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sgRNA for preparing skeletal dysplasia swine model and application thereof

A technology for bone development and abnormality, applied in DNA/RNA fragments, recombinant DNA technology, and stably introducing foreign DNA into chromosomes, etc., can solve the problems of bone structure and function differences in animal models of abnormal bone development in mice, and achieve good application value Effect

Pending Publication Date: 2019-09-24
ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is the defect that the existing mouse model with abnormal bone development and human beings have relatively large differences in bone structure and function.

Method used

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  • sgRNA for preparing skeletal dysplasia swine model and application thereof
  • sgRNA for preparing skeletal dysplasia swine model and application thereof
  • sgRNA for preparing skeletal dysplasia swine model and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of FGFR3p.G378R mutant pigs

[0053] 1. Experimental plan:

[0054] 1. Preparation of homologous recombination template

[0055] According to the sequence of the designed homologous recombination template, artificially synthesize single-stranded oligonucleotides (ssODN, ≤150bp) (Shanghai Sangong); use RNase-free ultrapure water to dissolve the synthesized ssODN freeze-dried powder at 500ng / uL The concentration is dissolved and subpackaged, and stored at -80 degrees Celsius for later use.

[0056] 2. Preparation of sgRNA

[0057] 1) Synthesize two complementary oligonucleotide single-stranded DNA (oligo DNA) containing sgRNA recognition sequence, sense strand (SEQ ID NO.6): 5-TAGGAAGCCCAGCCCGTAGCTG-3; antisense strand (SEQ ID NO.7 ): 5-AAACCAGCTACGGGCTGGGCTT-3. After the two complementary single-stranded DNAs are annealed into double strands, the ends should be complementary to the subcloning site after enzyme digestion on the sgRNA in vitro tra...

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Abstract

The invention belongs to the technical field of animal genome editing and particularly relates to sgRNA for preparing a skeletal dysplasia swine model and application thereof, and aims to solve the technical problem that existing rat skeletal dysplasia animal models have greater difference from human in skeleton structure and function. The technical means provides the sgRNA for preparing the skeletal dysplasia swine model. The sgRNA recognizes a target site located on an editing gene of a swine fibroblast growth factor receptor 3 and having a nucleotide sequence of 5'-GAAAGCCCAGCCCGTAGCTGAGG-3'. On the basis of obtaining the sgRNA, the matched donor DNA is designed, an Fgfr3 c.1132G>A precise mutant swine model can be prepared efficiently, an FGFR p.G378R mutant swine system with cartilage dysplasia is cultured, and a large animal model closer to human is provided for skeletal development study and clinical tretament of skeletal diseases.

Description

technical field [0001] The invention belongs to the technical field of animal genome editing, and in particular relates to an sgRNA for preparing a pig model with abnormal skeletal development and its application. Background technique [0002] Bone is one of the organs with the most developmental deformities and genetic diseases. Currently, the Online Human Mendelian Inherited Disease Database (OMIM) has included more than 400 single-gene skeletal genetic diseases. Common skeletal developmental deformities include dwarfism, spinal dysplasia, osteogenesis imperfecta, craniofacial deformities, hereditary bone metabolic diseases (osteosclerosis, rickets), etc. At present, the most commonly used animal model for the study of skeletal developmental diseases is genetically engineered mice, which have been used as model animals to carry out many studies on skeletal development and metabolic diseases. However, mice belong to rodents, and they are quite different from humans in term...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90A01K67/027
CPCC12N15/113C12N15/907A01K67/0275C07K14/71C12N2310/10C12N2310/20A01K2217/07A01K2227/108A01K2267/03
Inventor 王勇谢飞谢杨丽周晓杨王露露魏泓陈林郭科男
Owner ARMY MEDICAL UNIV
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