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Insulin and Brain Reward Systems
Published in André Kleinridders, Physiological Consequences of Brain Insulin Action, 2023
Brian C. Liu, Qingchen Zhang, Emmanuel N. Pothos
The knockout of IRs using the Cre/loxP recombination system in various tissues in mouse models has been an important tool for understanding the role of insulin. This is done by using a targeting vector with the mouse IR gene with a selection cassette surrounded by loxP sites upstream of exon 4 and another loxP site downstream of exon 4. Embryonic stem cells are then transfected with this targeting vector and then with a plasmid containing Cre cDNA, which results in the removal of the selection cassette. Mouse blastocytes are subsequently injected with these clones, and these mice are bred with C57BL/6J mice. When the offspring of this breeding is injected with the Cre recombinase, exon 4 of the IRlox allele is deleted, resulting in an IR-specific knockout (26).
Mitochondrial Dysfunction and DNA Methylation in Atherosclerosis
Published in Shamim I. Ahmad, Handbook of Mitochondrial Dysfunction, 2019
The relevance of mitochondrial function in atherosclerosis has been confirmed by two large-scale expression studies. First, proteomics analysis of different portions of early human aortic atherosclerosis revealed that differences in mitochondrial protein abundance is a predominant signature of affected aorta, although mitochondrial protein abundance greatly varies between aortic locations (Herrington et al. 2018). Another study exploited the Reversa model mouse, in which the hepatic microsomal triglyceride transfer protein gene can be silenced by Cre-lox recombination following interferon administration, thus blocking VLDL assembly and reversing hyperlipidaemia in a Ldlr-null/ApoB100 knock-in background (Lieu et al. 2003). A transcriptome analysis of the Reversa mouse revealed a causal relationship between mitochondrial protein abundance and hyperlipidaemia-induced atherosclerosis (Vilne et al. 2017).
Introductory Remarks
Published in Dongyou Liu, Laboratory Models for Foodborne Infections, 2017
Mice with conditional gene modifications are created with two different types of genetic alterations: one contains a conditional vector [through inserting recognition sequences for the bacterial Cre recombinase (loxP sites) using homologous recombination in ES cells], which functions as an “on switch” for the mutation, and the other contains specific sites (called loxP) inserted on either side of a whole gene, or part of a gene, that encodes a certain component of a protein that will be deleted. Similarly, mice with chromosomal rearrangement are created using the Cre/loxP recombination system to induce site-specific mutations that display defects resembling those caused by human chromosomal rearrangements (e.g., chromosome deletions, duplications, inversions, translocations, and nested chromosome deletions) [8].
Endothelial-to-mesenchymal transition in tumour progression and its potential roles in tumour therapy
Published in Annals of Medicine, 2023
Identifying EndoMT in human tumour tissues based on the coexpression of relatively specific protein markers of ECs and mesenchymal cells has the following disadvantages. On the one hand, it is only allowed to identify the potential EndoMT and quantify EndoMT ratio, and more comprehensive genomic, transcriptomic and proteomic profiles of any proposed EndoMT phenomenon cannot been provided. Using high-throughput techniques such as quantitative proteomic profiling and single-cell transcriptome analysis of TECs will be beneficial to clarify the global phenotypic plasticity of TECs and the heterogeneity of EndoMT and to discover the possible induction mechanism [36,37]. On the other hand, whether EndoMT cells originate from endothelial cells cannot be determined because of the phenomenon of mesenchymal-to-endothelial transition [38], and a complete transdifferentiation stage cannot be detected due to the probable total loss of endothelium-specific protein markers in newly emerged mesenchymal-like cells. The use of genetic engineering technology to construct a transgenic mouse model to trace cell lineages will effectively overcome the above problems. Using the conventional Cre-LoxP recombination system, Zeisberg et al. constructed Tie2-Cre; R26Rosa-lox-Stop-lox-LacZ transgenic mice to conduct endothelial cell lineage tracing, in which endothelial cells were irreversibly tagged with an expression of the LacZ transgene. They found that approximately 30% of the FSP1+ cells in the tumour stroma were β-galactosidase and FSP1 double positive, and 12% of the a-SMA+ cells revealed double positivity for a-SMA and β-galactosidase [25]. In another transgenic mouse model, in which endothelial cells were indelibly marked with ZsGreen expression, the colocalization of a-SMA and ZsGreen in TECs was also identified [39]. Combining a cell lineage tracing technique with high-throughput techniques will provide more precise tracing of endothelial cell lineage and facilitate the in vivo study of TECs plasticity in tumour progression. Moreover, expression of certain markers is not a reliable sign of a cell type conversion and functional assays must be provided to confirm the identity of the final cell populations. Elucidating functional changes accompanied by gene expression alterations of TECs undergoing EndoMT may intuitively reflect the cell type conversion and biological influence in tumour progression.