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Essay: Gene transfer techniques – pros and cons. Gene transfer is a technique to stably and efficiently introduce functional genes (that are usually cloned) into the target cells. Genetic transfer is the mechanism by which DNA is transferred from a donor to a recipient. Once donor DNA is inside the essay recipient, crossing over can occur. The result is a recombinant cell that has a genome different from either the donor or the recipient. A recombination event must occur after transfer in order that the change in the genome be heritable (passed on to the introduction next generation).The genes are the hemingway style blueprints essential to term paper, generate all the proteins in our bodies which eventually perform all the biological functions. Therefore, when a gene is hemingway, efficiently introduced into a target cell of the host, the protein which is encoded by that gene is produced . Gene transfer technologies developed initially as a research tool for studying the gene expression and its function. A range of techniques and naturally occurring processes are utilized for the gene transfer. Techniques are available for the introduction of essays on oil, DNA into many different cell types in culture, either to study gene function and regulation or to produce large amounts of recombinant protein. Cell cultures have therefore been used on a commercial scale to essay hemingway, synthesize products such as antibodies, hormones, growth factors, cytokines and viral coat proteins for immunization.The most important application of project, this technology is in vivo gene therapy, i.e. the introduction of DNA into the cells of essay style, live animals in order to treat disease.

The extraordinary potential of altered DNA molecule is to give rise to new life-forms that are better adopted for survival. Change in organ donation, base sequence of DNA leads to change in essay style, protein causing disease condition which can be corrected by manipulation of gene. Much emphasis has been given to the gene manipulation in cardiovascular diseases, parkinson’s disease, lysosomal disorders, ocular gene therapy and osteoarthritis. Overview of gene-transfer strategies: Gene transfer can be achieved essentially via three routes. The most straightforward is direct DNA transfer, the muslim paper physical introduction of foreign DNA directly into essay hemingway style, the cell. For example, in project, cultured cells this can be done by essay hemingway microinjection, whereas for cells in vivo direct transfer is often achieved by bombardment with tiny DNA-coated metal particles. Term? The second route is termed transfection, and this encompasses a number of techniques, some chemical and some physical, which can be used to persuade cells to take up DNA from their surroundings.

The third is to package the DNA inside an. animal virus, since viruses have evolved mechanisms to naturally infect cells and introduce their own nucleic acid. The transfer of foreign DNA into a cell by hemingway this route is termed transduction. The choice of on sports, methods of DNA transfer depends upon style, the target cells in which transformation will be performed. On Sports Violence? It also depends upon the objectives of essay, gene manipulation. The transfection may be either stable or transient. Although, choice of DNA transfer method is very important, the other important steps are selection of gene, isolation of muslim, gene, preparing recombinant DNA and selection of transformed cells. The regeneration of hemingway style, organism with new characteristics is also equally important. The delivery of genes in vitro can be done by treating the cells with viruses such as retrovirus or adenovirus, calcium phosphate, liposomes, particle bombardment, fine needle naked DNA injection, electroporation or any combination of these methods.

These are the powerful tools for research and have possible applications in gene therapy. FACTORS TO CONSIDER WHEN SELECTING A GENE DELIVERY METHOD. The advancement of new technologies has made gene delivery both more efficient yet more confusing given the formidable array of reagents and methods now available. Determining the best gene delivery approach for any particular experiment requires careful consideration of a number of organ donation, different factors as stated below. 1. Cellular Context or Environment – The context or environment of the cells (i.e., in vivo vs. in. vitro vs. ex vivo) to which nucleic acids will be delivered is the first factor to consider when. deciding upon a gene transfer method.

The obstacles and challenges posed by delivery to in. vitro cell cultures are very different than those posed by in vivo or ex vivo cells, and require. different tools and techniques for overcoming them. For example, targeting nucleic acids to. particular cells while avoiding other cells is usually a major concern for in vivo gene transfer. applications, sometimes a concern for style, ex vivo applications, but usually not a concern for in vitro. Another important issue that must be addressed when planning in vivo (but not in vitro) gene. delivery studies is the immunogenicity of the gene transfer system. This is particularly important when considering the muslim term paper use of certain viral vectors discussed previously, which may be highly immunogenic.

Also related to immunogenicity is the issue of general safety, both for essay, the experimenter and for the subject of any study utilizing transfection. Almost always, the use of viral vectors requires very extensive safety precautions than does the essays on sports violence use of essay hemingway style, synthetic transfection methods. 2. Cell or Tissue Type – The origin of the cells or tissue to which nucleic acids will be. transferred is typically the second parameter considered when selecting a gene delivery. method. For a number of different reasons, the difficulty of cellular nucleic acid delivery varies. from cell type to cell type. Because of this, it is always important to review the essay management literature to. determine if the cell type of hemingway style, interest has previously been utilized for essay introduction, gene transfer experiments, which exact methods have been used, and what delivery efficiencies were achieved, i.e., what. percentages of cells actually transfected or transduced, with each method.

Certain cell types are often described as ‘notoriously difficult to transfect’. These include primary cell cultures of many varieties. Successful gene transfer to essay hemingway style, such cells. usually requires a good deal of research before selecting any particular gene delivery method. 3. Delivery Efficiency – For most applications, transfecting or transducing as many cells as.

possible is preferred, especially when it is necessary to detect the presence of rare transgene. products. For Family? Thus, it is hemingway style, important to determine the level of delivery efficiency required for a given. application before deciding on a particular gene transfer method. In most cases, it is found that.

viral vectors provide the highest delivery efficiencies. Essay And Friends? For some commonly used and easily transfected cell lines, cationic lipids nearly match viral vectors in their gene transfer efficiency, while providing safer and hemingway much quicker procedures. 4. Stable vs. Transient Gene Delivery – Gene transfer procedures are frequently categorized. by whether the delivered gene remains separate from the essays host cell chromosome or whether it is integrated into the host cell chromosome. In the first case, known as transient gene delivery, expression of the transgene typically dissipates after a given period of time – usually within. several days – because the hemingway expression vector is either degraded or expelled from the host cell.

In the essays on oil second case, known as stable gene delivery, expression of the transferred gene is. prolonged, or stable, because the vector is integrated into the host cell chromosome. Obviously, different applications require different time periods of transgene expression. Thus, a careful. comparison between different gene delivery methodologies will allow a suitable choice for. generating the desired transient or stable expression. In the essay hemingway case of viral vectors, determining whether a particular virus is suited for stable or transient. transduction is simply a matter of learning the general characteristics of the vector. In the case.

of chemical or physical/mechanical gene transfer, determining whether a given method is. suitable for transient vs. stable delivery may be as easy as reading sales literature from the. manufacturer or checking primary research literature. 5. Type of Delivered Molecule – Another factor to essays on oil, consider when selecting a gene delivery. method is what type of nucleic acid or other molecule is being transfected or transduced. For. example, a reagent that efficiently delivers plasmids may not efficiently deliver DNA. oligonucleotides.

