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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

Pending Publication Date: 2020-08-07
JIANGSU TONEKER MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. At present, the research on HPV and E7 oncogenes mainly focuses on keratinocytes and epithelial tumor cells, but the research on HPV oncogenes represented by E7 in fibroblasts and tumor microenvironment is still insufficient
[0007] 2. At present, researchers mostly use traditional vector transfection and antibiotic screening to obtain fibroblasts with high expression of E7, which has the problems of poor transfection efficiency and long screening time; or use virus system packaging and infection system, combined with antibiotic screening In order to obtain fibroblasts with high expression of E7, the problems of poor transfection efficiency and long screening time of traditional liposomes have been improved to a certain extent. Yes, some occur at a single site, and in some cases are integrated at multiple sites on different chromosomes. The randomness may cause genome rearrangement, translocation deletion, and the insertion of exogenous E7 gene may destroy the endogenous gene of the cell; In addition, the number of integrated copies is not fixed, and multi-copy tandem integration may lead to low or no expression of exogenous genes, which is not conducive to controlling the expression level and function of genes

Method used

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  • Mouse fibroblast strain capable of conditionally overexpressing HPV E7 and application thereof
  • Mouse fibroblast strain capable of conditionally overexpressing HPV E7 and application thereof
  • Mouse fibroblast strain capable of conditionally overexpressing HPV E7 and application thereof

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses a mouse fibroblast cell strain capable of conditionally overexpressing HPV E7 and application thereof. The mouse fibroblast cell strain is constructed by the following steps: step 1, separating cKI mouse primary fibroblasts; step 2, immortalized induction and screening of cKI mouse primary fibroblasts; and step 3, carrying out immortalization-cKI mouse primary fibroblast Cre enzyme induction and identification. According to the invention, a promoter sequence is embedded into an H11 site of a mouse genome at a fixed point; only in the presence of a specific Cre enzyme, the target gene can be expressed in a specific cell or tissue; the mouse fibroblast strain capable of conditionally overexpressing HPV E7 and the application of the mouse fibroblast strain are providedthrough the steps of separation of a series of primary cells, immortalization and in-vitro Cre induction, and the problems existing in the prior art are effectively solved.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to a mouse fibroblast cell strain capable of conditionally overexpressing HPV E7 and its application. Background technique [0002] Cervical cancer is one of the most common malignant tumors in women, and its incidence is second only to breast cancer among gynecological malignancies. In recent years, the incidence of cervical cancer in my country has been on the rise, and tends to be younger. Human papillomavirus (HPV) belongs to the Papillomavirus A genus of the family Papovaviridae and is a spherical DNA virus. At present, more than 200 HPV subtypes have been identified, which can be divided into high-risk HPV (hrHPV) and low-risk HPV (1rHPV) according to their carcinogenicity. Persistent hrHPV infection is the main cause of high-grade cervical lesions and cervical cancer. More than 90% of cervical cancer tissues carry hrHPV (16, 18, 31 types) DNA, but...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0656C12N15/85C12N2509/00C12N2510/04
Inventor 袁奕唐林香张梦娇李昂何胜祥
Owner JIANGSU TONEKER MEDICAL TECH
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