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Animal biotech

Plasma Clot
In this technique, the explant is cultured on the surface of a clot consisting of chick (or any other) plasma as per the requirement and chick embryo extract contained in a watchglass. Hence, it is also called watchglass technique. The watchglass may or may not be closed with a glass lid sealed with paraffin wax. For studying morphogenesis in embryonic organ rudiments this has been the classical standard technique. This technique is well modified to study the action of hormones, vitamins, carcinogens, etc on adult mammalian tissues. A Plasma clot is prepared by mixing five drops of embryo extract with 15 drops of plasma in a watch glass placed on a cotton wool pad. The cotton wool pad is put in a Petri dish. Time to time cotton is moistened so that excessive evaporation should not occur. Thereafter, a small piece of organ tissue is placed on the top of the plasma clot present in the watch glass. In the modified technique the organ tissue is placed into raft of lens paper or Rayon. The raft makes easy to transfer the tissue, excess fluid can also be removed. The media used for a growing organ culture are generally the same as those used for tissue culture. The techniques for organ culture can be classified into (i) those employing a solid medium and (ii) those employing liquid medium. One of the chief disadvantages of all plasma clot methods is that the clot liquefies in the vicinity of explants so that they become partly or fully immersed in the medium. The duration of culture is rather short (less than 4 weeks) and biochemical analysis is not possible due to the complexity of the medium.

Raft method
In this method the explant is placed onto a raft of lens paper or rayon acetate, which is floated on serum in a watch-glass. Rayon acetate rafts are made to float on the serum by treating their four corners with silicone. Similarly, floatability of lens paper is enhanced by treating it with silicone. On each raft, four or more explants are usually kept. In a combination of raft and clot techniques, the explants are first placed on a suitable raft, which is then kept on a plasma clot. This modification makes media changes easy, and prevents the sinking of explants into liquefied plasma.

Agar Gel
The medium (consisting of suitable salt solution, serum, chick embryo extract or a mixture of certain amino acids & vitamins) is gelled with 1% agar. This method avoids the immersion of explants into the medium & permits the use of defined media. Generally the explants need to be subculutured on fresh agar gels every 5-7 days. The agar gels are generally kept in embryological watch glasses & sealed with paraffin wax. This method is used to study many developmental aspects of normal organs as well as tumors. Defined media with or without serum are also used with agar. The medium with agar provides the mechanical support for organ culture. It does not liquefy. Embryonic organs generally grow well on agar, but adult organ culture will not survive on this medium. The culture of adult organs or parts from adult animals is more difficult due to their greater requirement of oxygen. A variety of adult organs (e.g. the liver) have been cultured using special media with special apparatus (Towels II culture chamber). Since serum was found to be toxic, serum-free media were used, and the special apparatus permitted the use of 95% oxygen.

Grid method
Devised by trowel in 1954, this method utilizes 25 mm X 25 mm pieces of suitable wire mesh or perforated stainless steel sheet whose edges are bent to form4 legs of about 4 mm height. Skeletal tissues are generally placed directly on the grid but softer tissues like glands or skin are first placed on rafts, which are then kept on the grids. The grid themselves are then placed on a culture chamber filled with fluid medium up to the grid; the chamber is supplied with a mixture of O2 & Co2 to meet the high O2 requirement of adult mammalian organs. The modification of this method is widely used to study the growth and differentiation of adult and embryonic tissues.

Cryopreservation is a process where cells or whole tissues are preserved by cooling to low subzero temperatures, such as (typically) 77 K or 196 C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, when cryoprotectant solutions are not used, the cells being preserved are often damaged due to freezing during the approach to low temperatures or warming to room temperature. Cryopreservation is generally accomplished through the use of liquid nitrogen, a freezing agent that boils at approximately -300 degrees Fahrenheit (-169 Celsius or 77 Kelvin). Tissues are submerged in the liquid nitrogen, which freezes the water in the cells. In order to prevent cell death due to the freezing process, a vitrification agent is often used. It prevents the water from leaving the cells and allows the tissues to be thawed and used again. Cryopreservation is frequently used to freeze and store the elements used in fertility procedures. Both sperm (semen) and eggs can be successfully frozen and thawed for later use. Fertilized eggs, or embryos, can also be frozen for later use. These embryos, along with their component sperm and eggs, are frozen in stages to prevent cellular death. They are later thawed at about the same rate; allow the tissues to gradually come "back to life."