It is frequently easiest to simply review the literature to learn what methods have been used successfully, and to contact other researchers who have experience delivering the molecule of hemingway style, interest. 6. Cytotoxicity – Another issue that comes up frequently when choosing among different gene. delivery methods is the cytotoxicity of the various methods and reagents. Some methods, such. as electroporation and gene guns, can be extremely harsh on cells and often result in the death. of a majority of cells. Also, the DEAE-dextran can be quite harsh on some cell types, especially. when a DMSO or glycerol shock step is used as part of the procedure. In some applications, such as when a small number of transfectants are required from a recalcitrant cell type, this is.

not a problem. In the majority of applications however, a high percentage of research studies, cell death is simply. not acceptable. The plethora of essay hemingway, available cationic lipids and polymer reagents have varying. degrees of toxicity with different cell types, and marketing case their concentrations during transfection can be. optimized as needed to minimize cell death. 7. Suspension vs. Adherent cells – Most commonly transfected cells grow – and are. transfected – while attached to some type of support, whether that support is the tissue from. which they are derived, some inert membrane or scaffolding, or the surface of a tissue culture.

plate. Less common are cells that grow while suspended in medium, such as those derived from. blood and other bodily fluids. Suspension cells are generally more difficult to transfect; therefore, it may be necessary to rely on harsher gene delivery methods, such as. electroporation, gene guns, microinjection, or viral vectors for essay hemingway style, acceptable transfection levels. Also, some cationic lipids can effectively transfect suspension cells, and should be tried when.

possible due to their simplicity and cost-effectiveness. 8. Expertise – The degree of essays on oil, difficulty in performing the various transfection techniques can. vary significantly, and therefore the expertise and experience of the researcher is style, usually an. important factor to consider. For example, methods like microinjection viral transfection methods are invariably more complex than cationic lipids and polymer reagents. Also, of the chemical methods, calcium phosphate co-precipitation can be quite tricky due to paper, its sensitivity to hemingway style, slight changes in experimental conditions. 9. Time – Another important factor to consider is the time required for gene delivery.

Viral. methods, while highly efficient, can be quite time consuming, typically taking anywhere from two weeks to one month to complete. In contrast, chemical and physical/mechanical methods can usually be completed within a few days, but frequently provide lower transfection efficiencies. Thus, it is not uncommon to have to weigh the benefits of faster results and less labor against the benefit of higher efficiency. 10 Cost – Finally, cost can be a major factor when deciding on the best gene delivery. method for term, a given application. Hemingway Style? The most expensive methods tend to be the. physical/mechanical methods, such as electroporation and gene guns, due to the specialized.

equipment they require.Viral gene delivery is perhaps the second most expensive method, since it requires the vector reagents, extensive time and labor to complete lengthy vector preparation procedures, as well as proper disposal of viral waste materials. The third most expensive gene delivery methods utilize polymers and essay project cationic lipids, which do not require any specialized equipment or lengthy viral vector preparation steps. Such reagents typically cost in style, the hundreds of marketing research ethics, dollars, depending on the number of hemingway, transfections to project management, be completed. Essay? Finally, the least expensive methods utilize calcium phosphate, which is a cheap and abundant mineral, and naked DNA, which obviously requires no transfection reagent cost, but can be highly inefficient. GENE TRANSFER TECHNIQUES. Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the studies fusion of the essay style foreign DNA with the marketing native DNA resulting in essay style, the genetic expression of the received DNA. Transformation involves the uptake of “naked” DNA (DNA not incorporated into structures such as chromosomes) by competent bacterial cells. Cells are only competent (capable of taking up DNA) at a certain stage of their life cycle, apparently prior to the completion of cell wall synthesis . Transformation is organ donation introduction, usually a natural method of gene transfer but as a result of essay hemingway, technological advancement originated the artificial or induced transformation.

Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and essay with the help of the protein DNA translocase it enters the host cell. Essay Hemingway? The presence of essay project management, nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. Any DNA that is not integrated into the chromosome will be degraded. This single stranded DNA mingles with the host genetic material successfully. The artificial or induced method of essay, transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation..

Genetic engineers are able to essay, induce competency by putting cells in certain solutions, typically containing calcium salts. At the entry site, endonucleases cut the DNA into fragments of 7,000-10,000 nucleotides, and the double-stranded DNA separates into single strands. The single-stranded DNA may recombine with the host’s chromosome once inside the essay cell. This recombination replaces the gene in the host with a variant – albeit homologous – gene. DNA from paper, a closely related genus may be acquired but, in general, DNA is not exchanged between distantly related microbes.

Not all bacteria can become competent. Conjugation is a means of gene transfer in many species of hemingway style, bacteria. Cell-to-cell contact by a specialized appendage, known as the F-pilus (or sex pilus), allows a copy of an F- plasmid (fertility plasmid) to organ essay, transfer to a cell that does not contain the plasmid. On rare occasions an F-plasmid may become integrated in the chromosome of its bacterial host, generating what is known as an Hfr (high frequency of essay hemingway, recombination) cell. Such a cell can also direct the synthesis of a sex pilus. As the chromosome of the Hfr cell replicates it may begin to cross the pilus so that plasmid and chromosomal DNA transfers to organ introduction, the recipient cell. Such DNA may recombine with that of its new host, introducing new gene variants. Plasmids encoding genes for virulence factors antibiotic-resistance are passed throughout populations of bacteria, and between multiple species of bacteria by conjugation. In the laboratory, conjugation can be used to transfer disrupted genes on a self-transmissible plasmid, to develop a mutant strain. A gene of interest from hemingway, a recipient E. coli strain is cloned into a self-transmissible vector and maintained in a donor strain for genetic manipulation. The deleted gene construct is project management, then transferred by essay conjugation from the muslim paper donor strain back into the recipient strain.

Transduction is another method for transferring genes from one bacterium to another; the transfer is mediated by bacteriophages. A bacteriophage infection starts when the virus injects its DNA into a bacterial cell. The bacteriophage DNA may then direct the synthesis of hemingway style, new viral components assembled in the bacterium. Bacteriophage DNA is replicated and organ donation essay introduction then packaged within the essay phage particles. Early in the infective cycle the phage encodes an enzyme that degrades the DNA of the host cell. Some of these fragments of bacterial DNA are packaged within the bacteriophage particles, taking the place of phage DNA. As the number of on sports, phages increases it breaks open (lyse) the cell. When released from the infected cell, a phage that contains bacterial genes can continue to infect a new bacterial cell, transferring the bacterial genes.

Sometimes genes transferred in this manner become integrated into the genome of their new bacterial host by homologous recombination. Such transduced bacteria are not lysed because they do not contain adequate phage DNA for viral synthesis and these phages can enter an alternate life cycle called lysogeny. In this cycle, all the virus’s DNA becomes integrated into essay hemingway, the genome of the host bacterium. The integrated phage, called a prophage, can confer new properties to the bacterium. Researchers use transduction as a means of gene transfer as it requires a media like virus for transferring genes from one cell to the other. Viruses are thus as a tool to introduce foreign DNA from the selected species to the target organism. There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes. Viral vectors for gene transfer:

The use of viruses as vectors for transduction, i.e. the introduction of genes into animal cells by exploiting the natural ability of the virus particle, within which the transgene is packaged, to adsorb to muslim term paper, the surface of the cell and gain entry is being employed . Due to hemingway style, the efficiency with which viruses can deliver their nucleic acid into essay for family, cells and the high levels of replication. and gene expression it is possible to achieve, viruses have been used as vectors not only for gene expression in cultured cells but also for gene transfer to living animals. Four classes of viral vector have been developed for use in human gene therapy and have reached phase 1 clinical trials. These are the hemingway style retrovirus, adenovirus, herpesvirus and adenoassociated virus (AAV) vectors . Research Ethics? Before introducing the individual vector systems,we discuss some general properties of essay hemingway, viral transduction vectors. Transgenes may be incorporated into. viral vectors either by addition to the whole genome or by replacing one or more viral genes. This is generally achieved either by ligation (many viruses have been modified to incorporate unique restriction sites) or homologous recombination.

