Tet-Off Advanced human stably expressing high-transition nasopharyngeal carcinoma cell line S18, construction method thereof and application thereof

A technology for stable expression and construction method, applied in the field of genetic engineering, can solve the problem of application of nasopharyngeal cancer cells without tet-offadvanced system, and achieve the effect of sensitive and effective regulation, high regulation range, real-time timing and quantitative regulation

Active Publication Date: 2010-07-28
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there is still no technology to apply the tet-off advanced system to nasopharyngeal carcinoma cells

Method used

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  • Tet-Off Advanced human stably expressing high-transition nasopharyngeal carcinoma cell line S18, construction method thereof and application thereof
  • Tet-Off Advanced human stably expressing high-transition nasopharyngeal carcinoma cell line S18, construction method thereof and application thereof
  • Tet-Off Advanced human stably expressing high-transition nasopharyngeal carcinoma cell line S18, construction method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construction of human highly metastatic nasopharyngeal carcinoma cell line S18 stably expressing Tet-Off Advanced:

[0042] (1) Plasmid preparation: transform pTet-Off Advanced into JM109 competent bacteria, pick a single clone and amplify it, use the endotoxin-free plasmid extraction kit, extract 200 μg of plasmid, and take 10~50 μg of pTet-Off Advanced plasmid After digestion with 5μl-25μl restriction endonuclease ScaI for 2 hours, precipitate with alcohol.

[0043] (2) Cultivation of nasopharyngeal carcinoma cells: Nasopharyngeal carcinoma cell S18 was cultured in DMEM medium containing 10% fetal bovine serum for future use.

[0044] (3) Cell transfection: cells were cultured in a 24-well plate, and when the cell adhesion area reached 90% to 95%, 0.5 μl liposome and 0.8 μg pTet-Off Advanced plasmid per well were mixed and transfected in optimized serum. dye. At this time, the culture medium is 0.5ml.

[0045] (4) Screening of stable cell lines: after 48 hours of t...

Embodiment 2

[0053] A nasopharyngeal carcinoma cell line induced to express luciferase (Luc) was constructed on the basis of the human highly metastatic nasopharyngeal carcinoma cell line S18 stably expressing Tet-Off Advanced obtained in Example 1.

[0054] The S18Tet-Off Advanced cell line obtained in Example 1 with a 42-fold difference in expression fold was selected. pTRE-Tight-Luc was linearized with the restriction endonuclease ScaI, mixed with the genetic resistance gene hygromycin B DNA fragment at a molar ratio of 20:1, and transfected into S18Tet-Off Advanced cells with Invitrogen Lipofectamine 2000 liposomes , the transfection conditions were as in step (3) of Example 1. The medium was changed 6 hours after transfection, Dox was added to one part to make the final concentration 1 μg / ml, and Dox was not added to the other part. For transient detection, 48 hours after transfection, by luciferase analysis, the expression intensity without Dox is 42-250 times that of Dox. For stab...

Embodiment 3

[0058] Example 2 On the basis of the human highly metastatic nasopharyngeal carcinoma cell line S18 stably expressing Tet-Off Advanced obtained in Example 1, a nasopharyngeal carcinoma cell line that induces the expression of human ferritin was constructed.

[0059]The S18Tet-Off Advanced cell line obtained in Example 1 with a 42-fold difference in expression fold was selected. The reconnection (FTH) of the cloned human ferritin gene was cloned into the pTRE-Tight-BI-Luc plasmid to construct pTRE-Tight-BI-Luc-FTH after sequencing without mutation. The latter was linearized with the restriction endonuclease ScaI, mixed with the quality of the genetic resistance gene hygromycin B DNA fragment at 20:1, and transfected into S18 Tet-Off Advanced cells with Invitrogen Lipofectaime 2000 liposomes as the carrier. The transfection conditions As embodiment 1 step (3). The medium was changed 6 hours after transfection, Dox was added to one part to make the final concentration 1 μg / ml (+...

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Abstract

The invention discloses a Tet-Off Advanced stably expressing human high-transition nasopharyngeal carcinoma cell line S18, a construction method thereof and application thereof. The nasopharyngeal carcinoma cell line with high tetracycline induced and regulated target gene expression capacity is obtained by inducing a Tet-Off Advanced gene into a nasopharyngeal carcinoma cell, screening, resistance cloning and enlarged culture and identification. The nasopharyngeal carcinoma cell line constructed by the method is named Tet-Off Advanced human high-transition nasopharyngeal carcinoma cell line and is preserved in China Center for Typical Culture Collection, and the preservation number is CCTCC C200959. The cell line has stable and sensitive function of regulating the expression of a target gene. When some target gene is required to be studied, the nasopharyngeal carcinoma cell line is transfected with a pTRE-Tight plasmid of the target gene, and after screening, the obtained resistance clone gene is identified and thus the nasopharyngeal carcinoma cell line expressing the target gen under the induction of the tetracycline is obtained. The expression of the target gene can be regulated quantitatively at regular time by simply regulating the tetracycline content of an environment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a nasopharyngeal carcinoma cell line and its establishment method and application. Background technique [0002] Nasopharyngeal carcinoma is a malignant tumor that occurs in the nasopharyngeal mucosa. It occurs frequently in Guangdong, Guangxi, Fujian and other places in China, and there are more men than women. The age of onset is mostly middle-aged, and there are also adolescent patients. Nasopharyngeal carcinoma has a high degree of malignancy, and cervical lymph node metastasis can occur in the early stage. The cervical lymph node metastasis is about 60.3% to 86.1%, and half of them are bilateral metastasis. Cervical lymph node metastasis is often the first symptom of nasopharyngeal carcinoma (23.9% to 75%). In a small number of patients, the primary lesion cannot be found in nasopharyngeal examination, and cervical lymph node metastasis is the only ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/11C12N15/85A61K48/00A61P35/00A61P11/02A61P11/04C12R1/91
Inventor 李立钱朝南冯宇鹏
Owner SUN YAT SEN UNIV
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