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Cancer cell analysis gene circuit assembly and production method thereof

A gene circuit, cancer cell technology, applied in the field of genetic engineering, can solve the problem of inability to distinguish cancer cell subsets

Pending Publication Date: 2022-02-25
珠海中科先进技术研究院有限公司
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Problems solved by technology

[0003] However, the driving elements used in existing gene circuits are often composed of only a relatively single fixed microRNA and natural promoter expression patterns, and this set of expression patterns can only distinguish cancer cells from normal healthy cells, but cannot distinguish between cancer cells and normal healthy cells. Various subgroups of cancer cells appear in the total population due to the heterogeneity among cancer cells, and the heterogeneity among cancer cells is precisely the most critical factor for drug-resistant recurrence of cancer cells. For multiple states that exist at the same time Accurately identifying and judging the proportion of cancer cells will help to study the heterogeneity among cancer cells

Method used

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  • Cancer cell analysis gene circuit assembly and production method thereof
  • Cancer cell analysis gene circuit assembly and production method thereof
  • Cancer cell analysis gene circuit assembly and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0052] Material

[0053] cell:

[0054] MHCC97H human highly metastatic liver cancer cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0055] Drugs and reagents:

[0056] Doxorubicin (Adriamycin), specification: 10mg, batch number: KFS276, supplier: Biolab.

[0057] Sorafenib (Sorafenib) specification: 10mg, batch number: Bay 43-9006, supplier: Shanghai Ruihui Chemical Technology Co., Ltd.

[0058] Roseville-1640 (RPMI-1640) medium and fetal bovine serum (FBS) were purchased from GIBCO, USA.

[0059] Doxycycline (doxycycline hydrochloride), specification: 10mM / mL, batch number: ID0390-10mM*1mL (inWater), supplier: Suo Laibao.

[0060] Plasmids were synthesized from General Biology (Anhui), the classifier plasmid backbone used SWB-Blasticidin lentiviral backbone, and the synthetic promoter vector backbone used SWP-Puromycin.

[0061] Lipofectamine 3000, specification: 1mL, batch number: L3000015, supplier: ThermalFisher.

[0062]...

Embodiment approach

[0066] 1. Mix 8 plasmids of 8 phenotypic cell state identifiers in equal mass ratios as the target mixed plasmids, and use the third-generation lentiviral packaging system to co-transfect HEK293T cells with 4 plasmids in a 10cm cell culture dish. The state recognizer mixes plasmids for lentiviral packaging, and the transfection ratio is gag / pol:rev:vsv-g:target plasmid=5:2:3:8. Among them, gag / pol is genome integration protein, rev is reverse transcription protein, vsv-g is packaging protein.

[0067] 2. After the lentivirus is successfully packaged, collect the lentivirus supernatant and filter it with a 0.45 μm filter to remove cells and debris.

[0068] Note: It is recommended to use cellulose acetate or polyethersulfone (PES) filter membrane (low protein binding) as a filter, not nitrocellulose filter membrane.

[0069] 3. Mix the lentivirus supernatant (4 parts) and the 5X lentivirus concentrate (1 part) according to the volume ratio of 4:1, place at 4°C for 2 hours or o...

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Abstract

The invention provides a cancer cell analysis gene circuit assembly and a production method thereof, and belongs to the technical field of gene engineering. The cancer cell analysis gene circuit assembly comprises a first identification gene circuit and a second identification gene circuit which are both used for identifying cancer cells in the same cancer cell state. A microRNA sequence is inserted into the 3'UTR segment, and the microRNA is from cancer cells in the cancer cell state. The invention also discloses the production method of the cancer cell analysis gene circuit assembly. A positive synthetic promoter and a negative synthetic promoter are formed by obtaining a positive enhancer and a negative enhancer capable of representing different phenotypes of the tumor cells, and the bar code area capable of assisting judgment is combined, so that the cancer cell state can be accurately calibrated, the proportion information of the cancer cells in various states can be analyzed by means of high-throughput cell experiments and the like, and the analysis of heterogeneity among the cancer cells is facilitated.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a cancer cell analysis gene circuit assembly and a preparation method thereof. Background technique [0002] With the continuous evolution of medical methods and the rapid development of gene technology, medical workers have found that genetically modified cells can make highly specific detection of specific states of other cells, such as cancer cells, such as based on the specific state of HeLa cells. A synthetic gene circuit established by microRNA expression patterns that can distinguish human cervical cancer cells HeLa from normal human HEK293 cells. These synthetic gene circuits can play an excellent role in identifying cancer cells and targeting and killing cancer cells. [0003] However, the driving elements used in existing gene circuits are often composed of only a relatively single fixed microRNA and natural promoter expression patterns, and this set of exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
CPCC12N15/113C12N2830/001C12N2830/48
Inventor 余裕姜长安
Owner 珠海中科先进技术研究院有限公司
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