For many applications, it is favourable to essay for family and friends, use vectors from which all viral coding sequences have been deleted.These amplicons (also described as ‘gutless vectors’) contain just the cis-acting elements required for packaging and genome replication. The advantage of such vectors is their high capacity for foreign DNA and hemingway the fact that, since no viral gene products are made, the vector has no intrinsic cytotoxic effects. The choice of vector depends on the particular properties of the virus and the intended host, whether transient or stable expression is required and how much DNA needs to be packaged. Muslim Term Paper? For example, icosahedral viruses such as adenoviruses and retroviruses package their genomes into preformed capsids, whose volume defines the maximum amount of foreign DNA that can be accommodated. Hemingway? Conversely, rod-shaped viruses such as the essay project management baculoviruses form the capsid around the genome, so there are no such size constraints. There is no ideal virus for gene transfer ‘ each has its own advantages and disadvantages. In recent years, a number of hybrid viral vectors have been developed incorporating the beneficial features of two or more viruses. 1) viral vectors that especially efficient at transducing the intended cell type but not other cell types. 2) retroviruses are particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines. 1) cannot deliver genes to terminally differentiated cells. 2) viral vectors may be highly immunogenic.

TRANSFECTION BY CHEMICAL METHODS. One of the essay methods of gene transfer where the essays on oil genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and essay hemingway style the gene is transfection. Essays On Oil? This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. Essay Style? When transformation is carried out in eukaryotic cells it is termed as transfection. 1) Calcium phosphate mediated DNA transfer. The process involves a mixture of isolated DNA (10-100ug) with solution of. calcium chloride and potassium phosphate. . When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface .Cells are then incubated with precipitated DNA either in on sports violence, solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by a process not entirely understood but thought to be endocytosis. Transfection efficiencies using calcium phosphate can be quite low, in the range of essay style, 1-2 % . It can.

be increased if very high purity DNA is used and the precipitate allowed to form slowly. 1. Frequency is donation, very low. 2. Integrated genes undergo substantial modification. 3. Many cells do not like having the solid precipitate adhering to essay hemingway, them. and the surface of muslim term, their culture vessel. 4. Integration with host cell chromosome is random. 5, Due to above limitations transfection applied to somatic gene therapy. This technique is used for introducing DNA into mammalian cells. This process has been a preferred method of essay hemingway, identifying many oncogenes. 2)DNA transfer by essays on oil DAE-Dextran method:

DNA can be transferred with the help of essay style, DAE Dextran also. Ethics? DAE-Dextran may be used in the. transfection medium in which DNA is present. Essay Style? This is a polycationic, high molecular weight substance and is convenient for muslim paper, transient assays. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.

If DEAE (diethylaminoethyl )-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell can express the transferred gene. The advantage of style, this method is that, it is cheap, simple and can be used for transient cells. which cannot survive even short exposure of calcium phosphate. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and and friends induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. It does not appear to be efficient for the production of stable transfectants. For cells that grow in essay style, suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used. Transfer of organ donation essay, DNA by essay hemingway style polycation-DMSO:

As calcium phosphate method of DNA transfer is reproducible and efficient but there is narrow range of optimum conditions. Essays On Oil? DNA transfer by. Another polycationic chemical,the detergent Polybrene is carried out to essay hemingway style, increase the adsorption of DNA to the cell surface followed by a brief treatment with 25-30% DMSO to increase membrane permeability and enhance uptake of DNA. In this method no carrier DNA is required and stable transformants are produced. This method works with mouse fibroblast and chick embryo. LIPOSOME MEDIATED GENE TRANSFER. Liposomes are spheres of lipids which can be used to transport molecules into the cells. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. They are considered as spheres of lipid bilayers surrounding the muslim term paper molecule to be transported and promote transport after fusing with the cell membrane.

Liposomes for use as gene transfer vehicles are prepared by essay adding an appropriate mix of essays on oil, bilayer constituents to an aqueous solution of DNA molecules. In this aqueous environment, phospholipid hydrophilic heads associate with water while hydrophobic tails self-associate to essay hemingway, exclude water from within the lipid bilayer. This self-organizing process creates discrete spheres of continuous lipid bilayer membrane enveloping a small quantity of DNA solution. The liposomes are then ready to be added to paper, target cells. Hemingway Style? Germline transgenesis is possible with liposome mediated gene transfer.Lipofection is the most common and generally utilized gene transfer technique in the recent years and it utilizes cationic lipids. Ethics Case Studies? The combined DNA and cationic lipids act instantaneously to form structures called as lipoplexes that are more complex in structure than the simple liposomes. Style? Cationic lipids have a positive charge and these form cationic liposomes which interact with the negatively charged cell membrane more readily than uncharged liposomes.

This leads to a fusion between cationic liposome and the cell surface resulting in quick entry by endocytosis and the delivery of the DNA directly across the essays on oil plasma membrane. Style? Cationic liposomes can be produced from a number of cationic lipids that are commercially available and sold as an in vitro-transfecting agent, termed lipofectin. However this pathway would usually result in organ donation introduction, the fusion of lipoplexes with lysosomes and essay hemingway undergo degradation. Essays On Oil? This problem is overcome by essay utilizing the neutral helper lipids, which are generally included along with the cationic lipid. This allows entrapped DNA to escape the endosomes, reach the nucleus and project get access to essay, the cell’s transcriptional machinery.The endosomal structure is destroyed by increasing the osmotic pressure created by paper the lipids’ buffering action within the style endosomes and by the fusion of the lipid with the endosomal membrane. Essay? The ability of a lipid to destroy endosomes is one of the main characteristics of a transfection reagent. 2. Long term stability. 4. Hemingway? Protection of nucleic acid from degradation.

5. particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines. 6. cationic lipids effectively transfect suspension cells. 1) Cannot deliver genes to terminally differentiated cells. A rapid and simple technique for introducing genes into a wide variety of microbial, plant and animal cells, including E. coli, is electroporation. Electroporation uses electrical pulse to produce transient pores in the plasma membrane thereby allowing macromolecules into the cells. These pores are known as electropores which allow the molecules, ions and water to pass from one side of the studies membrane to another. The electropores reseal spontaneously and the cell can recover. Essay? The pores can be recovered only if a suitable electric pulse is applied .The formation of electropores depends upon the cells that are used and essays violence the amplitude and duration of the essay style electric pulse that is essay introduction, applied to them. When subjected to electric shock, cells take up exogenous DNA from the suspending solution.

Once inside the hemingway cell, the DNA is research ethics case, integrated and the foreign gene will express. A proportion of these cells become stably transformed and can be selected if a suitable marker gene is carried on the transforming DNA. The most critical parameters are the intensity and duration of the electric pulse, and these must be determined empirically for different cell types. Electric currents can lead to dramatic heating of the cells that can results in hemingway, cell death. Heating effects are minimized by using relatively high amplitude, a short duration pulse or by using two very short duration pulses. However, once optimal electroporation parameters have been established, the method is simple to carry out and highly reproducible. Many different factors affect the efficiency of essays on oil, electroporation, including temperature, various electric-field parameters (voltage, resistance and capacitance), topological form of the DNA, and various host-cell factors (genetic background, growth conditions) and post-pulse treatment. General applications of electroporation. 1. Introduction of exogeneous DNA into animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast.

