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CIC cell models targeting to CDKN2A and preparation methods of CIC cell models

A cell model and target cell technology, applied in biochemical equipment and methods, cells modified by introducing foreign genetic material, measurement/testing of microorganisms, etc., can solve the problem of inability to target specific genes, difficulty in obtaining in vitro research systems Lack of cell models etc.

Active Publication Date: 2018-09-28
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the existing CIC research, there is a lack of cell models regulated by specific genes, it is difficult to obtain in vitro research systems, and it is impossible to target specific genes.

Method used

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  • CIC cell models targeting to CDKN2A and preparation methods of CIC cell models
  • CIC cell models targeting to CDKN2A and preparation methods of CIC cell models
  • CIC cell models targeting to CDKN2A and preparation methods of CIC cell models

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, establishment of induced CIC cell model

[0043] The target cells in this example are HEK293 cells, and Western blot can detect the expression of CDKN2A gene-encoded protein in HEK293 cells ( figure 1 ), for the specific detection method, refer to Westernblot in Step 1-6 of this embodiment.

[0044] 1. Establishment of HEK293 / CDKN2A-cas9 knockdown cell line

[0045] 1. Design sgRNA oligos

[0046] According to the principle of adding PAM sequence after the target sequence, with the CDKN2A gene (GenBank: 21967752-21995043 in NC_000009.12, complement, 2018-3-26) as the target sequence, three CDKN2A-sgRNAs were designed as follows:

[0047] sgRNA-1: 5'-caccGCACCGAATAGTTACGGTCGG-3' (SEQ ID No.1);

[0048] 5'-aaacCCGACCGTAACTATTCGGTGC-3' (SEQ ID No. 2).

[0049] sgRNA-2: 5'-caccGACCGTAACTATTCGGTGCGT-3' (SEQ ID No.3);

[0050] 5'-aaacACGCACCGAATAGTTACGGTC-3' (SEQ ID No. 4).

[0051] sgRNA-3: 5'-caccGTGGGCCATCGCGATGTCGCA-3' (SEQ ID No.5);

[0052] 5'-aaacT...

Embodiment 2

[0077] Embodiment 2, the establishment of inhibition CIC cell model

[0078] The target cells in this embodiment are MCF10A cells and MCF7 cells, and Western blot cannot detect the expression of CDKN2A gene-encoded protein in MCF10A cells and MCF7 cells ( figure 1 ), for the specific detection method refer to Western blot in Step 1-6 of this embodiment.

[0079] 1. Establishment of MCF10A / CDKN2A overexpression cell lines

[0080] Using the retroviral vector pQCXIP-EGFP-N1 as the backbone, a CDKN2A overexpression vector was constructed, including its four different transcripts p16INK4a, p16γ, p12 and p14ARF. The nucleotide sequence of the gene encoding the transcript p16INK4a is specifically shown in SEQ ID No.7. The nucleotide sequence of the gene encoding the transcript p16γ is specifically shown in SEQ ID No.8. The nucleotide sequence of the gene encoding the transcript p12 is specifically shown in SEQ ID No.9. The nucleotide sequence of the gene encoding the transcript p1...

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Abstract

The invention discloses cell-in-cell (CIC) cell models targeting to CDKN2A and preparation methods of the cell-in-cell (CIC) cell models. For the methods for preparing the cell-in-cell relevant cell models provided by the invention, the cell-in-cell relevant cell models are prepared by regulating the expression of the CDKN2A gene in the target cell. An inducing cell-in-cell cell model can be prepared by inhibiting the expression of the CDKN2A gene in the target cell, and an inhibiting cell-in-cell cell model can be prepared by realizing overexpression of the CDKN2A gene in the target cell. Forthe method for promoting the formation of the CIC structure by interfering the expression of the CDKN2A gene provided by the invention, the complete tumor cell is expressed aiming at CDKN2A, throughthe method, the intracellular death mediated by CIC can be promoted, and the purposes of killing tumor cells and inhibiting tumor growth can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a cell-in-cell (CIC) cell model targeting CDKN2A and a preparation method thereof. Background technique [0002] Cell-in-cell (CIC) structure refers to a unique cell nest-like structure formed by one or more living cells inside another cell. The phenomenon of CIC widely exists in various species, including lower organisms amoeba, to metazoans C. elegans and mammalian systems. According to the different types of cells that form the CIC structure, it can be divided into homogeneous CIC, such as the CIC structure formed between tumors or epithelial cells of the same origin; and heterogeneous CIC, the CIC structure formed between cells of different origin. [0003] At present, tumor is the most active field of research on the biological significance of homogeneous CIC structure. Studies on clinical tissue samples have reported that CIC phenomenon is common in human tumor tissues, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12Q1/02
CPCC07K14/4703G01N33/5011
Inventor 孙强梁剑青牛祖彪黄红艳
Owner ACADEMY OF MILITARY MEDICAL SCI
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