Gene-ablation and other genetic technique

broker-deletion and former(a)wise agenttic proficiencyHow has the enjoyment of comp superstarnt- cut and other componenttic techniques in mice altered our approach to scientific problems in pharmacology and physiology? 280 pts (8-12 pages, 11pt Arial font, double spaced)A component is a region of desoxyribonucleic acid that curtails a discrete hereditary characteristic, usually corresponding to a atomic number 53 protein or ribonucleic acid1. Most scientists ar sidelineed in determining how components, and the proteins they encode, function in the intact organism. One of the most direct representations to find push through with(predicate) what a component does is to see what happens to the organism when that constituent is missing. Because changes layab unwrap trouble kioskular operationes, mutants often hold the key to thought component function. A well accepted strategy for evaluating gene function is to create and specify a gene arduous cringe. Virtual ly whatsoever(prenominal) gene in the germ line raft be mutated through gene object glassing in totipotent embryonic bowing (ES) carrels or to use gene deletion technology to knock-out every a circumstantial gene or a precise cell type in an intact animal2. Both of these methods base on balls almost unlimited possibilities for addressing moves concerning the molecular and cellular biota of growing, the relationship in the midst of contagiouss and biological function, unraveling the causes of malady, and enhance pharmaceutical research. ES cells be a versatile source of cells for repair of damaged and speculative meanders in the big(a) be2. Totipotent embryonic stem cells fool been employ in vitro to generated precursors for oligodendrocytes and astrocytes. transplantation of these ES cells into a puzzle pilfer of gentleman myelin ailment allowed the precursors to interact with soldiery neurons and efficiently myelinate axons in brain and spinal cord. fos ter investigation of this mechanism could play to pharmacological, physiological and therapeutic interventions. ES cells overly make possible the most precisely controlled forms of patrimonial modification, allowing animals to be created with closely any desired alteration introduced into their genome2. One way to produce a transgenic ( mantrap) animal is through gene targeting in totipotent ES cells. By incorporating a null allele into one allele of murine ES cells, and injecting these cells into early hook embryos, one tooshie create chimeric mice (heterozygous for the knockout allele) containing tissues derived from both host cells and ES cells2. Mating the chimeric mice allows one to patronise that the null allele is incorporated into the germ line and can tune these heterozygous chimeric mice to homogeneity producing progeny that ar homozygous knockout mice2. A knockout fawn generated to carry a mutating apolipoprotein E (apoE) gene through inactivation by gene targe ting in embryonic stem cells can be apply as a amaze for atherosclerosis. apoE is a constituent of very low density lipoprotein (VLDL) synthesized by the liver and of a subclass of high density lipoproteins (HDLs) pertain in cholesterol transport among cells. In humans a variant form of apoE, has been found to be defective in binding to the LDL sensory receptor that is participatord with familial type third hyperlipoproteinemia, a ailment characterized by elevated plasma cholesterol and premature coronary heart disease. apoE deficient mice develop severe hypercholeste affairmia receivable to a delayed clearance of large atherogenic wear outicles from the circulation. A study was through to analyze the genesis of atherosclerotic lesions, including the nature of the cells touch, the taking over of cellular fifty-fiftyts and the anatomical location of specific lesion types over time on mice fed a normal or a high fat diet. They found that the lesions were distri onlyed t heme the arterial tree and contained many features of the specialized, chronic, inflammatory-fibroproliferative solvent characteristic of atherosclerosis with age. This mouse model is apply to correct understand the physiological role of apoE in lipid metabolism, atherogenesis, and nerve injury. The model can alike be utilise as a way to test new pharmacological medicines in pre-clinical trials to steady down dose interaction, new therapeutics or to develop variations of current medicates to better meet the needs of the vast array of genetic variability seen in the human population today.Gene ablation involves the homologous genetic recombination of a gene in much(prenominal) a way that the gene has been do null. Genetic ablation is a genetic engineering technique utilise to suppress selectively the process of a specified cell line or cell type in an animal earlier than suppress the activity of an indivi duple gene2. Analyzing the in vivo function of cells can be accom plished through specific cell ablation. Using genetic ablation to produce a knockout mouse involves the gibe of one or more transgenes (usually a DNA segment bearing its own promoter) into the pronucleus of a fertilized mouse oocyte, which, after reimplantation into a foster mother, gives birth to a transgenic mouse bearing one of several(prenominal) hundred copies of the transgene3. These mice again can be bred to produce a homozygous colony.An over materialisation Sod2 transgenic mouse was generated through the use of