2. Essay Hemingway? Electroporation can be used to increase efficiency of transformation or transfection of bacterial cells. 3. Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1% by research this method. 4. Genes encoding selectable marker may be used to introduce genes using electroporation. 5. Hemingway Style? To study the transient expression of molecular constructs. 6. Electroporation of early embryo may result in donation introduction, the production of essay, transgenic animals. 7. Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B lymphocytes can be transformed by this technique. 8. Naked DNA may be used for gene therapy by applying electroporation device on animal cells. Advantages of electroporation. 1. Method is fast. 3. Applied for a number of muslim term, cell types.

4. Hemingway Style? Simultaneously a large number of cell can be treated. 5. High percentage of stable transformants can be produced. 1)Limited effective range of. 1 cm between the electrodes. 2)Surgical procedure is essays on oil, required to place the electrodes deep into the internal organs. 3)High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating. The technique has high input costs, because a specialized capacitor discharge machine is required that can accurately control pulse length and amplitude. 4) Larger numbers of cells may be required than for essay style, other methods because, in many cases, the most efficient electroporation occurs when there is up to 50% cell death. Sonication is the act of applying sound energy to agitate particles in essays on sports, a sample. Ultrasonic frequencies (20 kHz) are usually used, leading to the process also being known as ultrasonication . Sonoporation, or cellular sonication, is the use of these ultrasonic frequencies for modifying the permeability of the cell plasma membrane.

This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation. Sonoporation employs the acoustic cavitation of microbubbles to essay hemingway style, enhance delivery of such large DNA molecules. Although the early results could achieve only 10- to 30-fold increases in ethics case, transfection efficiency in vitro, technical refinements have lead to enhancements of up to several thousand fold in vitro ,sufficient to essay hemingway, encourage ultrasonication mediated gene transfer in vivo. 1) It is non-invasive and well tolerated, 2)extraordinary safety record over a wide range of term, frequency and intensity. 3)high levels of public acceptability and understanding. there are highly sophisticated, flexible, cost-effective and hemingway style readily available diagnostic and therapeutic systems that can achieve site-specific transfer of ultrasound energy almost anywhere in the body, except perhaps the lung. 4) The bioactivity of this technique is similar to, and in some cases found superior to, electroporation. 5) in vivo is relatively nontoxic.

1)Extended exposure to low-frequency (MHz) ultrasound has been demonstrated to result in complete cellular death (rupturing) as well as microvascular hemorrhage and management disruption of tissue structure, thus cellular viability must also be accounted for when employing this technique. 2) Whether ultrasound affects later steps in the transfection process, particularly plasmid entry into essay hemingway, the nucleus, remains unclear. Sonoporation is under active study for the introduction of foreign genes in research, tissue culture cells, especially mammalian cells. Hemingway Style? Sonoporation is also being studied for use in targetedGene therapy in vivo, in a medical treatment scenario whereby a patient is given modified DNA, and an ultrasonic transducer might target this modified DNA into specific regions of the patient’s body. In microinjection DNA can be introduced into cells or protoplast with the help of very fine needles or. glass micropipettes having the diameter of 0.5 to 10 micrometer. The DNA may be directly delivered into the nucleus or in the cytoplasm. Some of the DNA injected may be taken up by the nucleus.

The desired gene in the form of plasmid or alone is injected directly into the plant protoplast. Injection into ethics case, the nucleus is more efficient than that into the cytoplasm. Micro-injection is carried out with automatic equipments (robotics) using a micro-needle. Essay Hemingway Style? Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency of this technique. Advantages of microinjection. 1. Organ Introduction? Frequency of essay hemingway style, stable integration of DNA is far better as compare to other methods. 2. Marketing Research? Method is hemingway style, effective in transforming primary cells as well as cells in essay for family, established cultures. 3. Hemingway? The DNA injected in this process is subjected to less extensive modifications. 4. Mere precise integration of recombinant gene in limited copy number can be obtained. 2. Skilled personal required.

3.Has not been successful for many plant cells . 4. Embryonic cells preferred for manipulation. 5. Knowledge of mating timing, oocyte recovery is essential. 6. Method is useful for essay for family and friends, protoplasts and not for the walled cells. 7. Process causes random integration. 8. Rearrangement or deletion of essay hemingway, host DNA adjacent to site of integration are common. 9. The technique is laborious, technically difficult, and limited to the number of essay, cells actually injected. Applications of microinjection. 1. Process is essay hemingway, applicable for plant cell as well as animal cell but more common for marketing research studies, animal cells. 2. Essay Hemingway Style? Technique is paper, ideally useful for essay hemingway style, producing transgenic animal quickly. 3. Procedure is important for gene transfer to embryonic cells.

4. Applied to inject DNA into plant nuclei. 5. Method has been successfully used with cells and protoplast of. tobacco, alfalfa etc. Fig: microinjection in mouse egg. Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles to. deliver DNA or other molecules into intact tissues and cells. Essays On Oil? Biolistics transformation is relatively new and novel method amongst the physical methods for artificial transfer of exogenous DNA. Essay? The nucleic acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles like microscopic particles of tungsten or gold coated into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA becomes free to paper, integrate into the plant-cell genome.

The gene gun is a device that literally fires DNA into target cells . The DNA to be transformed. into the cells is coated onto microscopic beads made of essay hemingway, either gold or tungsten. For Family? Beads are carefully. coated with DNA. The coated beads are then attached to the end of the plastic bullet and loaded into. the firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun. towards the target cells that lie just beyond the end of the barrel.

When the bullet reaches the end of. the barrel it is caught and stopped, but the DNA coated beads continue on toward the target cells. Some of the beads pass through the cell wall into the cytoplasm of the target cells. Here the hemingway bead and. the DNA dissociate and the cells become transformed. Once inside the target cells, the DNA is. solubilised and may be expressed. 1. For Family And Friends? Biolistics technique has been used successfully to transform soyabean, cotton, spruce, sugarcane, papaya, corn, sunflower, rice, maize, wheat, 2. Genomes of subcellular organelles have been accessible to genetic. manipulation by hemingway biolistic method. 3. Mitochondria of plants and chloroplast of chlamydomonas have been.

4. Method can be applied to filamentous fungi and yeast (mitochondria). 5. The particle gun has also been used with pollen, early stage. embryoids, meristems somatic embryos, plant cells, root section, seeds and pollen. 6. This approach has been shown to be effective for transferring transgenes into mammalian cells in vivo . 1. Requirement of protoplast can be avoided. Unlike electroporation and organ essay microinjection, this technique does not require protoplasts or even single-cell isolations and is applicable to even intact plant tissue.

2. Walled intact cells can be penetrated. 3. Manipulation of genome of subcellular organelles can be achieved. 4. This is also a highly mechanized or robotic mediated technique in which the. speed of the micro-projectile particles is controlled by hemingway style foolproof mechanisms. 5) It is also gaining in use as a method for transferring DNA construct into whole animals.

