclei.
Applications of SCNT:-
Veterinary, Animal Science
• 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 human welfare, if integrated in breeding programs and these transgenic clones will be delivering the expected products.
• Conserving wild animals for next generation: This use can be effective to preserve and propagate endangered species that are being produced poorly in the zoos. Can also be helpful to even create extinct species, if any tissue or cells are available.
• Disease resistant animal production: Genes causing diseases can be manipulated in order to have healthier farm animals that live a lot longer.

Human Medicine

Human therapeutic proteins:
 human proteins are needed and are in demand for treatment of diseases. Purifying proteins from blood is an expensive procedure and also carries the risk of contamination by hepatitis C or HIV. Proteins can be produced in human cell but the output is small so transgenic animals like sheep, goats and cattle can be used to produce human proteins in milk.
• Xenotranplantation: Shortage of organs is a big problem considering the amount of patients needing them. Transplantation of organs is the solution for this. Genetically modified animals like pigs are being developed for this.
• Animal model for diseases: Experimental animals with altered disease causing genes can be tailor generated using SCNT, allowing better understanding of the complex pathogenesis of the disease.
• Cell therapy using dedifferentiated stem cells: This use is being developed for a range of diseases including heart attack, stroke and diabetes etc.