Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA. Several steps in the gene expression process may be modulated, including the transcription, RNA splicing, translation, and post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism. Transcription The gene itself is typically a long stretch of DNA and does not perform an active role. It is a blueprint for the production of RNA. The production of RNA copies of the DNA is called transcription, and is performed by RNA polymerase, which adds one RNA nucleotide at a time to a growing RNA strand. This RNA is complementary to the DNA nucleotide being transcribed; i.e. a T on the DNA means an A is added to the RNA. However, in RNA the nitrogen-containing base Uracil is inserted instead of Thymine wherever there is an Adenine on the DNA strand. Therefore, the mRNA complement of a DNA strand reading "TAC" would be transcribed as "AUG". RNA processing Transcription creates a primary transcript of RNA at the place where the gene was located. This transcript is often altered before being translated. RNA processing, also known as post-transcriptional modification, can start during transcription, as is the case for splicing, where the spliceosome removes introns from newly formed RNA. Introns are RNA segments which are not found in the mature RNA, although they can function as precursors, e.g. for snoRNAs, which are RNAs that direct modification of nucleotides in other RNAs. In some cases large aggregates of RNA and RNA processing factors are formed, notably in the nucleolus where ribosomal RNA is processed by snoRNAs and their partner enzymes. These cleave the primary ribosomal RNA transcripts into the correct segments and alter some of its nucleosides, for instance into pseudouridine. RNA export While some RNAs function in the nucleus, many other RNAs in eukaryotes are transported through the nuclear pores and into the cytosol, including all the RNA types involved in protein synthesis. In some cases RNAs are additionally transported to a specific part of the cytoplasm, such as a synapse; they are then towed by motor proteins that bind through linker proteins to specific sequences (called "zipcodes") on the RNA. Translation For most RNA, the mature RNA is the gene product (see non-coding RNA). In the case of messenger RNA however, the RNA is but an information carrier for the synthesis of a protein. Each triplet of nucleotides of the coding region of a messenger RNA corresponds to a binding site for a transfer RNA. Transfer RNAs carry amino acids, and these are chained together by the ribosome. The ribosome helps transfer RNA bind to messenger RNA and takes the amino acid from each transfer RNA and makes a structure-less protein out of it. Some proteins have parts that should be within a membrane, these parts are moved into the membrane by the signal recognition particle which binds to the ribosome when it finds a signal sequence on the nascent amino acid chain.

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Cleave DNA Using Restriction Endonulease:

In order to manipulate DNA you have to posses the ability to cleave DNA at specific sites by using bacterial enzyme, which is restriction endonulease. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. The name of the enzyme (such as BamHl, EcoRl, AluI, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul),GAATTC (for EcoRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence. Materials 1. 10X stock of the appropriate restriction enzyme buffer. 2. DNA to be digested in either water or TE (10 mM Tris-HCl pH 8.3, 1 mM ethylenediaminetetraacetic acid [EDTA]). 3. Bovine serum albumin (BSA) at a concentration of 1 mg/mL. BSA is routinely included in restriction digests to stabilize low protein concentrations and to protect against factors that cause denaturation. 4. Sterile distilled water. Good-quality sterile distilled water should be used in restriction digests. Water should be free of ions and organic compounds, and must be detergent free. 5. The correct enzyme for the digest. 6. 5X Loading buffer: 50% (v/v) glycerol, 100 mM Na2EDTA, pH 8, 0.125% (w/v) bromophenol blue, 0.125% (w/v) xylene cyanol. 7. 100 mM Spermidine. Digests of genomic DNA are dramatically improved by the inclusion of spermidine in the digest mixture to a final concentration of 1 mM since the polycationic spermidine binds negatively charged contaminants. Spermidine can cause precipitation of DNA at low temperatures, so it should not be added while the reaction is kept on ice. Methods 1. Thaw all solutions, with the exception of the enzyme, and then place on ice. 2. Decide on a final volume for the digest, usually between 10 and 50 microliters, and then into a sterile Eppendorf tube, add 1/10 vol of reaction buffer, 1/10 vol BSA, between 0.5 and 1 micrograms of the DNA to be digested, and sterile distilled water to the final volume. The amount of DNA to be digested depends on subsequent steps. A reasonable amount for a plasmid digestion to confirm the presence of an insertion would be 500 ng-l microgram, depending on the size of the insert. The smaller the insert, the more DNA should be digested to enable visualization of the insert after agarose gel analysis. 3. Take the restriction enzyme stock directly from the -20oC freezer, and remove the desired units of enzyme with a clean sterile pipet tip. Immediately add the enzyme to the reaction and mix. Stock restriction enzymes are very heat labile and so should be removed from -20oC storage for as short a time as possible and placed on ice. 4. Incubate the tube at the correct temperature (see Note 12) for approx 1 h. Genomic DNA can be digested overnight. Note that the incubation temperature for the vast majority of restriction endonucleases is 37oC but that this is not true for all enzymes. 5. An aliquot of the reaction (usually 1-2microliter) may be mixed with a 5X concentrated loading buffer and analyzed by gel electrophoresis. Hopefully this method can help your work.

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Therapeutic cloning

If reproductive cloning has few friends -- aside from some renegade scientists and cultists who insist they'll use it to help infertile couples -- a related technology poses much tougher ethical questions.

Therapeutic cloning does not strive to make whole humans. Instead, it makes embryos as a source of embryonic stem cells for therapeutic purposes. Because embryonic stem cells can grow into any body cell, they might be cultured into nerve cells, skin cells, even hair follicles for the bald. The obvious use of therapeutic cloning would be treating deadly diseases like diabetes and Parkinson's, where a specific type of cell has died. It's a good bet that replacing those cells would restore health.

