Cloned cell strain for determining biological activity of teriparatide
A biologically active and teriparatide technology is applied to animal cells, cells modified by introducing foreign genetic material, vertebrate cells, etc. It can solve the problems of cumbersome operation and unstable results, and achieve good repeatability and detection The effect of simple method and high application value
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0037] Example 1: Construction of cloned cell line UMR-106-GFP-CRE-Puro
[0038] (1) Determine the optimal screening concentration of puromycin: inoculate rat osteosarcoma cells UMR-106 into a 24-well cell culture plate, and add puromycin at different screening concentrations (concentration range: 0.1 μg / mL to 10 μg / mL ), cultured for 2 to 3 days, observed the growth morphology of the cells, and determined that the optimal concentration of puromycin for selection was 2 μg / mL;
[0039] (2) Plasmid construction: replace the NF-κB-RE element on the pNL3.2.NF-κB-RE[NlucP NF-κB-RE Hygro] plasmid with the cAMP-RE (ie CRE) element, and then replace the TurboGFP element and Insert the CRE-NlucP element into the pLVX-Puro vector to obtain the pLVX-GFP-CRE-Puro plasmid;
[0040] (3) Collect lentivirus: HEK-293T cells were plated overnight; use Lipofectamine 3000 reagent to transfect pLVX-GFP-CRE-Puro plasmid, pCMV-dR8.2 dvpr plasmid and pCMV-VSV-G plasmid into HEK-293T cells , and p...
Embodiment 2
[0044] Example 2: Verification of the activity and stability of No. 3 cloned cell line UMR-106-GFP-CRE-Puro
[0045] (1) Verification of cell density and reporter gene signal detection:
[0046] (1) Cell subculture and plating: the stable cell line was subcultured in a 5% complete culture medium in a carbon dioxide incubator at 37°C, the medium was changed every 3 days, collected into a sterile centrifuge tube, and centrifuged at 1000rpm for 2 minutes. Discard the supernatant, resuspend the cell pellet with fresh complete culture medium, transfer it to a cell culture bottle, and continue to culture; take the P2 generation cells in a good growth state for plating, centrifuge at 1000rpm for 2min, discard the supernatant, and add to the maintenance culture Cells were resuspended in solution, and the concentration of cells was adjusted to 2×10 5 pcs / mL, 5×10 5 a / mL and 1×10 6 1 / mL, added to 96-well culture plate at 100 μL / well, cultivated overnight;
[0047] (2) Adding sampl...
Embodiment 3
[0054] This embodiment provides a method for measuring the biological activity of teriparatide, the steps comprising:
[0055] Cells were cultured with complete culture medium (take 10 mL of heat-inactivated fetal bovine serum FBS, add 90 mL of DMEM culture solution, 10 μL of 20 mg / mL puromycin, mix well, and store at 4°C) at 37°C and 5% carbon dioxide. Cells in good condition are used for the test; under sterile conditions, trypsinize the cells, neutralize the trypsin in the complete medium and resuspend the cells, centrifuge at 1000rpm for 2 minutes, discard the supernatant, resuspend with complete culture medium and adjust the cell concentration up to 5×10 5 cells / mL, 100 μL per well was inoculated in a 96-well plate, and cultured overnight; the next day, the supernatant was absorbed, and the standard solution and sample solution of different concentrations diluted in DMEM were added, 100 μL per well, 37 ° C, 5% CO 2 Cultivate under conditions for 3 hours; absorb 50 μL of ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More