Rate of Cooling
The rate of cooling is important as this affects the formation of ice crystals. Programmable cell-freezing equipment allows a selection of variable rates for the different portions. Specially designed containers are used to accomplish the uniform rate of cooling. Various methods are used depending on the object being frozen. Uncontrolled cooling is done by placing the containers in a -60-degree C freezer for 90 minutes. Semicontrolled cooling is also conducted in a -60-degree C freezer. Controlled cooling uses a programmable cooling unit to cool the cells at 1 degree C per minute to -40 degrees C.

The process of thawing is rapid, and is done by placing the container in a cold water bath with gentle agitation during the warming phase to accelerate the thawing process while taking care not to damage the matter therein. Transferring the material to a fresh growth medium will avoid any exposure to the cryoprotective agent. Thawing at a water temperature of 37 degrees C is effective in thawing and stabilizing cells.

Slow-Freeze Procedure
The first step of the slow-freeze procedure is to expose the cell to cryoprotectant in a gradual stepwise fashion to slowly allow equilibration of the cell with the cryoprotectant while releasing water. Once the cells have been cleared of the majority of cellular water, the cells in the cryoprotectant fluid are placed into a container of some kind, such as a plastic straw, a glass ampule or a plastic vial. The volume of liquid surrounding the cells for slow freezing is typically less than a teaspoon and may only be a few drops. The pre-labeled container is filled, sealed and put in a programmable freezer, which slowly decreases the temperature of the container over a period of minutes or hours to very low temperatures. After a few minutes of cooling, starter ice crystals must be formed inside the container by "seeding" the container. Seeding is performed by using a tool (for example, forceps) which has been pre-chilled in liquid nitrogen to touch the container and cause ice crystal formation. This starter crystal will initiate controlled ice crystal formation in one spot, safely away from the cells. Once seeding is complete, the rest of the cooling ramps can proceed. When the container reaches temperatures between -30 and -85 C, the container holding the cells can be plunged directly into liquid nitrogen to complete the cooling to -196 C.

Researchers who have developed a new technique, vitrification, claims to provide the benefits of cryopreservation without damage due to ice crystal formation. In clinical cryopreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they lower the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution turns into an amorphous icei.e., it vitrifies. Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid drop in temperature (mega kelvins per second). The rate that is required to attain glassy state in pure water was considered to be impossible until 2005. Two conditions usually required to allow vitrification are an increase in the viscosity and a depression of the freezing temperature. Many solutes do both, but larger molecules generally have larger effect, particularly on

viscosity. Rapid cooling also promotes vitrification. Because freezing is so rapid, the duration of exposure to cryoprotectants is much less, and much higher concentrations of cryoprotectants can be tolerated by the cell. Warming of the cell to return it to normal metabolic functioning must also be incredibly rapid. Handling of vitrified samples is much more exacting than slow-frozen samples because even a brief exposure to a temperature above that of liquid nitrogen can start an unplanned warming process and refreezing, which is detrimental to the cell. In established methods of cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and depress freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as dimethyl sulfoxide, a common cryoprotectant, are often toxic in high concentration. One of the difficult compromises faced in vitrifying cryopreservation is limiting the damage produced by the cryoprotectant itself.

Liquid Nitrogen
Nitrogen does not exist as a liquid; it exists as a gas and is plentiful in the air. Under tremendous pressure, it becomes liquid nitrogen, the cold liquid. Doctors use it to freeze warts and skin lesions, and then scrape them off while they are frozen. Nitrogen has two advantages as a freezing agent. First, it is chemically fairly neutral, so it will not poison the life form that it preserves. Secondly, it is easily manufactured and stored at its liquid temperatures of--196 degrees centigrade, or 321 degrees Fahrenheit. Finally, it is quite cold; liquid oxygen is not nearly so cold, so is a poor freezing agent.

A few humans (including Ted Williams) have been cryogenically frozen. (Contrary to urban legend, Walt Disney is not.) In these cases, the head is removed from the body and cryogenically frozen. In order to revivify these people, several steps must be conquered: 1) curing whatever disease killed the patient; 2) finding a method of revivifying complex creatures; 3) mastering the head transplant; and 4) curing death. Each is a tall order, thus, anyone cryogenically frozen is simply unlikely to ever be revivified.