6) This technique is marketing research ethics, fast, simple and safe, and it can transfer genes to a wide variety of tissues. 7)there appears to be no limits to hemingway, the size or number of genes that can be delivered. 1. Integration is marketing research case, random. 2. Requirement of equipments. 3.It can be a challenge to obtain a sufficient number of cells modified by this method to see a biologically significant effect. A new technique is presented to incorporate exogeneous gene materials (DNA) into cells with a microbeam irradiation from an hemingway uv pulsed laser. A frequency-multiplied Laser, 355 m wavelength, 5 ns pulse duration, punches a self-healing hole of submicrometer aperture in cell membrane under selected irradiation conditions.

At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for essay management, a short time and fade spontaneously . It takes a fraction of a second for essay style, the aperture to muslim term, close, long enough to allow the foreign DNA, contained in the medium, to slip into the cell. It is well established that a strong pressure wave, known as a laser-induced stress wave (LISW), accompanies laser-induced plasma. We have extended their method to deliver macromolecules, such as genes. Plasmid DNA (circular DNA residing in bacterial cytoplasm) is used as a vector of the gene of essay hemingway style, interest. We inject the plasmid into and friends, target tissue, on which a laser target is essay style, placed, and essays on sports violence subject the target to irradiation with a high-intensity, nanosecond laser pulse to induce plasma and hence an LISW.

By placing optically transparent material on the target, the plasma is essay hemingway style, confined, resulting in an increase in the LISW’s impulse. Interaction of tissue cells with the LISW allows plasmid to essay management, enter the essay hemingway cytoplasm. 1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is essay for family, a very mild treatment virtually suitable for any cell type and. (3) it allows transfection of selected cells even in the presence of cells of hemingway, different type (providing that they are morphologically distinguishable). 4) . it is rapidly replacing virus mediated techniques as they have serious side effects caused by immune response and limited targeting characteristics rendering them inappropriate for clinical application. 5) The method offers a clear advantage over existing methods: increases the management success rate of DNA transfection as well as the efficiency of cell modification by style orders of magnitude. 6) . It enables minimally invasive tissue interaction. Thus there are a number of essay for family, ways by which the genes can be introduced into the cells. With the advent of molecular tools and technologies it is now comparatively easy to introduce gene into cells without losing its integrity and biological activity.

Moreover the recent development in molecular biology has made the transfer of gene with great accuracy to the target cell. The transfer of gene through different gene transfer technologies has cured a number of diseases. Research is on progress to cure those diseases which cannot be cured by using drugs. Moreover the treatment of essay style, diseases by gene transfer provides better result for a prolong period of time. It is the need of hour to discover new and cheap method of donation introduction, gene transfer technologies so to essay hemingway, make the treatment of the diseases a little easier and affordable.

Improvements in gene transfer are required in terms of transfection approaches to allow improved transgene uptake efficiencies. 1)Society of Applied Sciences. Gene Transfer Technologies and their Applications: K.H.KHAN. Medical Biotechnology Division, School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India. 2)Principles of on sports violence, gene manipulation: R.W.Old ,S.B. Primrose and R.M.Twymann ‘ sixth edition,chapter 10 and 11. 3) BIOTECHNOLOGY 2020-From the Transparent Cell to. the Custom-Designed Process–Prof.

Gerhard Kreysa Dr.-Ing. E.h. Dr.h.c. Dr. R??diger Marquardt. 4) INTRODUCTION TOBIOTECHNOLOGY AND GENETIC ENGINEERING.

5)From genes to genomes- concepts and applications of DNA technology–Jeremy W Dale, Malcom von Schantz. Search our thousands of essays: If this essay isn't quite what you're looking for, why not order your own custom Science essay, dissertation or piece of coursework that answers your exact question? There are UK writers just like me on hand, waiting to help you. Each of us is qualified to a high level in our area of expertise, and we can write you a fully researched, fully referenced complete original answer to style, your essay question. Just complete our simple order form and you could have your customised Science work in your email box, in organ essay introduction, as little as 3 hours. This Science essay was submitted to us by a student in order to help you with your studies. This page has approximately words. If you use part of this page in your own work, you need to provide a citation, as follows: Essay UK, Essay: Gene transfer techniques – pros and hemingway cons . Available from: http://www.essay.uk.com/essays/science/essay-gene-transfer-techniques-pros-and-cons/ [04-10-17]. If you are the muslim paper original author of this content and no longer wish to have it published on our website then please click on the link below to request removal:

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We hope that clears up any misconceptions you may have had. We invite you to scroll back to the top and choose from one of our many resume libraries, and essay hemingway style start writing. cover letter for nursing. Should i include collegiate sports in my resume? And if so where? It depends how much professional experience you have. On Sports Violence? If you are a recent college grad, then it is style, acceptable to include on your resume.

Good luck on the job hunt! Good luck on the job hunt! If the jobs are relevant to the ones you are applying for, then you can go as far back as you like. With regards to your military experience, check out essay project, our military to civilian resume guide: https://resumegenius.com/blog/go-shooting-guns-coffee-runs. Any of the templates in library 2 would be suitable for manufacturing careers.

Best of luck! I’ve worked in style the same industry for the past 13 years. Multiple employers with jobs lasting two to three years each. The jobs have been similar, so the experience looks a bit repetitive. I need to muslim term, find a template that highlights my experience without getting bogged down in the chronology, Any suggestions? It provides ample space for your professional experience, while also highlighting your top qualifications. Good luck on the job hunt! hi resume genius.. i need template resume that suitable for trainer and coach.. can u suggest to me with template is suitable.. Essay Style? #128578; I had a job for 7 years and during that time I wore many hats, Executive Admin, Purchasing, Vendor Management, Project Coordination, etc. How would I write that on and friends, my resume?

Perhaps the Company name and then all the style related roles under that and the times I did those jobs? I was always the essays on sports Executive Admin, but I did other jobs during that period. Yes, your suggestion is correct. Start with the company name and included the essay style related jobs with their own bullet points underneath. Good luck! Consider trying the ‘Job Hopper’ or the ‘Executive.’ They should able to fit all your jobs nicely. Ive never had a job so what should I use? Most of the templates above would suit your situation, but we suggest trying the Career Changer template because it emphasizes skills over the dates of your professional experience. (https://resumegenius.com/resume-templates/career-level-life-situation-templates#career-changer-templates) Best of luck! We suggest using the ‘Gatsby’ Template. Good luck with grad school!

As far as style, we suggest our ‘Professional’ template. In terms of format, if you want to include your restaurant experience, then you might want to consider using a functional format:https://resumegenius.com/resume-formats/functional-samples-writing-guide. Hope this helps! We suggest using our ‘Entry-Level’ template. Good luck with the essay project management internship! Good Day Resume Genius.I’m a midwife by profession an has worked in hemingway a military hospital for essay project management, 16 years in KSA. I’m trying to apply as a home based ESL educator and essay an email respondent . Since I’m from the medical profession, I’m having difficulty in essay project management choosing the hemingway style perfect resume.The skill I know is more on the medical.,clerical which involes data entry for appointments and summary, interpreter and my part time informal english lessons to marketing research ethics case, native speaking arabs. What template should I use? Try the ‘Murray’ template. Good luck!

Hello. Which is good for cabin crew applicant? I have no many work experience in service. So i want to highlight the other things. Thanks #128578; Take a look at our Flight Attendant resume sample: https://resumegenius.com/resume-samples/flight-attendant-resume-example You can download it and essay hemingway style input your own information. Which template would you recommend for a career in education? Check out our teacher resume samples: https://resumegenius.com/resume-samples/teacher-resume-example You can download them and input your own experience. Try using the ‘Freeman’ template. Best of essays on oil luck on hemingway, the promotion! Hi!

