We previously recognized a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia

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We previously recognized a novel syndrome in patients characterized by paraganglioma, somatostatinoma, and polycythemia. features [3]. Individuals with PacakCZhuang syndrome consistently fall Mavoglurant into Cluster 1 and are found to have high levels of normetanephrine (NMN) and norepinephrine (NE) [1]. Polycythemia is an irregular elevation of the hematocrit caused by either increased production or decreased damage of red blood cells (RBCs). Secondary polycythemia occurs as a consequence Mavoglurant of elevated circulating erythropoietin (EPO), while main polycythemia is due to intrinsic factors (e.g., somatic mutation and hereditary dominating mutations) of erythroid progenitors in the bone marrow and is EPO-independent [4]. Mixed polycythemia, such as Chuvash polycythemia caused by mutation, offers features of both main and secondary polycythemias characterized by elevated EPO and erythroid progenitors hypersensitive to EPO [5]. Elevated plasma EPO confirmed secondary polycythemia in the syndrome patients, but it is still unclear whether main polycythemia is present. Hypoxia signaling pathways have been established as essential to disease pathogenesis as well as normal development [6,7,8,9]. mutations were previously only found to cause familial polycythemia and pulmonary arterial hypertension [10,11,12]. This fresh syndrome of paraganglioma, somatostatinoma, and polycythemia provides a unique opportunity to study the effect of hypoxia signaling, specifically gain-of-function of HIF2, on tumorigenesis. In this study, we aimed to develop a transgenic mouse model to achieve the following aims: (1) to confirm mutations are causative gene mutations for the syndrome and (2) to Rabbit polyclonal to RAB14 use this model for further pathogenesis and therapeutic studies of the syndrome. 2. Results 2.1. Establishment of A Somatic Epas1A529V Animal Model The syndrome patients were found to carry somatic mutations in the ODD without other germline mutations [2]. We thus generated a transgenic mouse model with a somatic heterozygous mutation (corresponding to human point mutation is located in the 3 homology arm. G418-resistant ES cell colonies were picked up after co-electroporation of Mavoglurant TALEN expression vectors and A529V targeting vector into B6:129-mixed-background ES cells. Positive recombinant ES colonies were confirmed by PCR at both 5 and 3 ends (Figure 1B). Sanger sequencing also confirmed the presence of the A529V mutation (GCA GTA) in the positive ES colonies before injection into the blastocysts (Figure 1C). Chimera and subsequent germline-transmitted mice (mice. Open in a separate window Figure 1 Establishment of the animal model. (A) Schematic strategy of the mutant mice generation. (B) Positive embryonic stem (ES) colonies were confirmed by PCR at both 5 (F1/R1) and 3 (F2/R2) ends. (C) Sanger sequencing result of the F2/R2 PCR band. The mutant codon is labeled in red. To activate the expression of the A529V mutant allele, we mated mice with transgenic mice in C57BL/6 background and generated somatic heterozygous mutant mice (mutant mice (Figure 2B). To confirm the expression of the mutant allele, we extracted RNA from multiple cells from the mutant mice, including center, lung, liver organ, kidney, duodenum, adrenal gland, spleen, and testis, and performed invert transcription. Droplet digital PCR (ddPCR) with complementary DNA (cDNA) of every tissue verified high manifestation of in lung and center (Shape 2C,D), in keeping with a earlier record [13]. The percentage of mutant allele in cDNA different from 20.8% to 49.4% in various cells (Shape 2E). These total results verified Cre-mediated high expression of mutant allele in an array of tissues. Open in another window Shape 2 Successful manifestation of mutant allele in a variety of cells. (A) Mouse mating technique to generate the somatic mutant mice. (B) Genotyping PCR (F3/R3).