Molecular Biology For Surrogate Marker For Endoscopic

Conditional and inducible gene knockout technology

Describe about the Molecular Biology for Surrogate Marker for Endoscopic.

1. The conditional and inducible gene knockout is a type of technique which is used for the elimination of a particular gene from a certain tissue like the kidney and liver. The use of this technique is in the study and determination of the function of specific individual genes of the living organisms. This process is different from the traditional gene knockout because in this process only specific gene at specific time is being deleted (Li, Y et al. 2014). The traditional gene knockout may result in the embryonic death due to the gene mutation and thus the study of the gene in adult condition by the scientist gets prevented but the use of the conditional gene knockout technique eliminates all these side effects of the traditional gene knockout technique. There are several tissues which are not possible to be studied properly in the isolation, this proves that genes are inactive in some specific tissue and active in other tissues (George et al. 2015). However by the use of the this conditional gene knockout technology it is possible to eliminate the genes present at a particular stage of development and the study can also be done about the affect of the knockout of the gene of one tissue have over the genes of the other tissues.

The most common technique use for the conditional gene knockout technology is the Cre-lox recombination system. In this system the specific recombinase enzyme Cre recognizes the two lox that is the loci of the recombination sites present within the DNA and causes recombination among them (Kumar et al. 2016).

This technique of conditional gene knockout in mice is often used for the study of several human diseases because there are many genes present which produces similar phenotypes both the species. For studying kidney function and learning and memory in mice lacking Msc1 knockout mutation must be created in each of the embryonic stem cells that take part in the protein coding genes present in these tissues of the mice. The genes are made to be knockout because it is the best way to determine the accurate function of the genes and study more about the role of these genes in human disease. In this particular project it is to be focused that is there any deficiencies occurring in the formation of the wiring of the brain and development of  it  due to the elimination of the Msc1 and what happens if the targeted gene is not eliminated.  It also have to be determined that what changes occurs in the development of the memory cells due to the lack of these Msc1 (Put et al. 2016). Also the changes in the function of kidney due to the lack of the Msc1 have to be checked. This can be done by knockouting the Msc1 gene and after that monitoring the changes that are occurring in the mutated mice in comparison to the wild type mice.For the development of the mouse models comprising these diseases requires a new strategy for the generation of the animal lines with genetic changes that are identical. This opportunity is provided by the pluripotent stem cells isolated from the embryo of the mouse(Rotunno et al. 2015)..

2. Inflammatory bowel disease (IBD) along with the ulcerative colitis (UC)  andCrohn’s disease (CD) are disorders that are mediated  by the immune system  and these diseases are characterized by the relapsing and chronic inflammation  of the gastrointestinal tract. Though the cases of IBD were observed very rarely about century ago but in recent times the prevalence rates of the IBD have increased (D’Haens et al. 2012). Though it is clear that genetic susceptibility is responsible for this disease still the environment, diet and the resident microbial population of the intestinal area are also considered to play an essential role in the start and development of the disease. For the collection of the pure population of cells from the frozen tissue laser micro dissection technique can be used (Lutgens et al. 2013). This allows the molecular profiling of these cells which is also reflective of these cells in vivo state. The mouse model is used for the analysis of the gene expression present in the large intestine. This analysis is mainly done with the intact colon tissue and also gives insights as the molecular pathways that are involved in the maintenance and the initiation of inflammation. However the colon is type of a complex tissue which possessesdifferent distinct layers like the submucosa, mucosa, serosa and muscular layer.The epithelial layer present in the colon mucosa comprises goblet cells,enterocytes, intraepithelial lymphocytes and stem cells (Xuan et al. 2016).

Cre-lox recombination system

The main aim of this project is to depictthe global gene expression changes in complete colon tissue after and before the development of the inflammation in Il10^−/− mice.The total inflamed colon comprises a huge number of infiltration of the immune cells which can be considered to be associated with the damage of the tissue and the overall changes in the morphology of the intestine which occurs due to inflammation. The primary role of the epithelium in the process of inflammation improvement needs an additional understanding of the changes of the cell that are occurring in the IBD epithelial layer in vivo condition. The laser micro-dissection and subsequent microarray analysis of the epithelial cells of the colon make it possible to identify the pathways and the process that are related to the procedure of inflammation. For the investigation of this hypothesis interleukin 10 gene deficiency mouse that is (Il10^−/−) is used, which is responsible for the development of the inflammation of the intestine that is similar to IBD (Hua et al. 2013).

Utilizing creatures as a part of research has dependably been a subject of level headed discussion among mainstream researchers around the world. The choice to play out specific trials on live creature subjects is constantly taken after a progression of examinations were at that point performed, leaving this as a last necessity before live human testing. It ought to dependably be performed when it is resolved that it will give a huge progression in the field and will later outcome in a change to the human, social or natural prosperity. Verifiably, creatures have been utilized as a part of logical trials since old circumstances. Their utilization has differed significantly, thus did the ethical ramifications (Yipp et al. 2012). As pharmaceutical progressed as the centuries progressed, utilizing live or dead creatures was of vital significance for physiology or life systems tests. In cutting edge society, creature examinations are a privilege credited to skilled specialists who can guarantee others conscious care and utilization of the creatures in controlled situations, with as meager uneasiness as could be expected under the circumstances to the living creature. Mice were utilized as prime subjects for therapeutic examinations, particularly in pharmacokinetics studies and treatment (Jouan-Lanhouet et al. 2012).

