Mouse embryonic stem cell as well as preparation method and application thereof

A mouse embryo and stem cell technology, applied in the field of mouse embryo stem cells and their preparation, can solve the problem of not being able to respond well to hemophilia gene mutation and the like

Pending Publication Date: 2022-05-27
SHANXI MEDICAL UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the past, the mouse model of hemophilia knocked out the F8 / F9 gene, resulting in the synthesis of coagulation factors. However, there are many kinds of gene mutations in hemophilia, and this knockout mouse model cannot well reflect the specific genes of hemophilia. mutation

Method used

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  • Mouse embryonic stem cell as well as preparation method and application thereof
  • Mouse embryonic stem cell as well as preparation method and application thereof
  • Mouse embryonic stem cell as well as preparation method and application thereof

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Experimental program
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Embodiment 1

[0037]1. Mutation site: The mutation F9 c.223C>T was detected in a patient with hemophilia B in our hospital, which is a nonsense mutation.

[0038] 2. Plasmid construction: PCR amplification of the left arm (LA) from the genome of E14 cells, using site-directed mutagenesis to mutate the target site on the left arm and amplify the right arm; selection markers for puromycin + mCherry red fluorescent gene, purine P2A is used to connect the pycnogenol and mCherry, and a Loxp sequence in the same direction is added to each end. Use Goldgate cloning method (E1601 (BsaI-HFV2)) to assemble LA, RA and selectable markers (PGK-Puro, mCherry) into the PGGA plasmid to obtain a template plasmid, namely donor, such as figure 1 . Design sgRNA in the website (https: / / zlab.bio / guide-design-resources), screen through various methods, and finally obtain a suitable sgRNA (Guide RNA-2(px330)), which is constructed into the px330 plasmid.

[0039] Guide RNA-2 (px330): sgRNA was designed in the s...

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Abstract

The invention relates to a mouse embryonic stem cell strain SXMUe002-A-1, which has a preservation number of CGMCC No: 23035, and is preserved in the China Center for Type Culture Collection on September 28, 2021. The invention also provides an application of the mouse embryonic stem cell strain SXMUe002-A-1 in preparation of a mouse hemophilia model. According to the obtained mutant cell strain SXMUe002-A-1, the growth and reproduction capability and the cellular morphology of the mutant strain are highly similar to those of a wild type, no base is deleted and inserted on an exon, only one base is mutated, and the mutant cell strain SXMUe002-A-1 is closer to clinical cases. The mutant cell strain SXMUe002-A-1 has the characteristics of mouse embryonic stem cells, can be subjected to mouse embryonic stem cell chimeric injection to obtain a hemophilia mouse model, and is of great significance in further hemophilia nonsense mutation mechanism research, hemophilia drug screening and gene therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mouse embryonic stem cell and a preparation method and application thereof. Background technique [0002] Hemophilia is an X-linked recessive bleeding disorder. F8 gene defect causes hemophilia A (hemophilia A, HA), F9 gene defect causes hemophilia B (hemophilia B, HB), in the male population, the incidence is 1 / 5,000 and 1 / 25,000 respectively (Yang Renchi. Blood Chinese expert consensus on diagnosis and treatment of palliative diseases (2017 edition) [J]. Chinese Journal of Hematology, 2017, 38(05): 364-370). China is a populous country with an estimated 80,000–120,000 hemophiliacs, about 50% of whom are severe. The existing treatment methods for hemophilia are still mainly based on the replacement therapy of blood coagulation factor products, which causes a great burden on the family and society. [2] . In addition, the production of inhibitors caused by long-term infusion of ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/113C12N15/57C12Q1/02A01K67/027A61K49/00C12Q1/6883
CPCC12N5/0606C12N15/85C12N15/1137C12N9/644C12Y304/21022G01N33/5073A01K67/0271A61K49/0008C12Q1/6883C12N2510/00C12N2800/107C12N2310/20C12N2503/02G01N2500/10A01K2207/12A01K2227/105A01K2267/0381C12Q2600/156C12Q2600/112C12Q2600/118C12Q2600/136
Inventor 杨林花马艳春王刚孙雯雯姚明泽吴长新赵丽东
Owner SHANXI MEDICAL UNIV
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