pronuclear injection of the Sod2 gene into fertilized eggs. These mice overexpressed superoxide dismutase which catalyzes the conversion of superoxide anion to hydrogen peroxide and oxygen. superoxide anions are reactive oxygen species (ROS) generated in the respiratory chain as a by point of intersection of mitochondrial respiration. ROS cause oxidative damage to key mitochondrial components, such as protein, lipids and DNA. They are thought to lead to an overall decline in cellular function and ultimately mold the life span of an organism. Sod2 trangsenic mice have been used to test the inwardnessuate of overexpressing MnSOD on mitochondrial function, levels of oxidative stress or damage and live span in mice. They found that a ii fold increase of MnSOD throughout the life of the organism resulted in a slight decrease in oxidative damage and enhanced impedance against oxidative stress. Oxidative damage is excessively associated with arthritis, heart disease, gastrointestinal disorders, doubled sclerosis, cancer and neurological diseases. This model can be used to physiologically improve ones understanding of oxidative stress and to develop pharmaceutical agents that can enhance ones ability to decrease or protect against oxidative damage.Customized gene targeted animal models embarrass constitutive knockouts or knock-ins, or conditional knockouts or knock-ins of known genes, a gene family, mutant genes or artificially created repor ters and gene work ups. A constitutive knockout is a model that leave behinds a abundant overview of gene function, by the inactivation of the gene of interest at the genetic level, by haphazard transition or targeted insertion4. The advantage of development this model is that you get a total inactivation of the gene in any cell4. The disadvantage of this model is that the phe nonypes can be complex since all organs are partakeed4. A conditional knockout is a model that provides a research model of high potential, by targeting tissue-specific inactivation of the gene of interest, through the use of recombinase4. Recombinase is an enzyme that deletes the DNA fragment located between the two recombinase-specific sites4. This tissue-specific contemplation of the recombinase allows the inactivation of the gene of interest except in the tissue were the recombinase is expressed4. in that respect are two advantages to using this model, the first is the inactivation is restricted to specific tissues and the second is that several tissue-specific models can be made in parallel4. The disadvantages of using this model include the efficiency of the excision and the inability to validate the recombinase governance4. thither are a number of other strategies that can be used in place of, or to complement, conventional gene knockout technology. both such manikins are regulatable gene ablation and temporary gene ablation (gene knock downs). Ablation of a gene can be spatially or temporally regulated, that is only the organ of interest is affected or the mutation occurs only in one particular developmental state5. by the use of a regulator, the promoter can be regulated to control gene expression in the organ or particular tissue of interest. A gene knockdown refers to the technique by which the expression of one or more of an organisms genes is reduced, either through genetic modification or by treatment with a reagent such as a fiddling DNA or RNA oligonucleoti de with a sequence complementary to either an mRNA transcript or a gene6. These approaches offer a rapid and economical method to examine the specific actions of a gene product at a particular time point. Genes can be transiently down-regulated by targeted blockade of gene expression, either systemically or within a specific tissue. Two such approaches are antisense technology and the use of short-change interfering RNA (siRNA). Antisense technology is the use of short limited DNA sequences complementary to the mRNA of the gene of interest2. These sequences bind to the 5UTR and the offshoot of the coding sequence so preventing translation initiation and stoppage ribosomal interaction with the AUG start codon2. Antisense oligonucleotide genetic-code blocking medicines might control disease by inhibiting deleterious or die genes. siRNA on the other hand, utilizes small lengths of double stranded RNA knowing against the gene of interest and is introduced into the cell where t hey interact with intracellular machinery to form RNA-induced silencing complexes (RISCs)2. These complexes slow the siRNA strands and allow specific binding to the complementary mRNA sequence2. This binding causes segmentation and destruction of the now double stranded mRNA, and subsequently prevents translation2. siRNA can be used to study gene function, manipulating gene expression, as well as drug development. siRNA compounds can be created and delivered into human cells were they are able to whitewash genes and viruses responsible for human diseases.Two other genetic techniques that are used to approach gene investigation include suppressor synopsis and double back genetics. suppressor abstract is used to identify genes that are functionally connect to another gene of interest7. A suppressor mutation is a mutation that counteracts the do of another mutation thereby reducing its phenotypic effect7. Suppressor mutations whitethorn be intragenic (in the aforementioned(pr enominal) gene as the original mutation) or intergenic (in another gene)7. Intergenic suppressor mutations can be either information suppressors or function suppressors7. Of the two, function suppressors are most valuable for the genetic analysis of cellular processes7. Reverse genetics is a process in which specific mutations are introduced into a cloned gene for the purpose of identifying functional domains8. One way to conduct reverse genetics is to do RNA interference (RNAi)8. RNA interference is an in vivo gene knock-down, which is used to insert a random shRNA construct into the mouse genome4. Here, double-stranded RNAs inhibit corresponding gene expression by inducing degradation of its mRNA4. RNAi can be used as part of the immune response to viruses and other genetic material it can withal be used to look at downregulation and upregulation of genes4. RNAi has been used to evaluate whether insulin-like growth factor binding protein-3 (IGFBP3) modulates gastrointestinal stro mal tumor (GIST) cell response. Here, transfection of siRNA to knockdown IGFBP3 gene into GIST cells resulted in a significant loss of cell viability and therefore found that IGFP3 gene is required for GIST cell survival. IGFBP3 gene expression is lost in many cancer cells and reintroduction of the protein often results in cell death. On the other hand IGFBP3 has been linked to renal cell carcinoma, titty cancer, and metastatic melanoma, which suggests that IGFBP3 whitethorn contribute to tumorigenesis or disease progression. By canvass IGFBP3 and the IGF pathway one can explore IGF for prognostic and therapeutic cling to in GISTs and other cancers.In order to develop new treatment and preventative strategies for a human disease, we must first understand the biology and pathogenesis of the human disease itself10. A way to study human gene function is by mouse gene humanization. Humanization is the commutation of the murine gene by its human counter part4. Animal models of human d isease have been crucial in elucidating normal organ biology, pathogenic mechanisms of the disease and developing new therapeutic strategies10. Animal models are being used instead of human studies, because human studies are limited by a number of factors such as the variability seen in genetic accent and environmental models, the inability to readily obtain human tissues for molecular analysis and the small numbers of perseverings who may have a particular genetic abnormality10. Transgenic animals have already made valuable research contributions to studies involving regulation of gene expression, the function of the immune system, genetic diseases, viral diseases, cardiovascular disease, and the genes responsible for the development of cancer. Transgenic animal models are most effective if they have both construct validity and face validity. Construct validity is the degree to which the model corresponds to the clinical state in humans. Face validity is the animal model respond s in an appropriate manner to the effects of diametric therapeutic agents. An animal model that can recapitulate at least part of the neuropathology and the cognitive and behavioral impairment is the most effective representative of the disease. Several rodent models of Alzheimers disease (AD) have been created. The first example of such a model has been created to mime the cholinergic deficits of AD patients and to investigate the role of the central cholinergic system in the cognitive and behavioral deficits observed in AD9. The second example of such a model has been created with acute or chronic injections of starchy beta within the brain parenchyma of rodents, to overexpress AD-related mutated protein (amyloid deposits) in the central nervous system of rodents9. A third model of AD has been to create a transgenic mouse to express wild-type or mutant forms of the human APP and/or PS genes. all(prenominal) three of these models provide many insights into AD pathogenesis and approached to new therapies. By exploring and understanding of the neuropathological processes underlying the cognitive deterioration leading to dementia and investigating of the effects of potential new treatments on both AD neuropathology and cognitive deficits we go forth be able to test drugs that could potentially alleviate symptoms of the disease.Transgenic animals have overly been used to develop animal strains that secrete important proteins in milk2. These animals are used to synthesize recoverable quantities of therapeutically useful proteins. These proteins can be harvested from the animal by simply milking the animal and then using insularity techniques for protein purification2. For example cows have been used to purify collagen which can then be used to indicate burns and bone fracture2. Pigs have been used to purify human hemoglobin as a blood replacement for transfusion2. Goats have been used to make monoclonal antibodies for colon cancer2. In supplement sheep ha ve been used to make factor VIII and factor IX for hemophilia2. Despite the large amount of knowledge that can be gained by knockout studies, there are virtually important considerations to be interpreted when investigating the role of a particular protein or gene of interest. These considerations include but are not limited to redundancy, lethality, delineating systemic vs. local effects, crafty effects, and specific pathogen shrive conditions3. Gene redundancy is the existence of multiple copies of the alike(p) gene in the genome