The host and microbe relation-ship is very necessary for the normal development of the immunity of the gut mucosal and for the maintenance of the intestinal homeostasis and it also prevents the uncontrolled periods of the inflammation of the local portion. The colonization of the pathogens and the virulence is also prevented by the microbiota of the gut and at the same time it also promotes the function of the epithelial barrier by promoting the rejuvenation of the epithelial cells. the genetics of the host and the immune mechanism modulated the gene expression of the recognition of the microbial molecular pattern so that an influence over the diversity and the function of the local microbiota can be made. There are many genes which includes for the maintenance of epithelial barrier function are IBD5, ITLN1, DMBT1, DLG5, PTGER4, XBP1, and the genes which are included in the process of  beginning of secondary immune response are HLA-region, PTPN2, TNFSF15/TL1A, IRF5, PTPN22, NKX2-3, IL-18RAP, IL-12B,  MST1. Thus it can be concluded from here that alteration in the genetic level may influence the immunity of the organism just by promoting or suppressing the blooms of the pathogenic microbiota and in return of this it affects the integrity of the epithelial-barrier, inflammation and immunity of the host intestine.  The recent day’s studies shows that there is a quite good relationship between the process of inflammation, the gene Il10^−/− and the gut microbiota. The microbial status has a huge influence over the growth of inflammation and tumorigenesis. Due to the deficiency of the immunosuppressive cytokine IL-10 on the effector T cells the mice develops chronic intestinal inflammation. This chronic inflammation increases the chances of the tumorigenesis which is further initiated by colon specific carcinogen. Different hypothesis shows that inflammation sometimes alter the composition of the microbial community in the Il10^−/− mice which susceptible to colitis in comparison to the wild type (WT) mice (Devkota et al. 2012). Though the germ free mice exhibit to have an undeveloped immune system, this approach is perfect for the study of the microbiome because it diminishes the microbiota legacy. The samples of the stool and the samples of the distal colon represent the luminal and the microbiota which are mucosally adherent and these samples are composed from the Il10^−/− (colitis), AOM-treated Il10^−/− (colitis or cancer), AOM-treated WT (healthy) and WT (healthy). For the evaluation of the microbial population the Illumina HiSeq2000 sequencing of the V6 region of the bacterial 16S rRNA gene is performed.  This region of the bacterial gene is the hyper variable portion of the gene. The colitis susceptible Il10^−/−   mice shows a decrease in the microbial richness in comparison to the healthy WT mice. Studies shows that there is an abundance of Proteobacteria in the Il10^−/−   microbiota in comparison to the healthy WT mice. In the Proteobacteria the class of Gammaproteobacteria, Enterobacteriales order and the Enterobacteriaceae family were observed to be more significantly abundant in the Il10^−/−   mice (Teichmann et al. 2012).

Mouse models for studying human diseases

The IL-10-819C/T polymorphism is not expressively related to the colon cancer. However the recent studies suggested that IL-10 −819TT genotype can be considered as the protective factor for the cancer among the Asians especially in the case of gastric cancer. On the other hand CT genotype and the dominant model can be a risk factor for the ovarian and the cervical cancer (Yu, Z et al. 2013). The significance of the stratifying by the cancer type, ethnicity, sample size and study design  required to be standardize in the studies of the future along with the consideration of the association between the cancer riskand IL-10 −819C/T polymorphism. A broad variety of the usual cell as well as the tumor cells produces interleukin (IL)-8 and the principle role of it is the beginning and the intensification of the acute inflammatory reactions. The IL-8 is also being concerned in the processes of chronic inflammation. The IL-10 is one type of the immunoregulatory cytokine and the main biological function of it is the termination process of the inflammatory response. The proliferation and the differentiation of several types of immune cells are regulated by the IL-10 (Turner et al. 2014). This gives the evidence that IL-10 have dual role one is the antiangiogentic cytokine and the second one is the Immunosuppressive which promotes and inhibits the effect on the tumor enlargement and progression. Since the role of the IL-8 and IL-10 are not at all very well defined, so investigation is still going on the impact of the IL- and IL-10 on the sporadic colon cancer progression and development. Though an increased IL-8 and IL-10 expression is observed in various tumors and also some studies exhibits IL-8 and IL-10 levels related to tumor metastasis and progression. Under different pathological conditions over expression as well as deficiency of the IL-10 can be found. Also it is analyzed that there is a significant IL-8 and IL-10 protein expression in the cases of sporadic colon tumors (Galdiero and Mantovani, 2015).

Reference

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D’Haens, G., Ferrante, M., Vermeire, S., Baert, F., Noman, M., Moortgat, L., Geens, P., Iwens, D., Aerden, I., Van Assche, G. and Van Olmen, G., 2012. Fecal calprotectin is a surrogate marker for endoscopic lesions in inflammatory bowel disease. Inflammatory bowel diseases, 18(12), pp.2218-2224.

Role of inflammation and gene IL-10 in chronic intestinal inflammation and tumorigenesis

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