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Human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method

A human coagulation factor, in situ repair technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as bad, activating harmful genes, and destroying endogenous genes.

Active Publication Date: 2015-07-08
SHANGHAI PINPOINT MEDICAL TECH CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0015] c. If the plasmid is integrated into other endogenous genes, it may destroy endogenous genes or activate harmful genes that were not originally expressed, resulting in bad consequences

Method used

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  • Human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method
  • Human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method
  • Human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0254] (1) Construction of TALENs expression plasmid

[0255] ①Pick colonies after each ligation transformation, and use primers TAL-J-F and TAL-J-R for preliminary screening by bacterial liquid PCR. The size of the PCR product is about 100×(number of TALE modules)+200bp. Such as figure 2 , the size of the PCR product of the doublet-positive colony should be close to 400bp, and the size of the product of the hexaplex and heptad should be about 800bp and 900bp respectively, and the PCR-positive samples should be further sent for sequencing verification;

[0256] ②The connection of the octamer and the hexademer needs to be digested and gel-purified in the previous step, otherwise the positive rate will be relatively low. Such as image 3 A, The two plasmids to be ligated were completely digested with the same enzyme digestion system, one of the two adjacent lanes completely excised the TALE module, and the other only excised a dozen bases to linearize the plasmid. The TALE m...

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Abstract

The invention discloses a human blood coagulation factor VIII gene intron 22 inversion mutation in-situ remediation plasmid, kit and method. The method constructs specific in-situ remediation treatment and can specifically repair the type of F8 mutation, and the in-situ remediation strategy is verified in hemophilia patient specific iPSCs by combination with a TALEN technology. The in-situ remediation strategy is a first remediation strategy for remediation of intron 22 inversion as common HA mutation. The in-situ remediation strategy utilizes a sequence accurate fixed-point introduction method, is relatively safe and has no nondeterminacy of the prior art.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an in situ repair plasmid, a kit and a method for the inversion mutation of the No. 22 intron of the human blood coagulation factor VIII gene. Background technique [0002] Hemophilia A (Hemophilia A, HA) is the most common X-linked hemorrhagic genetic disease with a high incidence rate. Severe hemophilia is disabling and fatal, and there is no radical cure. HA has always been considered by the academic community as one of the most likely genetic diseases to be cured by gene therapy. Its cause is the deficiency or functional defect of human coagulation factor VIII protein (FVIII) due to the defect of human coagulation factor VIII gene (hereinafter referred to as F8) , thus causing coagulation dysfunction, the incidence rate is about 1 / 5000 in male babies born. The main symptom of HA is spontaneous bleeding or bleeding after trauma. Repeated bleeding in the joints can of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/55
Inventor 梁德生吴涌邬玲仟
Owner SHANGHAI PINPOINT MEDICAL TECH CO LTD
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