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Method for obtaining driving element

A technology of driving elements and promoters, applied in the field of genetic engineering, can solve the problems of inability to distinguish cancer cell subsets, systemic recurrence of patients, etc.

Pending Publication Date: 2022-02-15
珠海中科先进技术研究院有限公司
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  • Abstract
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Problems solved by technology

[0003] However, the driving elements used in existing gene circuits are often composed of a relatively single fixed mi croRNA 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 cancer cell subpopulations appear in the total cell 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. A small part of stubborn heterogeneity Sexual cancer cell subsets can enter the blood circulation system through high metastatic ability, which is likely to cause systemic recurrence of patients in the future

Method used

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  • Method for obtaining driving element
  • Method for obtaining driving element
  • Method for obtaining driving element

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0075] The preparation process in step 3 and step 4, such as the means of knockout, insertion and functional connection at specific sites, such as CRISPR technology, CRISPR-Cas9 technology, etc., are well known to those skilled in the art and will not be repeated here.

[0076] In step 5, when the selected cancer cell line is a human highly metastatic liver cancer cell line with the registration number MHCC997H, the specific implementation method can be to uniformly mix different cell phenotype state evaluators in the form of lentiviral vectors and then infect them to be studied For the target cells, use Blasticidin to screen out stable liver cancer cell lines. Of course, when the cancer cell lines used are other cancer cell lines, the corresponding screening methods should be adjusted accordingly, which will not be repeated here.

[0077] In step 6, after the constructed synthetic promoter lentiviral test vector is used to infect the stable cancer cell line containing the cel...

Embodiment

[0108] Material

[0109] cell:

[0110] MHCC97H human highly metastatic liver cancer cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0111] Drugs and reagents:

[0112] Doxorubicin (Adriamycin), specification: 10mg, batch number: KFS276, supplier: Biolab.

[0113] Sorafenib (Sorafenib) specification: 10mg, batch number: Bay 43-9006, supplier: Shanghai Ruihui Chemical Technology Co., Ltd.

[0114] Roseville-1640 (RPMI-1640) medium and fetal bovine serum (FBS) were purchased from GIBCO, USA.

[0115] Doxycycline (doxycycline hydrochloride), specification: 10mM / mL, batch number: ID0390-10mM*1mL (inWater), supplier: Suo Laibao.

[0116] 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.

[0117] Lipofectamine 3000, specification: 1mL, batch number: L3000015, supplier: ThermalFisher.

[0118]...

Embodiment approach

[0122] MHCC97H liver cancer cells were cultured in RPMI-1640 containing 10% FBS, 0.1 μM Doxorubicin and 5 μM Sora fenib at 37°C, 5% CO 2 cultured in an incubator. Passage once every 3 days, and take cells in the logarithmic growth phase to perform high-throughput functional screening of drug-resistant synthetic promoters to obtain multiple drug-resistant cancer cell subpopulations.

[0123] Screen the candidate promoters with positive (activity from none) or negative (activity to no) activity indication reactions from the obtained drug-resistant cancer cell subpopulations, and label the screened candidate promoters sequentially For sample 1 to sample 10 packs containing such as figure 2 Promoter test lentiviruses for the indicated gene sequences.

[0124] After successful packaging of the lentivirus, the lentiviral supernatant was collected and filtered through a 0.45 μm filter to remove cells and debris.

[0125] Mix lentiviral supernatant (4 parts) and 5X lentiviral conc...

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Abstract

The invention provides a method for obtaining a driving element, and belongs to the technical field of genetic engineering. The method for obtaining the driving element comprises the following steps: step 1, preparing a drug-resistant cancer cell subgroup under the condition of combined culture of a chemotherapeutic drug and a targeted drug; 2, preparing at least one preselected promoter; 3, constructing a promoter test lentivirus; 4, constructing a cell phenotype state evaluator; 5, obtaining a stably transferred cancer cell line; step 6, obtaining a dual stable cancer cell line; and step 7, screening based on the survival rate of the dual stable transformation cancer cell line to obtain the driving element. On the basis of the drug-resistant culture result of a double stably transferred cancer cell line, the type of the preselected promoter capable of highly recognizing various subtype cancer cell lines is judged, so that the preselected promoter can be used as the driving element in a subsequently constructed gene circuit.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for obtaining driving elements. 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 mi croRNA 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 a relatively single fixed mi croRNA and natural promoter expression patterns, and this set of expression patterns can only distinguish can...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/65C12N15/113
CPCC12N5/0693C12N5/067C12N15/86C12N15/65C07K14/47C12N2740/15043C12N2510/00
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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