What resume template would you recommend for a college freshman trying to case studies, apply for a competitive summer program with the USDA and hemingway South Dakota State University? Sound like the ‘Entry-Level’ template would be a good fit for what you’re trying to do. Good luck with the summer program. Hi! Which resume template would you recommend for essay for family, someone trying to tap into the finance and accounting market. Looking for essay hemingway style, an entry-level position. You should go with the ‘Entry-Level’ template. Good luck with the job hunt. I have worked 32+ years as a nurse, the last 4 years taking care of my elderly father and online work. Now seeking to get back into the job market for extra income, not necessarily in the health field, just to earn some income and socialize.

What resume do you suggest? Try the ‘Job Hopper’ template. Good luck with your job search! Hi! What resume template would you recommend for essay, a 9th grader trying to apply for a doctor (any)?? Apparently, resume making and interviewing is our project for the fourth quarter this year.

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Good luck! Hey there Margaret, In order to best understand which template works, it’s a good idea to check out hemingway, which resume format fits your particular needs; then you can take it from there. https://resumegenius.com/resume-formats. All of the donation essay introduction templates were created by hemingway, professional resume writers, so it’s hard to go wrong with any of them — it just depends on your preference. Good luck! It really depends on what job you’re applying for. Project Management? Since you have substantial work experience, try quantifying that in essay hemingway style your resume (think: any numbers that a hiring manager can look at and better understand what you accomplished during your time working there). Check out this page and choose the one you find most fitting, that should be a good start: https://resumegenius.com/resume-formats. Good luck on the job hunt! Hey there hbil036, This way, you can focus on essays on sports, your skills qualifications critical to essay style, the job application.

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When you receive the completed work, you are able to use it in the following ways: As a source for additional ideas and research on the particular subject As a better understanding of the subject and assignment itself that you are required to essay hemingway style complete As gain of new knowledge and exchange of any kind of experience The proper paraphrasing is acceptable.

contest essay rule [Note: The MEE will change for essay hemingway style the July 2007 exam. The exam will have nine essays and will test nine subjects, three of which are MBE topics.] The Multistate Essay Examination (the MEE) is a three-hour, six-essay-question exam, given on project the Tuesday before the last Wednesday in February and July of each year (the day before the essay hemingway style MBE). Corporations and Limited Liability Companies, Decedents' Estates, Family Law, Federal Civil Procedure, Sales, Secured Transactions, and Trusts and term paper, Future Interests. Essay. Some essays include more than one topic. You should average 30 minutes per essay. There is no uniform national grade on the MEE. Each jurisdiction using the exam grades the on sports exam and determines its own policy. as to weighing the MEE, the MBE and any other portion of its bar exam. According to the National Conference of Bar Examiners: (2) separate material which is relevant from that which is not; (3) present a reasoned analysis of the relevant issues.

in a clear, concise, and well organized composition; and (4) demonstrate an understanding of the fundamental legal principles. relevant to the probable solution of the issues raised by the factual situation. The primary distinction between the MEE and the Multistate Bar Examination (MBE) is that the MEE requires the applicant. to demonstrate an ability to communicate in writing effectively. Do not break the seal on this booklet until you are told to begin. You will have three hours in which to write your answers to six of the hemingway seven questions contained in this booklet; you will be told which of the questions you are to essays on oil answer. Each question is designed to be answered in thirty minutes.

There will be no break once the formal testing session begins. You may answer the questions in any order you wish. Do not answer more than one question in each answer booklet. If you make a mistake or wish to revise, simply draw a line through the material you wish to delete. Read each fact situation very carefully and do not assume facts which are not given in the question. Do not assume that each question covers only a single area of the law; some of the questions may cover more than one of the areas you are responsible for knowing. Demonstrate your ability to reason and analyze.

Each of your answers should show: an understanding of the facts; a recognition of the issues included; the applicable principles of law; and the reasoning by which you arrive at your conclusion. The value of your answer depends not as much upon your conclusions as upon the presence and quality of the elements mentioned above. Clarity and conciseness are important, but make your answer complete. Do not volunteer irrelevant or immaterial information. Some jurisdictions instruct applicants to answer MEE questions according to the law of the jurisdiction. Absent such an instruction, you should answer the questions by applying fundamental legal principles rather than local case or local statutory law. As Prepared by the National Conference of Bar Examiners. The following outlines indicate the examination’s potential scope of coverage. The outlines are not intended to list each aspect of each topic mentioned.

The questions on each test will be taken from at least six of the ten subject areas listed below. Some questions may require analysis of more than one subject area. The particular areas covered will vary from exam to exam. I. Essay Hemingway Style. Agency relationships. B. Apparent authority.

C. Inherent agency power. B. Duty of loyalty. C. Duty of obedience. V. Creation of partnerships. A. General partnerships. B. Limited partnerships. C. Limited liability partnerships. VI. Term Paper. Power and hemingway, liability of partners. VII. Muslim Term Paper. Rights of partners among themselves.

A. Profits and losses. B. Management and control. D. Essay. Duty of loyalty. A. Distinguished from marketing research ethics case studies winding up and termination. B. Rightful versus wrongful.

C. General partnerships, limited partnerships, or limited liability partnerships. IX. Special rules concerning limited partnerships. A. Disclosure requirements. B. The control limitation. C. Economic rights of limited partners. Commercial Paper (Negotiable Instruments) (*All citations to the UCC are to the 1999 Official Text.) I. General UCC principles* A. Rules of construction and hemingway style, application (§ 1-101, et seq.) B. General definitions and principles of interpretation (§ 1-102, et seq.) II.

General provisions and definitions (§ 3-102, et seq.) A. Definitions (§ 3-103) B. Negotiable instrument, issue, unconditional promise or order (§§ 3-104 through 3-107) C. Payable on demand or at definite time (§ 3-108) D. Payable to order or bearer; identification of payee (§§ 3-109 through 3-110)

E. Other terms (§§ 3-111 through 3-113) F. Essay Introduction. Contradictory terms; incomplete terms (§§ 3-114 through 3-115) G. Joint and essay hemingway, several liability; contribution (§ 3-116) H. Ethics Case Studies. Other agreements affecting instrument (§ 3-117) I. Statute of limitations; notice of right to defend (§§ 3-118 through 3-119) III. Negotiation, transfer and indorsement (§ 3-201, et seq.) A. Negotiation, rescission and transfer (§§ 3-201 through 3-203) B. Indorsements (§§ 3-204 through 3-206) C. Reacquisition (§ 3-207)

IV. Essay. Enforcement of instruments (§§ 3-301 through 3-311) A. Muslim Term Paper. Person entitled to enforce; holder in due course (§§ 3-101 through 3-102) B. Value and consideration (§ 3-103) C. Overdue instrument (§ 3-304) D. Defenses, claims in recoupment and claims to an instrument (§§ 3-305 through 3-306) E. Notice of breach of fiduciary duty (§ 3-307)

F. Essay Style. Proof of signatures and status as holder in due course (§ 3-308) G. Essays On Oil. Enforcement of lost, destroyed or stolen instrument (§ 3-309) H. Effect of instrument on obligation for which taken; accord and satisfaction (§§ 3-310 through 3-311) V. Liability of parties (§ 3-401, et seq.) A. Signature; signature by representative; unauthorized signature; impostors and fictitious payee (§§ 3-401 through 3-404) B. Employer's responsibility for employee's fraudulent indorsement (§ 3-405)

C. Alteration; negligence contributing to hemingway style forgery or alteration (§§ 3-406 through 3-407) D. Essay Project Management. Drawee not liable on unaccepted draft (§ 3-408) E. Acceptance of essay hemingway style, draft, certified check; acceptance varying draft (§§ 3-409 through 3-410) F. Refusal to organ pay cashier's, teller's and certified checks (§ 3-411) G. Obligations of issuer, acceptor, drawer, indorser (§§ 3-412 through 3-415) H. Transfer and presentment warranties (§§ 3-416 through 3-417) I. Payment or acceptance by mistake (§ 3-418) J. Instruments signed for accommodation (§ 3-419) K. Conversion of essay hemingway style, instrument (§ 3-420) VI.

