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Construction method of human CD55 transgenic large animal

A construction method and animal technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of declining animal health, inappropriate insertion sites of foreign genes, animal death, etc.

Inactive Publication Date: 2021-07-23
成都中科奥格生物科技有限公司
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0004] In order to reduce the human body's immune rejection of heterologous organs, technicians have tried to gene-edit large animals to express CD55 in their genomes. However, previous studies have found that introducing CD55 genes into large animals will lead to animal health conditions decline, eventually leading to animal death
This phenomenon is common in gene-edited animals, and the main reason for this phenomenon may be that the insertion site of the foreign gene is inappropriate, but how to avoid this phenomenon is still an insurmountable technical problem in this field

Method used

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  • Construction method of human CD55 transgenic large animal
  • Construction method of human CD55 transgenic large animal
  • Construction method of human CD55 transgenic large animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0256] Example 1, Preparation of Transgenic Pigs Highly Expressing Human Complement Regulatory Protein CD55

[0257] Use single-stranded guide RNA (single guide RNA, sgRNA) to design sgRNA (sgRNA: gctccttctcgattatgggc) that specifically recognizes the target sequence DNA at the safe harbor Rosa26 site in the pig genome, and connect it into the pX458 vector recovered by Bbs I digestion to construct Rosa26 Target knockout vectors. Synthesize the CDS (Coding sequence) sequence (Gene ID: NM_000574) of the human (Homo sapiens) complement regulatory protein CD55 (Cluster of differentiation 55), and construct Rosa26 with about 800 bp homology arms on both sides by means of Overlap PCR and TA cloning Target site-directed integration vector.

[0258] According to different linearization methods, the carrier for linearization in vivo is the HMEJ site-directed integration vector ( figure 1 ), and contains the Hyg resistance gene; the in vitro linearized vector is the Tild-CRISPR site-d...

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Abstract

The invention relates to the technical field of animal transgenosis and xenotransplantation, in particular to a construction method of a CD55 transgenic large animal. The invention provides sgRNA for site-specific integration of an hCD55 gene at a safe harbor Rosa26 site of a large animal genome, provides an integration plasmid comprising the sgRNA, constructs a transgenic cell for site-specific integration of a human complement regulatory protein CD55 at the safe harbor Rosa26 site through a site-specific integration technology, further obtains a cloned embryo, and obtains a transgenic hCD55 gene pig after pregnancy. The transgenic pig obtained by the method disclosed by the invention is good in healthy state and can survive to be adult. The expression quantity of the hCD55 is high, the hCD55 can be used for xenotransplantation, stable inheritance and normal breeding can be realized, and verification is carried out in the F1 generation.

Description

technical field [0001] The invention relates to the technical field of animal transgene and xenotransplantation, in particular to a method for constructing a human CD55 gene transgenic large animal. Background technique [0002] Organ transplantation is an effective treatment for end-stage organ failure in humans. There are about 300,000 patients waiting for organ transplantation due to end-stage organ failure each year, but the number of organ transplants is only about 20,000 cases each year, and there is still a large gap. In this case, it is expected that the corresponding organs from other animals will be transplanted into the human body in need of organ transplantation to solve the problem of shortage of donor organs. [0003] Immune rejection is an important obstacle to clinical xenotransplantation. After xenotransplantation, donor tissue antigens will be recognized by antibodies in the recipient, and complement-mediated cell killing will occur. Complement inhibitors...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/12A01K67/027
CPCC12N15/113C12N15/8509C07K14/70596A01K67/0278C12N2310/10A01K2227/108A01K2267/025
Inventor 潘登科杜嘉祥
Owner 成都中科奥格生物科技有限公司
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