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The Value of Animal Models in Endotoxin Research
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
The relatively recent availability of both transgenic mice and gene knockout mice have greatly facilitated the study of endotoxin activities in this animal species. Transgenic mice allow for the detailed investigation of a single selected gene from humans or other animal species, which has been inserted into the genome of mice. This allows for the evaluation of the specific transgene product in comparison with isogenic, wild-type mice. The ability to isolate specific genes in this fashion can be supplemented by targeted deletion of the selected gene of interest by gene knockout techniques (16–18).
Approaches to Studying Polycystic Kidney Disease in Zebrafish
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Reverse genetics, including knockdown and knockout, are widely used in studying the function of genes. In the zebrafish field, as the RNAi technique is not working, we use morpholinos (MO), which are DNA analogues and knockdown gene function by blocking translation or splicing of the target transcripts. Although this technique has been recently replaced by the CRISPR/Cas9-mediated gene knockout technique, due to the off-target issue by the MO, it is still widely used. For example, if we want to block the maternal deposit of a gene transcript and the maternal-zygotic mutant is difficult to obtain, we use MO. To avoid the nonspecific effects of the MO, we should follow the guidelines.20 Basically, the criteria for a reliable MO are that the phenotypes caused by MO knockdown should phenocopy the mutants and can be rescued by its mRNA overexpression.
Gene Targeting Models of Epilepsy: Technical and Analytical Considerations
Published in Steven L. Peterson, Timothy E. Albertson, Neuropharmacology Methods in Epilepsy Research, 2019
Given estimates that 30,000 genes are expressed in the mammalian brain,21 it is likely that the proliferation of new mutant mouse strains will include many that are relevant to the epilepsies. The first members of this new wave of epilepsy models illustrate the wide variety of genes that participate in the regulation of neuronal network excitability. It is likely that, in the future, an abundance of mechanisms involved in the regulation of excitability will be discovered. An advantage of pursuing this work in gene knockout models is that the genetic lesions are known, providing molecular points of reference for these studies. Furthermore, these models will provide candidate genes for studies aimed at uncovering the genetic bases of seizure susceptibility in humans.
MMP-9-mediated regulation of hypoxia-reperfusion injury-related neutrophil inflammation in an in vitro proximal tubular cell model
Published in Renal Failure, 2021
Yan Dong, Hong Zhao, Jiangwei Man, Shengjun Fu, Li Yang
Some limitations of our study should be considered. The application of gene knockout technology may validate our findings on the role of MMP. However, our experimental design is based on clinical treatment, and pharmacological inhibitors have established applications. If we study the molecular mechanism in the future, we will consider designing a gene knockout model. We know that if the aforementioned techniques are adopted, the role of MMPs in this process can be clarified. Another limitation of our study is that ECM is difficult to characterize using standard methods. In view of the close relationship between the ECM and MMPs, we indirectly indicate changes in the ECM based on changes in MMPs. The purpose of the experiment is not to reveal the molecular mechanism, although the authors will explore it in future experiments, if possible.
Getting a good view: in vitro imaging of platelets under flow
Published in Platelets, 2020
Oluwamayokun Oshinowo, Tamara Lambert, Yumiko Sakurai, Renee Copeland, Caroline E. Hansen, Wilbur A. Lam, David R. Myers
Animal models have long been advantageous in the study of both hemostasis and thrombotic disorders as the highly complex in vivo environment has increased our mechanistic understanding and knowledge of disease outcomes that are relevant to human health. Murine platelets have played a vital role in hemostasis and thrombosis studies because of their genetic and functional similarity to human platelets, despite differences in size and structure. As such, combining in vitro microfluidic studies with genetically modified animal models has played an imperative role in illuminating biochemical and/or biophysical adhesion and aggregation processes that are difficult to discern in vivo. Gene knockout models have provided insight into the importance of specific gene mutations and various mechanistic processes. Various microfluidic systems have leveraged gene knockouts in order to specifically decipher the importance of α2β1 and PAR4 [53], the importance of the PI3 K signaling pathway [54], and the importance of talin1 [55] in adhesion and aggregation of platelets to collagen and fibrinogen. Utilizing a commercial microfluidic system, an ADAMTS13 knockout mouse model has been used to better understand the pathophysiology of thrombotic thrombocytopenia purpura [56]. One study employed both knockout models of mice lacking GPVI and ex vivo inhibitors of αIIbβ3 and Src kinase to reveal the existence of two potential routes of platelet adhesion to collagen [57].
Efficacy and safety of sotagliflozin in treating diabetes type 1
Published in Expert Opinion on Pharmacotherapy, 2018
The sotagliflozin program was driven by outstanding scientific technology. Lexicon mastered high throughput approaches to development of gene knockout modeling. They then exhaustively phenotyped resultant knockout lines for several therapeutically relevant physiologic processes such as glucose tolerance. They created one of the largest gene knockout libraries in the world. Their selection of the SGLT2 knockout mouse for further analysis paralleled more conventional SGLT2 inhibitor development pursued by multiple large pharmaceutical companies. They went further, however, in a thorough exploration of the process by which SGLT2 mediates UGE. They demonstrated that SGLT2 contributes primarily to UGE, although SGLT1 also is involved to a lesser extent. SGLT1 inhibition by sotagliflozin appears to be primarily local at the intestines, since the serum levels achieved are not high enough to affect renal SGLT1 action. They found that increased intestinal glucose resulting from inhibition of intestinal SGLT1 stimulated secretion of high levels of GLP1. In further studies of SGLT1 heterozygous mice, they found that these mice had delayed intestinal glucose absorption and increased intestinal GLP-1 release without any signs of the severe malabsorption syndrome that affects homozygous SGLT1 knockout mice. As a result of their observations, they decided to synthesize and test agents for their ability to inhibit both SGLT1 and SGLT2. Consequently, they pursued the synthesis of a dual SGLT1/SGLT2 inhibitor with moderate effect on SGLT1.