Regulation of functional nucleic acid sites corresponding to activation of thermogenic genes
A gene expression and target technology, applied in the field of molecular biology, can solve the problems of adverse side effects and high risks of surgery, and achieve the effect of great application prospects.
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Embodiment 1
[0057] Example 1 Ucp1 Mining of potential sequence for G quadruplex formation in promoter region
[0058] for research Ucp1 Whether there is a G quadruplex element in the promoter, use the G quadruplex prediction software QGRS Mapper to determine Ucp1 The sequence of the promoter before the transcription start site (TSS) was analyzed ( figure 1 A). Predicted by G4Hunter web application (http: / / bioinformatics.ibp.cz) Ucp1 Potential G quadruplex elements in the promoter region, five sequences indicating high prediction scores for G quadruplex formation were obtained ( figure 1 B). In order to prove that the selected sequence does have G quadruplex formation potential, the corresponding sequence was synthesized and diluted to 100 μM in IDTE buffer (10 mM Tris, pH=7.5, 0.1 mM EDTA). Then dilute to the desired concentration with 100 mM KCl, 10 mM tris buffer (Tris-HCl, pH=7.5) to prepare the working solution, heat the working system to 95°C for 5 min, and then cool to over...
Embodiment 2
[0060] Example 2 carry Ucp1 Promoter region psiCHECK2- Ucp1 Construction of p-dual luciferase recombinant plasmid vector
[0061] The genome of HepG2 cells was extracted with the TIANamp Genomic DNA Kit genome extraction kit from Tiangen Biotechnology Co., Ltd., and the position of G454 sequence relative to the transcription start site of the genome -141--110 bp was taken as human Ucp1 Upstream of the promoter region, designed to amplify a full-length 1801bp Ucp1 Amplification primers corresponding to the promoter sequence, and the homologous sequences of about 15-30bp at the two ends of the psiCHECK2 backbone vector after XhoI / NotI double enzyme digestion and linearization at the 5' end of the primers, the specific sequences are: Ucp1 p-upstream primer: 5'-AATTCTAGGCGATCGCTCGAGAGGCCGCAAGGTGCCTGC-3', as shown in SEQID No.6, the bold sequence is a homologous sequence; Ucp1 p-Downstream primer: 5'-ATTTTATTGCGGCCAGCGGCCGCAATCGATTTTGCATACTTTTTTTCTATG-3', as shown in SEQ ID No.7...
Embodiment 3
[0062] Example 3 Intracellular Natural Functional Small Molecule Pair Ucp1 Validation of the regulatory effect of the promoter region
[0063] Preadipocyte 3T3-L1 cells are a type of preadipocytes with the ability to proliferate and differentiate into adipocytes, which provides a better research platform for the study of human metabolism. Therefore, 3T3-L1 cells were selected for research. When the adherent density reached about 90% of the entire 10cm culture dish, cells were plated on a 24-well plate 18-24 hours before transfection, and an appropriate volume of fresh culture medium was added to each culture well. base (DMEM high-glucose medium + 10% FBS), placed in 37 ° C, 5% CO 2 cultured in a constant temperature incubator. When the density of adherent cells reached about 80% after splitting, the cells were transfected according to the operating instructions of the EZ Trans cell transfection kit from Liji Biotechnology Co., Ltd. Set psiCHECK2- Ucp1 The p recombinant vec...
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