Biological Samples
Not all cryopreservation is meant to revivify the subject; sometimes, a scientist wishes to preserve a tissue sample (like a biopsy) for later study. Or, the scientist may wish to freeze a sample to take a snapshot; when using a bioreactor to grow a particular type of cell, the scientist may wish to count the cells in a given sample to determine the rate of reproduction. Some frozen blood samples dating back to the 1950s revealed that AIDS has been present in Africa for several decades.

Natural cryopreservation
Water bears (Tardigrada), microscopic multi cellular organisms, can survive freezing at low temperatures by replacing most of their internal water with the sugar trehalose. Sugars and other solutes that do not easily crystallize have the effect of limiting the stresses that damage cell membranes. Trehalose is a sugar that does not readily crystallize. Mixtures of solutes can achieve similar effects. Some solutes, including salts, have the disadvantage that they may be toxic at high concentrations.

Somatic Cell Hybridization

In Somatic Cell Hybridization, the body cells are taken from two varieties of a species. Their cell walls are removed by certain chemicals. Then the protoplasts are fused by chemicals or by very brief high electric current. The resulting fused nucleus is called heterokaryon. Then they are cultured and treated chemically to regenerate cell walls and then they begin to divide to produce plantlets (little plants). Such somatic hybrids have been produced either between the different varieties of the same species (e.g. between non-flowering potato plants an flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale to produce Triticale) Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g. to study the control of cell division and gene expression, to investigate malignant transformations, to obtain viral replication,

for gene or chromosome mapping and for production of monoclonal antibodies by producing hybridoma (hybrid cells between an immortalized cell and an antibody producing lymphocyte), etc.

Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes are fused with mouse continuous cell lines. When human and mouse cells (or cells of any two mammalian species or of the same species) are mixed, spontaneous cell fusion occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet inactivated Sendai (parainfluenza) virus or polyethylene glycol (PEG). These agents adhere to the plasma membranes of cells and alter their properties in such a way that facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cell with two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus.

Generalized scheme
Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouse and 'hybrid' cells) are plated on a selective medium, e.g., HAT medium, which allows the multiplication of hybrid cells only. Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic and appropriate biochemical analyses for the detection of enzyme/ protein/trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a human chromosome in the hybrid clones. If there is a perfect correlation between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait is taken to be located in the concerned chromosome. The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis of purines and pyrimidines from simple sugars and amino acids. However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a salvage pathway, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acids. Hypoxanthine is converted into guanine by the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase (TK); both HGPRT and TK are enzymes of the salvage pathway. On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) cannot divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium). For using HAT medium as a selective agent, man cells used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme of this pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) with TK deficient mouse cells (denoted as TKHGPRT+). Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+ (due to the mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion.

Organismal Cloning
Organismal cloning, the subject of this entry, is the production of genetically identical organisms and, as such, can be used to produce genetically identical copies of livestock or may be used to produce new members of endangered or even extinct species. Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species, including most plants

(see vegetative reproduction) and some insects. Genetic manipulation of whole organism has been happening by sexual reproduction since the beginning of time. The evolutionary progess of almost all linving creatures has involved active interaction between the genomes and the environment. Active control of sexual reproduction has been practiced in agriculture for decades.In more recent times it has been used with several industrial micro-organims , eg, yeasts. It involves selection, mutation, sexual crosses, hybridization etc. However, it is a very random process and can take a long time to achieve the desired results-if at all in some cases. In agriculture, the benefits have been immense, with much improved species of plants and animals, while in the biotechnological industries there have been greatly improved productivities. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows. There is a lot of ethical debate over whether or not cloning should be used. However cloning, or asexual propagation, has been common practice in the horticultural world for hundreds of years.

