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Diabetes Mellitus is a disease characterized by hyperglycemia associated with neuropathic, macrovascular and microvascular complications. It is a disease in which the human body doesn't secrete, or use insulin properly . The hormone produced in the organ of the body called pancreas, an organ proximal to the stomach. Insulin is necessary to turn glucose and other food elements into energy used by the body. There are two conditions in Diabetes Mellitus, either your body doesn't make enough insulin for body use or it can't use its own insulin, or both. This condition leads to build up of too high sugar in the blood .
Diabetes mellitus Type 2 is characterized by fasting blood glucose of more than 126 mmg per deciliter (mg/dL). A condition in which blood glucose levels are higher than normal is called pre-diabetic but not necessarily diabetes mellitus. This type of DM is adult onset or maturity onset type affecting majority of obese people over 40 years old. The signs and symptoms of Diabetes Mellitus includes polyuria, polydypsia, polyphagia, glucosuria, weight gain but usually asymptomatic. This condition will lead to complication called Hyper Osmolar Non-Ketotic Coma. The diagnosis of Type 2 DM can
be associated to symptoms and blood glucose >200mg%, fasting blood glucose >126mg%, 2-hr postprandial blood glucose >200mg%, FBS is increased; 3 consecutive times with signs or polyuria, polydypsia, polyphagia and glucosuria confirmatory for DM .
The main organ involve in this disease process is pancreas. The function of hormones of the pancreas is the production of insulin, gastrin, somatostatin and glucagon which plays an important function in maintaining balance of salt and sugar in our bodies, and therefore, any alterations in the regulation or production of these hormones will lead to problems or manifestations with blood glucose and fluid / salt imbalances .
Type 2 Diabetes Mellitus is related to metabolic syndrome because this condition or illness manifests several cluster of signs and symptoms such increased insulin levels, as - elevated blood excess and abnormal cholesterol level - that eventually manifest together that increases the risk of stroke, heart disease and diabetes mellitus. Type 2 Diabetes Mellitus is also defined as metabolic disorder characterized by non utilization of carbohydrates, protein and fat metabolism .
Because metabolic syndrome are composed of cluster manifestations, DM type 2 having more than one of these manifestations can be diagnosed as metabolic syndrome, that will contribute to serious illness development. Considerably, more than one metabolic conditions will potentates each other that will also increases the risk of complications .
Establishment of Diabetes mellitus model System
In order to develop model system for diabetes mellitus, a Tet29 group of rats were carefully treated with 5 µg/ml of Doxycycline (DOX) solution daily for about eight days the until level of blood glucose became 300 mg/dL. After the treatment, the concentration of the solution was modified to 1 µg/mL solution of Doxycycline (DOX) for additional five weeks. This kind of long term treatment facilitated with low doses of DOX produced in medium blood insulin and glucose levels enhancements and of the commendable volume in transgenic rats. Moreover, a continuous body weight loss was noted in the diabetic animals in chronic condition. In these rats, the observation detected a high shRNA expression and efficient regulation of down Insulin Receptor (IR) .
As a proof-of-principle target in this experiment for establishing reversible and inducible gene knockdown and model systems in rats, insulin receptor (IR) mRNA was the choice in order to develop a model system for type 2 diabetes mellitus. Increasing incidents of Type 2 Diabetes in the world population and its complications is observed presently. Animal model for this kind of disease type yielded numbers of insights into its paths and pathogenesis and opened new fields in drug development and discovery. The frequently used model for type 2 diabetes mellitus is according to the animal treatment with streptozotocin, which destructs the pancreatic β-cells and develops a state of type 1 diabetes mellitus. Presently, there is no available inducible rat model developed and established for type 2 diabetes mellitus characterized by the tissues resistance to insulin .
The utilization of transgenic for medical purposes dated back hundreds of years but genetic engineering to manufacture or produce desired biopharmaceuticals is much more recent. Model system for diabetes mellitus is the production in transgenic systems and has huge potential for the therapeutic and anti-cancer antibodies production .Transgenes make fully functional in terms of recombinant animal or human antibodies. As the biopharmaceuticals demands is expected to rise, it would be appropriate to ensure that there will be available DM model systems in significantly larger amounts, on a cost-efficient basis. Presently, the cost limits their availability. Expression systems of trangenes offer important advantages over transgenic cell-based and animal-based systems. Unlike bacteria, animals can perform the complex processing steps of proteins necessary to produce eukaryotic proteins in active form .
