Application of gRNA target combinations in construction of hemophilia model pig cell line

A hemophilia and cell line technology, applied in the field of gene editing, can solve the problems of difficulty, low primate cloning efficiency, late primate sexual maturity, etc., to achieve good applicability and improve gene editing efficiency.

Active Publication Date: 2021-03-05
NANJING KGENE GENETIC ENG CO LTD
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Problems solved by technology

However, rodents such as mice differ greatly from humans in terms of size, physiology, pathology, etc., and cannot simulate human diseases well. In fact, more than 95% of the drugs that have been proven to be effective in mice are ineffective in human clinical trials
However, there is no report on any artificially constructed large animal hemophilia model
As far as large animals are concerned, primates are the closest relatives to...

Method used

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  • Application of gRNA target combinations in construction of hemophilia model pig cell line
  • Application of gRNA target combinations in construction of hemophilia model pig cell line
  • Application of gRNA target combinations in construction of hemophilia model pig cell line

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Experimental program
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Embodiment Construction

[0095] 1. Construction of Cas9 high-efficiency expression vector and detection of application effect

[0096] 1.1 Construction of Cas9 high-efficiency expression vector

[0097] The pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (pKG-GE3 for short) was transformed from the addgene (Plasmid#42230, from Zhang Feng lab) pX330-U6-Chimeric_BB-CBh-hSpCas9 vector. Nuclear localization signal, adding WPRE sequence to enhance translation, adding green fluorescence and resistance screening puro gene, the map is as follows figure 1 , the base sequence is shown in SEQ ID NO:1.

[0098] The structure of the original vector pX330-U6-Chimeric_BB-CBh-hSpCas9 used is as follows figure 2 As indicated, purchased from addgene (Plasmid #42230, from Zhang Feng lab).

[0099] The build steps are as follows:

[0100] (1) Remove excess short gRNA backbone

[0101] pX330-U6-Chimeric_BB-CBh-hSpCas9( figure 2 ) was digested with BbsI and XbaI, and the vector fragment (about 8313bp) was recovered, an...

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Abstract

The invention discloses application of gRNA target combinations in construction of a hemophilia model pig cell line. The application comprises three gRNA target combinations, and specifically comprises the following steps: respectively designing a pair of target sequences aiming at pig F8 and F9 genes, constructing three CRISPR/Cas9 systems, respectively transferring the three CRISPR/ Cas9 systemsinto pig fibroblasts, and screening gene mutant strains, thereby obtaining the A, B and A & B hemophilia model pig cell line. According to the application disclosed by the invention, the gene editing efficiency is obviously improved by transforming the Cas9 expression vector. The editing efficiency is also obviously improved by adjusting the molar ratio of the gRNA expression vector to the Cas9 expression vector. The A, B and A & B hemophilia model pig can be obtained by applying a somatic cell nuclear transplantation cloning technology and is used for performing drug screening, drugefficacy evaluation, pharmacology and toxicology, disease pathology, gene therapy, cell therapy and other researches, effective experimental data is provided for further clinical application, and a powerful experimental means is provided for successfully treating human hemophilia.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to the application of gRNA target combination in the construction of A, B and A&B hemophilia model pig cell lines. Background technique [0002] Hemophilia (heamophilia) usually refers to a group of X-chromosome-linked recessive hereditary hemorrhagic diseases caused by the deficiency of coagulation factors caused by gene mutations, and there is currently no effective cure. According to the results of epidemiological surveys, as of 2018, the number of hemophilia patients in my country is about 130,000, but only 20% of the patients can really do preventive treatment. The blood coagulation process is a limited hydrolysis process of a series of proteins, generally divided into three stages: thromboplastin formation, thrombin formation and fibrin formation. Hemophilia is generally in the first stage of blood coagulation, that is, thromboplastin is impaired. According t...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/85C12N5/10
CPCC12N15/907C12N9/22C12N15/85C12N5/0656C12N2510/00C12N15/90C12N5/10C12N15/11C12N15/113
Inventor 牛冬汪滔王德华王磊程锐曾为俊马翔
Owner NANJING KGENE GENETIC ENG CO LTD
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