Mouse fibroblast strain capable of conditionally overexpressing HPV E7 and application thereof
A fibroblast, overexpression technology, applied in the fields of molecular biology and biomedicine, can solve the problems of low expression rate of exogenous genes, lack of translocation, poor liposome transfection efficiency, etc., and achieves good practical value. Effect
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Embodiment 1
[0035] Unless otherwise specified in the following examples, the reagents and biological materials used are all commercially available. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The molecular biology experimental methods not specifically described in the examples are carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or carried out according to the kit and product instructions .
[0036] A mouse fibroblast cell strain that can conditionally overexpress HPV E7, and the specific construction of the mouse fibroblast cell strain includes the following steps:
[0037] Step 1, HPV16 E7-H11 conditional knock in (cKI) mouse primary fibroblast isolation
[0038] Step 1-1. Take one littermate female mouse (wild type and HPV16 E7-H11 cKI mouse) identified by genotype for 6-8...
Embodiment 2
[0068] The application of a mouse fibroblast cell line conditionally overexpressing HPV16 E7, using the constructed MF-HPV16E7-Cre cell line, the application of the mouse fibroblast growth rate.
[0069] The operation steps of the application include:
[0070] Step (1) On the first day, take MF-HPV16 E7-Cre and control cells in the logarithmic growth phase, and adjust the cell concentration to 3*10 4 Cells / mL, spread in a 96-well plate, set 3 replicate wells at each time point, a total of 100 μL / well;
[0071] Step (2) After the cells adhere to the wall, take 3 wells for each group of cells, add 10 μL of toxicity detection solution CCK8 to each well, and place in the incubator to continue culturing for 1 hour. Measure the OD value at 450 nm wavelength with a microplate reader, and detect in the same way Blank medium well OD 450nm Value, and the difference from the blank group is recorded as the reading value at 0 for each group;
[0072] Step (3) At 24h, 48h, and 72h, respe...
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