In this model system, rat is a significant animal model system used in biomedical research, but targeting technology using gene is not fully established for this kind of species . Therefore, aiming to produce transgenic knockdown in rats using shRNA pronuclear microinjection and technology is the major component of this model system. To this goal, the process utilized an expression system using a tetracycline-inducible shRNA that target the insulin receptor (IR). The treatment using Doxycycline (DOX) for the resulting transgenic rats led to a increased and reversible blood glucose rise and dose-dependent caused by ubiquitous inhibition of IR signaling and expression. The system for this model neither detects a response from interferon nor microRNA processing disturbances after treatment using DOX and excluding shRNA expression's toxic effects. Moreover, Low dosage of DOX treatment stimulated a chronic diabetes mellitus state .
Figure 2 the construc of transgene, pTet-shIR (A), have two different expression cassettes: First, presents shRNA against the insulin receptor (shIR) under the the human H1 promoter control that carries a sequence of tetracycline operator (tetO. The second cassette contains of a cDNA tetracycline repressor (tetR) bounded by a polyadenylation site (pA) and drives by the promoter CAGGS. A probe protection assay (RPA) by RNase was developed for loop and antisense binding hairpin strand.Adapted from .
This model system was developed using a technology that facilitates the inducible, specific, and reversible suppression of any genes of interest present in the rat. The first transgenic rat line generated for this model system and using this kind of method covers an inducible diabetes mellitus model .. The rat is the selected animal for this model and in other research areas neural and cardiovascular biology. However, because of insufficiency in gene targeting technology for this species, the rat has considered a lost ground in comparison to the mouse as experimental model animal in the past decades . The RNAi technology advent has created new ways to gain gene knockdown in mammals. Specifically, new animal models were generated with blunted gene-of-interest expression by the utilization of cassettes expression for small hairpin RNA (shRNA). However, these types of methods were based on stem cells of germline-competent embryonic type, which were not common and available in rats until recent times. These consequences seemed to be overpowered by two different independent studies in which constructs of pronuclear-delivered shRNA were victoriosly utilized to for genes knockdown in mice. However, also technology failure has been observed such as lack of germline transmission and toxic effects. In the year 2006, the descriptions of the first heritable and stable shRNA-based knockdown for an endogenous rat's gene using transgene (lentiviral) delivery were recorded. However, the downside of viral transgenesis is the mosaicism of transgene and multiplicity integration into the specific genome rendering genetically breeding of pure tedious lines and is time-consuming .
The Introduction of foreign DNA into the host cells has three major considerations. First of all identification and isolation of the DNA that to be introduced. Second step is the identification of the vector and construction of recombinant vector. Finally the identification of the suitable expression system to receive the DNA
The vectors could either be integrating (becomes part of the host's chromosomes) or extrachromosomal (Ikeno et al, 1998). They could be in copies varying from one to several hundreds.
Selected rats were individually maintained and ventilated (Tecniplast) cages under standardized and controlled conditions (a humidity of 65±5%, temperature of 21±2°C and with a 12 h artificial dark/light cycle) with free standard chow access (SSNIFF; 0.25% sodium) and ad libitum drinking water. Sprague-Dawley rats (SD) were taken from a commercial Taconic animal breeder .
In order to generate a transgenic rats, a 4-kb fragment of DNA containing pTet-shIR was cut out with KpnI and PacI restriction enzymes from the exchange pIR5-tet vector, drained from the QIAquick Gel Extract Kit gel using a (Qiagen), carefully dissolved at 3 ng/µl with buffer microinjection (pH 7.4, 8 mM Tris-HCl, 0.15 EDTA mM), and carefully injected fertilized eggs of SD rats in a microinjection process according to established techniques. Transgene integration was detected through the use of PCR on genomic DNA separated from biopsies of tail with the TetRfor primers. Two of thirty one rat newborns were transgene positive and were breed to produce the transgenic rat lines, Tet29 and Tet14. Estimated, 2 to 5 months old rats were utilized in all the process of experiments; littermates with negative were used as control wild type (WT) .
These vectors are to be faithfully replicated and distributed to daughter cells during cell division (Ikeno et al, 1998). Use of artificial chromosomes was widely used in DNA introduction. Available systems of expression, the eukaryotic and prokaryotic systems are the two major categories of systems in expression . The prokaryotic systems are generally easier to manage and are higher rate of satisfactory. But, there are major limitations in using this method for the production of some eukaryotic proteins. For instance, numbers of the eukaryotic proteins undergo a variety method in modifications of post-translational like correct folding, glycosylation, creation of disulphide bridges and phosphorylation. There is no adapted universal expression system for Type 2 Diabetes. All expression systems being used have its advantages as well as some limitations that should be considered as criteria in selection. Model system of rats is the most widely used host, its popularity is due to the available and information body of knowledge about its characteristics, genetics, physical formation, physiology and the complete genomic sequence which facilitates cultivation and gene cloning greatly .
In isolating the DNA, the mini-prep was used because it is quick method for isolating small amounts of DNA (about 1-5 ug/ml) and is often used to screen for transgenes after experiments. The DNA is rapidly isolated from a culture by any number of methods and in the same day may be digested with restriction enzymes and run out on an agarose gel .