Therapeutic cloning research would end in this country, however, if restrictive legislation passes the Senate. Sen. Sam Brownback, for example, writes that "The prospect of creating new human life solely to be exploited and destroyed in this way has been condemned on moral grounds by many as displaying a profound disrespect for life."

But society is already willing to tolerate the death of lab-created embryos during in-vitro fertilization, says medical ethicist Dan Wikler. "Anyone who would says we should not embark on this kind of therapeutic cloning would, on pain of inconsistency, be opposed to routine IVF, where embryo are created in advance, with big chance of being destroyed as surplus."

Wikler maintains that the quest to save existing lives deserves moral standing. "Anyone who would says that the chance to save a life through therapeutic cloning is wrong, would have to explain why they have not been upset by the practices that go on under IVF, which is basically the same thing."

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Therapeutic cloning

If reproductive cloning has few friends -- aside from some renegade scientists and cultists who insist they'll use it to help infertile couples -- a related technology poses much tougher ethical questions.

Therapeutic cloning does not strive to make whole humans. Instead, it makes embryos as a source of embryonic stem cells for therapeutic purposes. Because embryonic stem cells can grow into any body cell, they might be cultured into nerve cells, skin cells, even hair follicles for the bald. The obvious use of therapeutic cloning would be treating deadly diseases like diabetes and Parkinson's, where a specific type of cell has died. It's a good bet that replacing those cells would restore health.

Therapeutic cloning research would end in this country, however, if restrictive legislation passes the Senate. Sen. Sam Brownback, for example, writes that "The prospect of creating new human life solely to be exploited and destroyed in this way has been condemned on moral grounds by many as displaying a profound disrespect for life."

But society is already willing to tolerate the death of lab-created embryos during in-vitro fertilization, says medical ethicist Dan Wikler. "Anyone who would says we should not embark on this kind of therapeutic cloning would, on pain of inconsistency, be opposed to routine IVF, where embryo are created in advance, with big chance of being destroyed as surplus."

Wikler maintains that the quest to save existing lives deserves moral standing. "Anyone who would says that the chance to save a life through therapeutic cloning is wrong, would have to explain why they have not been upset by the practices that go on under IVF, which is basically the same thing."

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Cloning

Cloning Cloning in biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. More generally, the term refers to the production of multiple copies of a product such as digital media or software. Cellular cloning Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media. A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells which have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonally distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

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Cell culture

Cell culture is the process by which prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Applications of cell culture Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants. Tissue culture and engineering Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo. Vaccines Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,or the use of adjuvants.

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Types of cells

Cells are often categorized by their source: * Autologous cells are obtained from the same individual to which they will be reimplanted. Autologous cells have the fewest problems with rejection and pathogen transmission, however in some cases might not be available. For example in genetic disease suitable autologous cells are not available. Also very ill or elderly persons, as well as patients suffering from severe burns, may not have sufficient quantities of autologous cells to establish useful cell lines. Moreover since this category of cells needs to be harvested from the patient, there are also some concerns related to the necessity of performing such surgical operations that might lead to donor site infection or chronic pain. Autologous cells also must be cultured from samples before they can be used: this takes time, so autologous solutions may not be very quick. Recently there has been a trend towards the use of mesenchymal stem cells from bone marrow and fat. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve. A large number of cells can be easily and quickly isolated from fat, thus opening the potential for large numbers of cells to be quickly and easily obtained. Several companies have been founded to capitalize on this technology, the most successful at this time being Cytori Therapeutics. * Allogeneic cells come from the body of a donor of the same species. While there are some ethical constraints to the use of human cells for in vitro studies, the employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin. * Xenogenic cells are these isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants. * Syngenic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models. * Primary cells are from an organism. * Secondary cells are from a cell bank. * Stem cells (see main article: stem cell) are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. According to their source stem cells are divided into "adult" and "embryonic" stem cells, the first class being multipotent and the latter mostly pluripotent; some cells are totipotent, in the earliest stages of the embryo. While there is still a large ethical debate related with the use of embryonic stem cells, it is thought that stem cells may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs.

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Tissue engineering

Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. While most definitions of tissue engineering cover a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning. The term has also been applied to efforts to perform specific biochemical functions using cells within an artificially-created support system (e.g. an artificial pancreas, or a bioartificial liver). The term regenerative medicine is often used synonymously with tissue engineering, although those involved in regenerative medicine place more emphasis on the use of stem cells to produce tissues. A commonly applied definition of tissue engineering, as stated by Langer and Vacanti, is "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ".Tissue engineering has also been defined as "understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use." A further description goes on to say that an "underlying supposition of tissue engineering is that the employment of natural biology of the system will allow for greater success in developing therapeutic strategies aimed at the replacement, repair, maintenance, and/or enhancement of tissue function." Powerful developments in the multidisciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices ("scaffolds"), cells, and biologically active molecules. Among the major challenges now facing tissue engineering is the need for more complex functionality, as well as both functional and biomechanical stability in laboratory-grown tissues destined for transplantation. The continued success of tissue engineering, and the eventual development of true human replacement parts, will grow from the convergence of engineering and basic research advances in tissue, matrix, growth factor, stem cell, and developmental biology, as well as materials science and bioinformatics.

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Biochemical engineering

Biochemical engineering is a branch of chemical engineering or biological engineering that mainly deals with the design and construction of unit processes that involve biological organisms or molecules, such as bioreactors. Biochemical engineering is often taught as a supplementary option to chemical engineering or biological engineering due to the similarities in both the background subject curriculum and problem-solving techniques used by both professions. Its applications are used in the food, feed, pharmaceutical, biotechnology, and water treatment industries.

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