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Protocol for evaluating the activity of R2 retrotransposons in mammalian cells.
STAR Protoc
January 2025
Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China. Electronic address:
R2 retrotransposons can be harnessed to insert genes at targeted sites by all-RNA delivery, presenting a new technology for next-generation biotherapeutics. Here, we report a protocol for evaluating the gene integration activity of R2 retrotransposons in mammalian cells. We describe the construction of vectors separately expressing R2 protein and donor, the process of liposome transfection, and flow cytometry.
View Article and Find Full Text PDFPostmenopausal osteoporosis is a chronic inflammatory disease characterized by decreased bone mass and increased bone fracture risk. Estrogen deficiency during menopause plays a major role in post-menopausal osteoporosis by influencing bone, immune, and gut cell activity. In the gut, estrogen loss decreases tight junction proteins that bind epithelial cells of the intestinal barrier together.
View Article and Find Full Text PDFBackground: Cachexia is defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC), and negatively impacts patient outcomes. Nutritional supplementation cannot fully reverse tissue wasting, and the mechanisms underlying this phenotype are unclear. This work aims to define the relative contributions of catabolism and anabolism to adipose wasting in PDAC-bearing mice.
View Article and Find Full Text PDFApplications of genetic code expansion in live cells are widespread and continually emerging, yet they have been limited by their reliance on the supplementation of non-standard amino acids (nsAAs) to cell culturing media. While advances in cell-free biocatalysis are improving nsAA synthesis cost and sustainability, such processes remain reliant on multi-step processes of product isolation followed by supplementation to engineered cells. Here, we report the design of a modular and genetically encoded system that combines the steps of biosynthesis of diverse phenylalanine derivatives, which are the most frequently used family of nsAAs for genetic code expansion, and their site-specific incorporation within target proteins using a single engineered bacterial host.
View Article and Find Full Text PDFThe chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation.
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