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A zinc finger nuclease that inhibits the expression of human bcr-abl fusion gene or causes the function loss of human bcr-abl gene and its application

A technology of bcr-abl and zinc finger nuclease, which is applied in the translation products of oncogenes, nucleic acid vectors, genetic engineering, etc., can solve the problems of unproven completeness, long TALENs fragments, potential safety hazards, etc.

Active Publication Date: 2021-06-11
重庆医科大学国际体外诊断研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CRISPR / Ca-s nuclease technology started relatively late and is the easiest to operate, however, its potential integrity issues have not yet been proven
The main advantage of TALENs is that it is not limited by the characteristics of the target sequence, but the TALENs fragment that specifically recognizes the target sequence is too long, which is not conducive to the subsequent loading and expression of the vector, and the introduction of a large number of repetitive sequences into the target cell is also an unknown safety hazard

Method used

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  • A zinc finger nuclease that inhibits the expression of human bcr-abl fusion gene or causes the function loss of human bcr-abl gene and its application
  • A zinc finger nuclease that inhibits the expression of human bcr-abl fusion gene or causes the function loss of human bcr-abl gene and its application
  • A zinc finger nuclease that inhibits the expression of human bcr-abl fusion gene or causes the function loss of human bcr-abl gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Design and Construction of ZFN Plasmid and Donor Plasmid for Specific Target Site Sequence of Human bcr-abl Gene

[0017] 1. Design and synthesis of a zinc finger nuclease targeting the specific target site sequence of the human bcr-abl gene

[0018] According to the query and splicing of NCBI, the gene sequence of the human bcr-abl gene (as shown in SEQ ID No: 1) was determined, and the design of ZFNs was completed through the method of bioinformatics, and the zinc finger nucleic acid of the human bcr-abl gene was determined. The specific target site sequence for enzyme action is:

[0019] GGCGTCGACGGCgactacGAGGACGCCGAG (as shown in SEQ ID No: 2); the middle part sequence (gactac) is a FokI endonuclease cleavage site, that is, the target site sequence of ZFN specific knockout, and the FokI endonucleic acid used in the present invention The enzyme is an obligate heterodimerization Fok I plasmid, purchased from Addgene, which can avoid non-specific cleavage c...

Embodiment 2

[0079] Example 2. Zinc finger nuclease site-directed cutting of bcr-abl gene and insertion of NotI restriction site to promote homology-directed repair

[0080] 1. Recovery and culture of cryopreserved cells

[0081]Take out the cryopreservation vial containing K562 cells from the liquid nitrogen, put it into warm water at 37°C and shake it quickly until the cryopreservation solution is completely melted; complete rewarming within 2 minutes; transfer the cell suspension into a sterile centrifuge tube, add Gently blow 5mL of culture solution; centrifuge the cell suspension at 1000r / min for 5min, discard the supernatant; add 1mL of complete medium to the centrifuge tube containing the cell pellet.

[0082] 2. Cell culture conditions and subculture:

[0083] cell culture conditions

[0084] Medium composition: 1640 (Gibco Lot: 1737734)

[0085] 10% FBS (BI Lot: 1616756)

[0086] Subculture:

[0087] (1) K562 cell density reaches about 80%;

[0088] (2) Repeatedly blow and b...

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Abstract

The invention discloses a DNA molecule whose sequence is shown in SEQ ID No: 2, and also discloses the application of the DNA molecule as a target in inhibiting the expression of human bcr-abl fusion gene or causing the loss of human bcr-abl gene function Inhibition of human bcr-abl gene expression or loss of human bcr-abl gene function is brought about by zinc finger nuclease-mediated bcr-abl fusion gene homologous recombination technology. The ZFN plasmid constructed according to the specific target sequence site in the bcr-abl sequence of the present invention can efficiently target the bcr-abl gene to cause a DNA double-strand break, and repair it in an HDR manner so that the bcr-abl is destroyed by mutations such as a frameshift, As a result, the potential for malignant transformation such as promoting proliferation and inhibiting apoptosis was lost, and the feasibility of ZFNs targeting and destroying the bcr-abl gene to kill and inhibit CML cells was confirmed.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a zinc finger nuclease for inhibiting the expression of human bcr-abl fusion gene or causing the function loss of human bcr-abl gene and application thereof. Background technique [0002] The root cause of chronic myeloid leukemia (CML) is the bcr-abl fusion gene formed by c-abl gene and bcr gene due to balanced translocation of t(9;22)(q34;q11). The BCR-ABL fusion protein encoded by the fusion gene has strong tyrosine kinase activity, continuously activates downstream RAS / MAPK, PI3K / AKT, STAT5 and other pro-proliferation and anti-apoptosis signals, leading to malignant transformation of cells. The clinical first-line drug imatinib is a tyrosine kinase inhibitor (TKIs), which can make nearly 70% of CML patients in the chronic phase achieve complete remission of hematology and even genetics. However, in addition 30% of patients are resistant to drugs due to m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/90
CPCC07K14/47C07K14/82C07K2319/00C12N15/907C12N2800/80
Inventor 冯文莉高淼黄宁姝黄峥兰袁颖
Owner 重庆医科大学国际体外诊断研究院
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