Reproductive cloning generally uses "somatic cell nuclear transfer" (SCNT) to create animals that are genetically identical. This process entails the transfer of a nucleus from a donor adult cell (somatic cell) to an egg that has no nucleus. If the egg begins to divide normally it is transferred into the uterus of the surrogate mother. Such clones are not strictly identical since the somatic cells may contain mutations in their nuclear DNA. Additionally, the mitochondria in the cytoplasm also contains DNA and during SCNT this DNA is wholly from the donor egg, thus the mitochondrial genome is not the same as that of the nucleus donor cell from which it was produced. This may have important implications for cross-species nuclear transfer in which nuclear-mitochondrial incompatibilities may lead to death. Artificial embryo splitting or embryo twinning may also be used as a method of cloning, where an embryo is split in the maturation before embryo transfer. It is optimally performed at the 6- to 8-cell stage, where it can be used as an expansion of IVF to increase the number of available embryos. If both embryos are successful, it gives rise to monozygotic (identical) twins.

1. Artificial Embryo Twinning Artificial embryo twinning is the relatively low-tech version of cloning. As the name suggests, this technology mimics the natural process of creating identical twins.In nature, twins occur just after fertilization of an egg cell by a sperm cell. In rare cases, when the resulting fertilized egg, called a zygote, tries to divide into a twocelled embryo, the two cells separate. Each cell continues dividing on its own, ultimately developing into a separate individual within the mother. Since the two cells came from the same zygote, the resulting individuals are genetically identical. Artificial embryo twinning uses the same approach, but it occurs in a Petri dish instead of in the mother's body. This is accomplished by manually separating a very early embryo into individual cells, and then allowing each cell to divide and develop on its own. The resulting embryos are placed into a surrogate mother, where they are carried to term and delivered. Again, since all the embryos came from the same zygote, they are genetically identical. 2. Somatic Cell Nuclear Transfer Somatic cell nuclear transfer, (SCNT) uses a different approach than artificial embryo twinning, but it produces the same result: an exact clone, or genetic copy, of an individual. This was the method used to create Dolly the Sheep. To make dolly (first artificially cloned organism) researchers isolated a somatic cell from an adult female sheep. Next, they transferred the nucleus from that cell to an egg cell from which the nucleus had been removed. After a couple of chemical tweaks, the egg cell, with its new nucleus, was behaving just like a freshly fertilized zygote. It developed into an embryo, which was implanted into a surrogate mother and carried to term. The lamb, Dolly, was an exact genetic replica of the adult female sheep that donated the somatic cell nucleus to the egg. She was the first-ever mammal to be cloned from an adult somatic cell. The fertilization of an egg by a sperm and the SCNT cloning method both result in the same thing: a dividing ball of cells, called an embryo.

An embryo is composed of cells that contain two complete sets of chromosomes. The difference between fertilization and SCNT lies in where those two sets originated. In fertilization, the sperm and egg both contain one set of chromosomes. When the sperm and egg join, the resulting zygote ends up with two sets - one from the father (sperm) and one from the mother (egg). In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the nucleus from a somatic cell, which already contains two complete sets of chromosomes. Therefore, in the resulting embryo, both sets of chromosomes come from the somatic cell.

Embryo Splitting
A very simple method of splitting the embryo into two halves at the later morula or early blastocyst stage of development has recently been devised. The blastocysts are divided in such a way that each half contains inner cell mass (ICM) and trophoectoderm cells. ICM is the mass of cells which develop into foetus. Trophoectoderm is a single layer of trophoblast cells lining the inner side of the zone surrounding the blastocoels. The split embryos are then transferred to females to produce identical twins. Thus embryosplitting technology has increased the rate of pregnancy. Embryo splitting can be done as follows: Blastocyst stage embryos are transferred for a few minutes into the cell culture medium consisting of hypertonic sucrose and bovine serum albumin (BSA). The medium of a high osmotic strength enters into the cell membrane of the embryo (zona pellucida) and cells contract due to exosmosis. The BSA attaches to the zona pellucida and provides negative charges to the embryo. Then it is transferred into a plastic petri dish containing standard cell culture medium. The outer membrane of the embryo is negatively charged and the petri dish is positively charged. The embryo sticks to the surface of the petri dish due to the development of electrostatics interaction of the two charges. The Petri dish is kept on the stage of an inverted microscope. A micromanipulator equipped with a fine surgical blade is then used to cut the embryo roughly into two halves while caring that only minimal damage is done to the cells. The hemisphere mass of embryo is referred to as sphere. The half or semi embryo is transferred into the recipients oviduct. At this stage, it is essential to study the successful embryo implantation, which is possible with chemical signals from the trophoblast.