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Cancer cell state identification gene circuit group and preparation method thereof

A gene circuit and cancer cell technology, applied in the field of genetic engineering, can solve problems such as difficult precise grouping of multiple cancer cell subgroups, complexity of subgroups, gene expression sparsity, etc.

Pending Publication Date: 2022-02-25
ZHUHAI INST OF ADVANCED TECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, due to the sparsity of gene expression obtained by current single-cell sequencing technology and the complexity of subgroups, there are many difficulties such as the difficulty in realizing accurate grouping of multiple cancer cell subgroups based on signaling pathways. , phenotypically characterized tumor subpopulations

Method used

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  • Cancer cell state identification gene circuit group and preparation method thereof
  • Cancer cell state identification gene circuit group and preparation method thereof
  • Cancer cell state identification gene circuit group and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0090] Material:

[0091] cell:

[0092] MHCC97H human highly metastatic liver cancer cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0093] Drugs and reagents:

[0094] Doxorubicin (Adriamycin), specification: 10mg, batch number: KFS276, supplier: Biolab.

[0095] Sorafenib (Sorafenib) specification: 10mg, batch number: Bay 43-9006, supplier: Shanghai Ruihui Chemical Technology Co., Ltd.

[0096] Roseville-1640 (RPMI-1640) medium and fetal bovine serum (FBS) were purchased from GIBCO, USA.

[0097] Doxycycline (doxycycline hydrochloride), specification: 10mM / mL, batch number: ID0390-10mM*1mL (inWater), supplier: Suo Laibao.

[0098] Plasmids were synthesized from General Biology (Anhui), the classifier plasmid backbone used SWB-Blas ticidin lentiviral backbone, and the synthetic promoter vector backbone used SWP-Puromycin.

[0099] Lipofectamine 3000, specification: 1mL, batch number: L3000015, supplier: ThermalFisher.

[010...

Embodiment approach

[0104] 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, where gag / pol is the genome integration protein and rev is the reverse transcription protein , vsv-g is packaging protein.

[0105] 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.

[0106] Note: It is recommended to use cellulose acetate or polyethersulfone (PES) filter membrane (low protein binding) as a filter, not nitrocellulose filter membrane.

[0107] 3. Mix lentiviral supernatant (4 parts) and 5X lentiviral concentrate (1 part) in a volume ratio of 4:1, place at 4°C for 2 hours or overnight, mix ...

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Abstract

The invention provides a cancer cell state identification gene circuit group and a preparation method thereof, and belongs to the technical field of gene engineering. The cancer cell state identification gene circuit group 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 of the positive promoter, and the positive promoter, the negative promoter and the microRNA are from cancer cells in a cancer cell state. The invention also discloses a preparation method of the cancer cell state identification gene circuit group. The positive promoter, the negative promoter and the microRNA capable of representing different phenotypes of tumor cells are obtained, so that the first identification gene circuit and the second identification gene circuit are formed, and the bar code area capable of assisting judgment is combined, so that the cancer cell state can be accurately calibrated, and the accuracy of cancer cell detection is improved. And the proportion information of the cancer cells in various states can be analyzed by means of high-throughput cell experiments and the like, so that 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 state identification gene circuit set 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 discovered that compared with traditional identification methods, artificially synthesized gene circuits have the advantages of higher accuracy and stronger modulability, and can accurately Various types of cancer cells are identified. These synthetic gene circuits can play an excellent role in identifying cancer cells and targeting and killing cancer cells. [0003] The heterogeneity of tumor cell subgroups has always been a key difficulty in tumor diagnosis, prognosis and effective treatment, and it is also a hot spot in the basic research of tumor drug resistance. Although the current single-cell sequencing technology can distinguish s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N15/113C12N15/64
CPCC12N15/86C12N15/65C12N15/113C12N2740/15043C12N2800/107C12N2310/141
Inventor 余裕姜长安
Owner ZHUHAI INST OF ADVANCED TECH
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