of an organism. This problem can be over come after to some extent by the use of multiple knockouts or in some situations where multiple ligands bind to the same receptor, a dominant negative receptor approach can knockout the effects of all ligands at once5. Lethality phenotypes naughtily compromise the ability of the organism to function. Some knockouts are developmentally lethal, which heart and soul that the genetically altered embryos cannot gr ow in adult mice. Knocking out a gene may also fail to produce an plain (phenotypic) change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. In some instances, a particular organ of interest can be transplanted into a healthy wild type host in order to conduct the analysis on that organ. Local effect refers to an unfavourable health effect that takes place at the point or area of contact, for example the respiratory tract11. Systemic effect refers to an adverse health effect that takes place at a location distant from the bodys initial point of contact and assumes absorption has taken place, i.e. absorption into the blood stream11. sharp effects are those that are so slight as to be difficult to detect or describe. For example, small changes in nutrient words to the fetus can program post-natal and adult metabolic status and lead to increased susceptibility to a range of adult onset disease, including stroke, hypertension and non-insulin dependent diabetes5. Specific pathogen free conditions refers to the fact that laboratory mice in research institution are generally housed in a specific pathogen free environment, and are therefore not challenged with the array of pathogens most mice and humans are exposed to.When comparing transgenic models to humans a key difference could be in the initiation of the development or process in the mouse that may not have a similar mechanism as the human development or process it is thought to represent. The presence of a specific mutation in the mouse from the time of its conception may enable a distinction between phenotypic changes due to the mutation itself and changes caused by adaptation and compensation for the mutation12. A gene can also be expressed in different tissues where it may have different functions its alteration may induce unexpected consequences. Also, the function of two genes may overlap or a mutation in a single gene might not reveal an abnormal phenotype. There is also a possibility of random incorporation into genome, which may inadvertently sever other genes, or have a variable degree of gene silencing success. The gene may also induce gross morphologic or physiologic abnormalities that affect the gene directly. Note also, these animal models also only play a minor role in drug development, as relatively few human diseases are monogenetic12. In most cases, gene ablation and other genetic techniques must address the basic question as to whether a particular gene, wild type, or mutant is involved in the pathogenesis of a particular disease. If not a particular disease, it may also address whether a gene product which plays a role in a specific signaling process contributes to disease in conjunction with other predisposing conditions. Animal models are crucial in providing a unified understanding of how drugs work, developing new therapeutic agents, and obtaining critical data needed to advance comp ounds into clinical trails12. Animal models are an important scratch for scientist to investigate human diseases, especially in order to conduct time-course studies or when studying early disease. Transgenic and knockout animals are also used to determine the molecular mode of action of a gene product and the downriver consequences of its misexpression on normal processes. Assessing the role of a specific gene can know be accomplished through the use of transgenic animals whose genomes have been manipulated to under- or overexpress a target gene, or express it in a modified or defective, nonfunctional form12. Animal models represent an attempt to imitate the pathologies associated with human disease states in a preclinical setting12. ReferencesMolecular Biology of the Cell. Alberts, et. al. fourth edition 2002 Garland Science New York, NY.Crommelin, D.J.A. and Sindelar, R.D. (1997). Pharmaceutical Biotechnology basic principle and Applications, Third Ed. Taylor and Francis. Phil adelphia, PA.Seidman, J.G. (2009) Manipulating the Mouse Genome. new Protocols in Molecular Biology. January 01.Reliable and forward-looking Solutions for Transgenesis. Accessed on 11/4/2009. Ingman, WV. And Jones, RL. (2008). Cytokine knockouts in reporduciton the use of gene ablation to dissect roles of cytokines in reproductive biology. Human Reproductive Update 14(2) 179-192.Gene knockdown. (2009, June 8). In Wikipedia, The Free Encyclopedia. Retrieved 1353, November 8, 2009, from Fay, D. and Johnson, W. (2006). Genetic mapping and manipulation Chapter 10-Suppressor mutations. Accessed on 11/5/2009. Bio-Medicine. Accessed on 11/4/2009. Dodart, J.C. and May P. (2005) Overview on Rodent models of Alzheimers Disease. Current Protocols in Neuroscience. November 01.Lind J. and Semsarian C. (2006) Overview of Model Systems for the Analysis of Human Disease. Current Protocols in Human Genetics. February 01.Local vs. Systemic Health Effects. Accessed on 11/3/2009. Moser, P. (2009) Ani mal Models of Disease. Current Protocols in Pharmacology. September 01.Katz, D.A. (2007) Overview of Pharmacogenetics. Current Protocols in Pharmacology. March 01.Barash, CI. (2001)Ethical Issues in Pharmacogenetics. Accessed on 11/4/2009. Frequently Asked Questions virtually Pharmacogenetics. Accessed on 11/4/2009. Bustle, O., Jones, K.N., Learish R. D., et.al.