Dishonor (presentment, dishonor, notice, excuse, evidence of dishonor) (§§ 3-501 through 3-505) VII. Discharge and payment (§§ 3-601 through 3-605) A. Discharge and effect of discharge (§ 3-601) B. Payment; tender of payment (§§ 3-602 through 3-603) C. Discharge by cancellation, renunciation (§ 3-604) D. Discharge of indorsers, accommodation parties (§ 3-605) Conflict of for family, Laws. A. Meaning and legal consequences. B. State's law by which determined.

II. Jurisdiction of courts. A. Types of jurisdiction. 2. In rem and quasi in rem. B. Bases of jurisdiction. C. Hemingway. Notice and opportunity to be heard. D. Limits on exercise of jurisdiction. 1. Term Paper. Traditional limitations. a. Choice of forum by essay hemingway style, agreement. b. Fraud, force, and privilege. c. Forum non conveniens.

2. Constitutional limitations (due process) III. Choice of law. A. Basic concepts. 1. Essays On Oil. Legal characterization. 4. Proof of foreign law. B. Choice of law theories. 1. Traditional vested rights approach. 2. Hemingway Style. Contemporary policy approaches (including the interest analysis approach and the substantial relationship approach of Restatement, Second, of Conflict of Laws)

C. Marketing Case. Application in specific areas. 5. Worker's compensation. 7. Substance vs. procedure. D. Defenses against application of foreign law. 1. Local public policy. E. Constitutional limitations. 2. Full faith and credit. 3. Privileges and immunities.

F. Federal-state conflicts. 1. Federal supremacy. 2. Erie doctrine. IV. Recognition and enforcement of other states' judgments and foreign judgments. A. Full faith and credit. B. Effect: claim and issue preclusion. C. Defenses to recognition or enforcement. D. Family law judgments. Corporations and Limited Liability Companies.

I. Formation of organizations. A. Articles of incorporation. C. Articles of organization; certificates of formation. D. Operating agreements. II. Pre-organization transactions. A. Promoters: contracts and fiduciary duties. B. Subscriptions for shares. III. Piercing the veil.

IV. Essay. Financing the organization. A. Sources of finance. B. Essays On Oil. Securities issuance and characteristics. C. Dividends and distributions. D. Redemptions and repurchases. V. Management and control. 1. Meetings: annual, notice, and quorum. 2. Voting: eligibility, cumulative, voting, proxy voting, class voting, voting trusts, and shareholder voting agreements. 1. Meetings: quorum and notice.

2. Essay Hemingway Style. Action by written consent. 3. Action by committee. 4. Director's objections to muslim term paper actions. 2. Officer's liability on corporate obligations. D. Members and hemingway, managers. VI. Fiduciary duties. A. Directors, officers, and shareholders. B. Managers and members. VII. Term Paper. Close corporations and special control devices.

A. Share transfer restrictions. B. Special agreements allocating authority. C. Resolutions of hemingway style, disputes and deadlocks. D. Option or buy/sell agreements. VIII.

Organizational structure including relationships between parents and subsidiaries. 1. Articles of and friends, incorporation and bylaws. 2. Articles of organization, certificates of formation, and operating agreements. B. Hemingway. Mergers and consolidations. C. Sales of substantially all assets. E. Exchanges of securities. F. Dissolution of organization. IX. Shareholder and member litigation: direct, derivative, and class litigation.

I. Intestate succession. A. Share of the surviving spouse. B. Share of children and more remote descendants. 1. Adopted children. 2. Child born out of wedlock. C. Share of ancestors and collaterals. E. Simultaneous death. A. Execution requirements. 1. Governing law. 2. Wills complying with law of domicile.

3. Foreign wills. 4. Holographic wills. 5. Interested witnesses. B. Integration of wills. D. Incorporation by reference. E. Facts of independent significance. 1. Dependent relative revocation. 2. Revocation due to changed circumstances. 3. Revocation by physical act. 4. Partial revocation. H. Contractual wills.

I. Studies. Construction problems. 1. Lapsed legacies. 6. Essay Hemingway Style. Slayer statutes. 8. Simultaneous death. 10.Classification of on sports, legacies and style, devises. 11.Gifts to classes. 12.Gifts to children and essays on oil, issue. J. Will contests. 1. Age requirement. 2. Mental capacity.

3. Undue influence. 6. No contest clauses. 7. Standing to contest. K. Essay Hemingway Style. Non-probate transfers. 1. Inter vivos gifts. 2. Joint tenancy. 3. Tentative trusts and pay-on-death accounts.

4. Other non-probate transfers. L. Powers and duties of personal representative. III. Family Protection. A. Spouse's forced or elective shares. 2. Assets subject to share. B. Share of after-born or pretermitted child. C. Homestead and exempt property.

D. Limitations on charitable bequests. IV. Living wills and durable health care powers. A. Execution requirements. C. Individuals eligible to be agent or attorney-in-fact. D. Authority of agent or attorney-in-fact. I. Getting married.

A. Marketing Research Ethics. Controversies arising in anticipation of marriage. B. Limitations on essay hemingway who may marry. C. Procedural requirements. D. State of for family and friends, mind requirements. E. Common-law marriage and other curative or mitigative doctrines. F. Premarital contracts. II. Being married.

A. Rights and responsibilities of spouses. B. Family privacy. 1. Common-law doctrine. 2. Constitutional privacy. 3. Reproductive choices. 4. Essay. Evidentiary privileges.

C. Remedies for tortious interference with the marital relationship. III. Separation, divorce, dissolution, and annulment. A. Grounds and defenses. B. Jurisdiction and recognition of decrees. C. Preliminary interlocutory and final orders. D. Division of property. E. Maintenance or alimony. F. Child support. G. Modification of maintenance and child support.

H. Enforcement of awards. I. Mediation and other alternative means of dispute resolution. J. Separation agreements. IV. Child custody. A. Standards for decision. C. Joint custody. E. Procedural issues.

1. Jurisdiction to decide custody. 2. Research Ethics Case. Child's preference. 3. Counsel for the child. G. Mediation and other alternative means of dispute resolution. V. Rights of unmarried cohabitants.

A. Rights of cohabitants inter se. B. Unmarried parents and their children: illegitimacy. 1. Constitutional limits on discrimination. a.Unfavorable treatment of illegitimate children. b.Unfavorable treatment of unmarried parents. 2. Presumption of legitimacy. 3. Establishing paternity. VI. Parent, child, and state.

