Animal biotechnology is a broad field encompassing the polarities of fundamental and applied research, including molecular modeling, gene manipulation, development of diagnostics and vaccines and manipulation of tissue. It accounts for the use of biotechnology tools, including molecular markers, stem cells, and tissue engineering. Molecular markers are increasingly being used to identify and select the particular genes that lead to desirable traits and it is now possible to select superior germ plasma and disseminate it using artificial insemination, embryo transfer and other assisted reproductive technologies. These technologies have been used in the genetic improvement of livestock. Transgenesis offers considerable opportunity for advances in medicine and agriculture. In livestock, the ability to insert new genes for such economically important characteristics as fecundity, resistance to or tolerance of other environmental stresses would represent a major breakthrough in the breeding of commercially superior stock. Another opportunity that transgenic technology could provide is in the production of medically important proteins such as insulin and clotting factors in the milk of domestic livestock. A comprehensive evaluation of strategies for developing, testing, breeding and disseminating transgenic livestock in the context of quantitativeimprovement of economic traits is being done. Genetic improvement of livestock depends on access to genetic variation and effective methods for exploiting this variation. Genetic diversity constitutes a buffer against changes in the environment and is a key in selection and breeding for adaptability and production in a range of environments. Animal cell culture technology in today's scenario has become indispensable in the field of life sciences, which provides a basis to study regulation, proliferation, differentiation, and to perform genetic manipulation. It requires specific technical skills to carry out successfully. Application of tissue culture includes the study and understanding of intracellular activity, intracellular flux, pharmacology, cell-cell interaction, cell products, toxicology, tissue engineering, genomics, and immunology. Knowledge acquired from these studies can be used in the biomedical applications.
· Culture Media: The culture medium is the most importantcomponent of the culture environment, because it provides the necessary nutrients, growth factors, and hormones for cell growth, as well as regulating the pH and the osmotic pressure of the culture. Although initial cell culture experiments were performed using natural media obtained from tissue extracts and body fluids, the need for standardization, media quality, and increased demand led to the development of defined media. The three basic classes of media are basal media, reduced-serum media, and serum-free media, which differ in their requirement for supplementation with serum.
· Media Components Balanced Salt Solutions: A balanced saltsolution (BSS) is composed of inorganic salts and may include sodium carbonate and, in some cases, glucose. Commercial complete media will list which BSS formulation was used.
Serum: Serum is vitally important as a source of growth andadhesion factors, hormones, lipids and minerals for the culture of cells in basal media. In addition, serum also regulates cell membrane permeability and serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell. However, using serum in media has a number of disadvantages including high cost, problems with standardization, specificity, variability, and unwanted effects such as stimulation or inhibition of growth and/or cellular function on certain cell cultures. If the serum is not obtained from reputable source, contamination can also pose a serious threat to successful cell culture experiments. Always check new batches of serum before use. The quality and the composition can vary greatly from batch to batch. Serum is inactivated by incubating it for 30 min at +56oC. Originally, heating was used to inactivate complements for immunoassays, but it may also have other, undocumented effects.
· Other Supplements: In addition to serum, tissue extracts and digestshave traditionally been used to supplement tissue culture media. The most common ones are amino acid hydrolysates (from beef heart) and embryo extract (chick embryo).
· Basal Media: The majority of cell lines grow well in basal media,which contain amino acids, vitamins, inorganic salts, and a carbon source such as glucose, but these basal media formulations must be further supplemented with serum.
· Reduced-Serum Media: Another strategy to reduce the undesiredeffects of serum in cell culture experiments is to use reduced-serum media. Reduced-serum media are basal media formulations enriched with nutrients and animal-derived factors, which reduce the amount of serum that is needed.
Serum-Free Media: Serum-free media (SFM) circumvents issues withusing animal sera by replacing the serum with appropriate nutritional and hormonal formulations. Serum-free mediaformulations exist for many primary cultures and cell lines, including recombinant protein producing lines of Chinese Hamster Ovary (CHO), various hybridoma cell lines, the insect lines Sf9 and Sf21 (Spodopterafrugiperda), and for cell lines that act as hosts for viral production (e.g., 293, VERO, MDCK, MDBK), and others. One of the major advantages of using serum-free media is the ability to make the medium selective for specific cell types by choosing the appropriate combination of growth factors. Using serum in a medium has a number of disadvantages: the physiological variability, the shelf life and consistency, the quality control, the specificity, the availability, the downstream processing, the possibility of contamination, the growth inhibitors, the standardization and the costs. Using serum-free media and defined media supplements (Nutridoma-CS, Nutridoma-SP and Transferrin) offers three main advantages: The ability to make a medium selective for a particular cell type. The possibility of switching from growth-enhancing medium for propagation to a differentiation-inducing medium.The possibility of bioassays (e.g., protein production) free from interference with serum proteins (easier downstream processing).
Media Recommendations: Many continuous mammalian cell linescan be maintained on a relatively simple medium such as MEM supplemented with serum, and a culture grown in MEM can probably be just as easily grown in DMEM or Medium 199. However, when a specialized function is expressed, a more complex medium may be required. Information for selecting the appropriate medium for a given cell type is usually available in published literature, and may also be obtained from the source of the cells or cell banks. If there is no information available on the appropriate medium for your cell type, choose the growth medium and serum empirically or test several different media for best results. In general, a good placeto start is MEM for adherent cells and RPMI-1640 for suspension cells. The conditions listed below (Table 1) can be used as a guide line when setting up a new mammalian cell culture. Insect cells are cultured in growth media that are usually more acidic that those used for mammalian cells such as TNM-FH and Grace’s medium.
BME: Basal Medium Eagle; DMEM: Dulbecco’sModified EagleMedium; FBS: Fetal Bovine Serum; GMEM: Glasgow Minimum Essential Medium; IMDM: Iscove’s Modified Dulbecco’s Medium; MEM: Minimum Essential Medium; NEAA: Non-Essential AminoAcids Solution; TNM-FH:Trichoplusiani Medium-Formulation Hink (i.e. Grace’s Insect Medium, Supplemented)
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