(1999) Embryonic Stem Cell-Derived Glial Precursors A source of Myelinating Transplants. Science 5428(285) 754-756 Piedrahita, J.A., Zhang, S.H, Hagaman, J.R, Oliver, P.M, and Maeda N. (1992) Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. USA. 894471-4475. Nakashima, Y, clump AS, Raines E.W, Reslow J.L, and Ross R. (1994) ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arteriosclerosis and Thrombosis 14133-140. Jang Y.C et.al (2009). Overexpression of Mn Superoxide Dismutase Does Not I ncrease Life Span in Mice. Journal of GerontologyBiological Sciences. 64(11) 1114-1125. Dupart JJ. Et.al (2009). Insulin-like growth factor binding protein-3 has dual effects on gastrointestinal stromal tumor cell viability and sensitivity to the anti-tumor effects of imatinib mesylate in vitro. Mol Cancer Nov 108(1)99 (Epub ahead of print) PMID 19903356 Genes associate with drug responses can be grouped into three broad categories those involved in pharmacokinetics (i.e. drug metabolism, transport), those encoding pharmacological targets (i.e. drug-target pathways) and those with physiological associations (i.e. homeostasis pathways)13. Pharmacokinetic pathways mediate drug absorption, distribution, metabolism and excretion13. Pharmacological pathways such as transcription factors and phosphorylation targets whose genes encode other proteins are also candidates for pharmacogenetic analysis13. Understanding the sensitivity of a particular drug and the genes that encode the drug meta bolizing enzymes can help determine how the particular drug is transported and metabolized within the body. Understanding the gene variants and how they influence drug concentrations at the target site is a possible way to explain the differences in response to a particular drug dose among individuals. Pharmacogenetics is the study of how an individuals genetic differences influence drug action, usage and dosing2. The molecular compositions of enzymes are currently being set through the use of the Human Genome Project. Scientists are able to study these enzymes and determine correlations between genotypic and phenotypic variability14. These current advances will allow scientists to use the molecular knowledge of enzymes to detect individuals who are likely to experience adverse reactions to medicines without having to use potentially dangerous methods of trial and error14. An offshoot of pharmacogenetics is pharmacogenomics, which attempts to understand not only the molecular com position of genetic variants associated with drug response but also the behavior of those variants14. Pharmacogenomic knowledge can be used to authority the disease before it occurs in a patient, increase drug capacity upon pharmacotherapy, and reduce drug toxicity2. It could also facilitate the drug development process to improve clinical development outcomes, reducing overall cost of drug development and lead to development of new diagnostic tests that impact on therapeutic decisions2. Pharmacogenomics can be used by twists to identify the best dose and medicine for each individual patient with greater readiness and safety. As the dosage would be based on factors such as age, weight, diet, lifestyle, liver and kidney function. By using a pharmacogenetic test a doctor can determine the right dose for individual patients. There are phomacogenetic tests for cancer, HIV, depression, and cardiovascular disease15. These tests take in the factors which are most likely to affect the absorption and effectiveness of the drug. There are many benefits that can come from pharmacogenetics and pharmacogenomics. Pharmaceutical companies can use information obtained to create more mighty medicines based on proteins, enzymes, and RNA molecules associated with genes and diseases15. Again, doctors would be able to analyze a patients genetic profile and prescribe the best available drug therapy from the beginning instead of the standard trial and error method of matching patient with the right drugs. A more appropriate drug dosage could be determined by not only taking in a persons weight and age but also accounting for their individual body processes, such as the time it takes for their body to metabolize the drug. Pharmacogenetics could also provide advance screening for disease, better vaccines, improvements in the drug discovery and approbation process and decrease the overall cost of health forethought15. Pharmacogenomics is still in the development stage in the res earch field. There are several factors that need to be overcome before many of the pharmacogenomic benefits can be realized. These include the complexity of finding gene variations that affect drug response, the boundary of current drug alternatives, disincentives for drug companies to make multiple pharmacogenomic products, and educating health care providers14. There are also ethical issues to consider, such as inadequacy of data privacy, possibility that adverse reactions to drugs can occur during treatment, the cost of such designer drugs will be to expensive for everyone, and genetic testing can provide an array of diagnostic results (predisposal test) which may or may not be what the patient wants to know14.