A. Legal disabilities of childhood. B. Duty to support. C. Intra-family immunities. D. Claims for loss of consortium. E. Parent's right to control child's upbringing and limitations on parental autonomy. F. Style. Custodial disputes between parents and third parties. B. Agency versus independent placements. C. Parental consent. VIII. Alternatives to adoption. A. Artificial insemination by donor.

B. Surrogacy arrangements. C. In vitro fertilization, gestational surrogacy, and embryo transplantation. Federal Civil Procedure. I. Jurisdiction and venue. A. Subject matter jurisdiction. 1. Federal courts. B. Jurisdiction over parties. C. Jurisdiction over property. D. Management. Service of process and notice. E. Venue, forum nonconveniens, and transfer. II.

Law applied by federal courts. A. State law in federal court. B. Federal common law. III. Injunctions and provisional remedies. IV. Pretrial procedures. B. Abstention doctrines. C. Joinder of parties and claims (including class actions) E. Adjudication without a trial. F. Pretrial conference and order.

V. The trial process. B. Nonjury trials. C. Jury instructions. VI. Verdicts and judgments. A. Jury verdicts. B. Judicial findings and conclusions. C. Directed verdicts and nonsuits. D. Post-trial motions. E. Effect; claim and issue preclusion. F. Appealability and review.

(*All citations to the UCC are to the 1999 Official Text.) I. Hemingway Style. General UCC principles* A. Rules of construction and application (§ 1-101, et seq.) B. General definitions and principles of interpretation (§1-201, et seq.) II.

Scope and applicability (§ 2-101, et seq.) A. Applicable to transactions in goods (§ 2-102) B. Definitions—merchant, between merchants, goods (§§ 2-104, 2-105) C. Definitions relating to existence and formation of contracts (§ 2-106) III.

Form, formation, and readjustment of contracts (§ 2-201, et seq.) A. Statute of frauds and exceptions (§ 2-201) B. Parol evidence (§ 2-202) C. Formation (§§ 2-204, 2-206, 2-207) D. Firm offers (§ 2-205) E. Course of project, performance or practical construction (§ 2-208) F. Modification, rescission, waiver (§ 2-209)

G. Delegation and assignment (§ 2-210) A. Essay Hemingway. Unconscionability (§ 2-302) B. Open price term (§ 2-305) C. Term. Output/requirement contracts (§ 2-306) D. Mode and style, terms of delivery and particulars relating to performance (§ 2-307 through 2-311) 1. Including tender of delivery, shipment and payment (§§ 2-503 through 2-505, 2-507 through 2-508; 2-511 through § 2-512)

2. Including risk of loss (§ 2-509 through § 2-510) 3. Including right of inspection and preserving evidence of goods in dispute (§§ 2-513, 2-515) E. Warranties, including waivers, disclaimers, and modifications thereof (§ 2-312 through § 2-317) F. Sale on approval, consignment sales and incidents thereof (§§ 2-326, 2-327) G. Sale by auction (§ 2-328) V. Passage of title (§ 2-401, et seq.) A. Identification of the goods (§ 2-401), including manner of identification (§ 2-501) B. Marketing Studies. Good faith purchaser; entrusting (§ 2-403) VI.

Breach, repudiation, and excuse (§ 2-601, et seq.) A. Buyer's rights on improper delivery (§ 2-601) B. Essay Hemingway Style. Rejection of goods; rights, obligations, and consequences (§ 2-602 through § 2-605) C. Acceptance, revocation of acceptance, and consequences (§ 2-606 through § 2-608) D. Adequate assurances; anticipatory repudiation(§ 2-609 through § 2-611) E. Installment contract (§ 2-612) VII. Remedies (§ 2-701, et seq.)

A. Seller's remedies in general (§ 2-703) 1. Seller's rights to deal with goods as yet unde- livered (§ 2-704 through § 2-706) 2. Damages—measure, incidental damages (§ 2-708 through § 2-710) B. Buyer's remedies in general (§ 2-711) 1. Essays Violence. Cover; nondelivery, repudiation, noncon- forming goods, incidental damages, deduction of damages from price (§§ 2-712 through § 2- 715; 2-717) 2. Specific performance (§ 2-716) C. Liquidated damages (§ 2-718) D. Contractual modification, limitation of remedies (§ 2-719) (*All citations to the UCC are to the 1999 Official Text.) I. General UCC principles* A. Rules of construction and application (§ 1-101, et seq.) B. General definitions and principles of interpretation (§1-201, et seq.)

II. Applicability and definitions (§ 9-101, et seq.) A. Subject matter of Article 9 (§ 9-109) B. Perfection of security interests in multiple state transactions (§ 9-301) C. Excluded transactions (§ 9-109) D. Definitions; account; purchase money security interest; control (§§ 9-102 through 9-107) E. Classification of goods (§ 9-102) F. Including sufficiency of essay hemingway style, description (§ 9-108) G. Including security interests arising under Article 2 (§ 9-110) H. Priority of consignments (§ 9-103, 9-324)

III. Validity of security agreements and rights of parties (§§ 9-201, et seq.) A. Title to collateral immaterial (§ 9-202) B. Enforceability (§ 9-203) C. After-acquired property; future advances (§ 9-204) D. Use or disposition of collateral by debtor (§ 9-205) E. Collateral in essays on oil, secured party's possession (§ 9-207, 9-208)

F. Request for accounting (§ 9-210) IV. Hemingway Style. Rights of third parties; perfected and unperfected security interests; rules of priority (§ 9-301, et seq.) A. Essays On Oil. Priority over unperfected security interests (§ 9-317) B. Essay Style. Requirement of filing and steps to be taken for perfection (§§ 9-308 through 9-316; §9-501, et seq.); assignment of security interest (§§ 9-514, 9-519) C. Protection of buyers of goods and chattel paper (§ 9-320, 9-330), including protection of. holders and purchasers of negotiable instruments (§ 9-331) D. Priority of liens arising by law (§ 9-333)

E. Alienability of debtor's rights (§ 9-401) F. Priority among conflicting security interests (§§ 9-322 through 9-329) G. Fixtures (§ 9-334) H. Accessions; commingling (§§ 9-335, 9-336) I. Subordination (§ 9-339) J. Defenses against assignee; modification of contract (§§ 9-404 through 9-406) K. Termination statement (§ 9-513); release of collateral (§ 9-512)

V. Default (§ 9-601, et seq.) A. Rights and remedies on default (§§ 9-601 through 9-606) 1. Muslim. Including secured party's collection rights (§ 9-607) 2. Including secured party's right to style take possession and dispose of collateral (§§ 9-608 through 9-624) B. Debtor's rights (§§ 9-625 through 9-628) Trusts and Future Interests. A. Classification of trusts. B. Creation of trusts. 1. Studies. Requirement of trust res. 2. Requirement of beneficiary.

3. Requirement of trustee. C. Types of trusts. 1. Revocable trusts. 2. Irrevocable trusts. 3. Testamentary trusts. 4. Pourover trusts. 5. Charitable trusts. D. Essay Hemingway. Alienability of trust interests.

E. Protective trusts. 1. Discretionary trusts. 2. Management. Support trusts. 3. Spendthrift trusts. F. Powers of invasion. G. Modification of trusts. H. Termination of trusts.

I. Powers and duties of trustees. 1. Prudent person rule. 3. Duty of loyalty. 4. Essay. Duty to act impartially. 5. Principal and income allocations. II. Future interests.

A. Classification of reversions, remainders and executory interests.