Various methods have been developed for rapid small-scale isolation of plasmid DNA the most notable are alkaline lysis and boiled lysis methods. In most cases the plasmid DNA is maintained and carried in a bacterial host. In today's practical we shall be isolating the plasmid pBlueScript (pBS) along with a recombinant plasmid (ie pBS carrying a cloned cDNA insert). The bacterial strain carrying both plasmids is a common laboratory stain of E. coli known as DH5. Selection pressure for the maintenance of this plasmid is conferred in insulin. The vector pBS contains the gene lactamase, a periplasmic enzyme that cleaves insulin, thereby yielding cells resistant to this insulin .
This model system utilized a microinjection using a pronuclear technology, which is an established and modern methodology in rats, for the shRNA-induced establishment gene knockdown in this rat species. However, the first attempts for this model using constructed transgene driven by an active U6 promoter permanently were unsuccessfully done . Since the process reasoned out that toxicity of embryo because of high shRNA expression may be the primary failure cause, by this result, the process was directed to the use of H1 promoter constructs with an insertion block of an tetO operator controlled by the (tetR) tetracycline-repressor and the ubiquitous and simultaneous and expression of a tetR-codon-optimized. The activation system of tetracycline from E. coli was developed for culture of cells and used in fungi plants, and protozoa. It was based on wild-type tetR originally, which in the antibiotic tetracycline absence or its doxycycline (DOX) derivative binds to tetO and gene transcription represses . Later, this model system was modified by the tetR fusion with a domain transactivator resulting in 2 completely different activation systems of gene, 'tet-off' and 'tet-on'. Recently, the model systems allow tetracycline inducible microRNA or shRNA or expression to have been proven to be controllable, safe, and effective materials for inhibition of gene in mice .
During treatment of DOX, the blood was taken from the rats' tail vein for blood glucose and plasma insulin measurement. Drastic rise of blood glucose and plasma insulin were detected after 3 days of treatment using DOX in rats Tet29 and 1 day later also in rats Tet14. Levels of blood glucose reached three fold levels higher than in control rat animals. Correspondingly, the levels of plasma insulin were more enhanced than seven fold. The insulin levels and plasma glucose of untreated and WT transgenic rats were not distinguished. While untreated transgenic rats body weight was not different from the WT animals, it was reduced markedly in both rat transgenic lines after three of DOX treatment days .
Results of the model:
Here the model is showing that the tetracycline-inducible promoter model system maintains shRNA a tight control expression in all transgenic rats' tissues. When released after the treatment of DOX, the ubiquitously expressed shRNA conforms to a knockdown of IR effectively in all examined tissues, to resistance to insulin, and to type tow diabetes mellitus. These are reversible effects after withdrawal of DOX indicating that the process established a reversible and inducible system allowing the gene-of-interest knockdown in the rat .
Table1 Tissue Specificity knockdown of Tissue. Different tissues of both transgenic lines,Tet14 and Tet29, and WT were analysed for expression of IR by Western Blot after treatment with doxycycline.Quantification of the protein band intensities was carried out by the program TINA 2.08e and percentages of reduction of expression were calculated (WT,100%). WAT, white adipose tissue; BAT, brown adipose tissue. Adapted from(16)
Blood glucose level was analyzed in a tail-vein blood drop from conscious and freely feeding mice using Roche AccuChek Sensor. Insulin concentration in the Plasma has been quantified utilizing the Mouse/mouse Insulin ELISA kit according to the protocol of the manufacturer. To identify the sensitivity of insulin the blood glucose was quantified in the same rat before and fifteen min after insulin injection (10 U/kg) or control saline solution .
Furthermore, this type 2 diabetes model system also tested whether the expression of shRNA triggers a response of interferon in treated DOX transgenic rats. For this purpose, the process utilized Western blotting to detect and observed protein kinase R (PKR), a specific inducible interferon Ser/Thr protein kinase. No protein kinase R up regulation was observed and detected in all tissues tested, such as WAT, BAT and brain, after acute high dose treatment with DOX as well as in the heart after chronic low dose treatment .
During the course of the experiment, all rats were checked regularly for their status, body weights, drinking volume and insulin and blood glucose levels. All experimental and control rats were maintained alive and since there are not many other appropriate insulin resistance-associated models for diabetes mellitus in rats, these rats represented an attractive selection for further studies and investigations on the disease pathogenesis and its complications and for the assessment of novel concepts for therapeutic targeting in IR signalling. The expected outcome of the transgenic animal is geared towards the physical manifestations of the disease condition and other related factors. This contribution is significant for the field and this transgenic rat line developed with this method creates an inducible diabetes mellitus. Model and serve as a milestone for future studies.