Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.
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PMID:Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080). 812 28

Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of alpha 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.
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PMID:Effects of clinorotation on COL1A1- EGFP gene expression. 1552 76

Mutations in COL1A1 or COL1A2 genes lead to osteogenesis Imperfecta (OI) in humans. There are three possiblities to successfully treat OI including (1) gene therapy, (2) mesenchymal stem cell (MSC) therapy, or (3) a combination of both. The aim of this study was to develop a model for combined gene/cell OI therapy by targeting Col1a1 and Col1a2 genes with isogenic sequences from corresponding human genes in rat bone marrow (BM)-derived MSCs. The recombination efficacy was tested for five different rat-human-rat hybrid DNAs with rat fragments that were 1 to 4 kb long. For selection of transfected clones a neomycine resistance gene was cotransfected, and clones resistant to G418 (G418(+)) were recovered and screened for integration of specific gene loci in the rat genome. Over 90% of G418(+) clones correctly integrated the rat-human-rat hybrid DNAs, and both OI loci in the rat genome were targeted to a similar degree. Longer homologous sequences integrated into rat collagen genes approximately 10 times more efficiently. Based on our data the nonviral gene targeting technology could be potentially employed to repair collagen genes in OI patients.
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PMID:Optimization of genetic engineering and homologous recombination of collagen type I genes in rat bone marrow mesenchymal stem cells (MSC). 2069 69