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Specific sequence of hCD46 genetically-modified Wuzhishan pig and detection method adopting specific sequence

A technology for Wuzhishan pigs and transgenic pigs, applied in biochemical equipment and methods, microbiological measurement/inspection, recombinant DNA technology, etc., can solve the problems of unidentified specific insertion positions and establishment of accurate detection methods for hCD46 transgenic Wuzhishan pigs, etc. Achieve high practical value, good specificity, and high experimental conditions

Inactive Publication Date: 2016-09-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific insertion position of the hCD46 miniature gene expression vector in the Wuzhishan minipig genome has not been identified
In other words, an accurate detection method for the obtained hCD46 transgenic Wuzhishan pigs has not been established

Method used

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  • Specific sequence of hCD46 genetically-modified Wuzhishan pig and detection method adopting specific sequence
  • Specific sequence of hCD46 genetically-modified Wuzhishan pig and detection method adopting specific sequence
  • Specific sequence of hCD46 genetically-modified Wuzhishan pig and detection method adopting specific sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of hCD46 transgenic Wuzhishan pig

[0040] 1. Preparation of Donor Cell Lines

[0041] A healthy wild-type primary Wuzhishan WPF19 boar with obvious physical characteristics was selected as the primary clone. The ear tissue of individual WPF19 was collected for the construction of a nuclear ear fibroblast cell line: the ear tissue was cut into about 1mm 3 Use a pipette to transfer the small pieces of tissue with a size of 1 to a T25 culture bottle, spread it on the bottom, and add about 5 mL of DMEM complete medium containing 20% ​​FBS to the bottle, and put it in a standard incubator for culture. The medium was changed every two days, and the fibroblast cell line grew to reach 80% confluency, and the donor ear fibroblasts for passage and cryopreservation were obtained.

[0042] 2. Construction of hCD46 minigene expression vector

[0043] see image 3 , to construct a hCD46 miniature gene expression vector of about 9.5kb. The constructed hCD46...

Embodiment 2

[0065] Example 2 Accurate detection of hCD46 transgenic Wuzhishan pig

[0066] 1. Obtaining the specific sequence of hCD46 transgenic Wuzhishan pig

[0067] Based on the second-generation resequencing method, the whole genome data of hCD46 transgenic Wuzhishan pig were obtained, and the data were compared with the published whole genome sequence of wild type Wuzhishan pig (http: / / gigadb.org / dataset / 100031) , found the transformation-specific region where the exogenous sequence joined the genome, and finally retrieved the sequence of SEQ ID NO:1 in the resequenced genome data (see figure 1 ), by comparison, it was found that 1-479bp of the SEQ ID NO:1 sequence was the transformation vector sequence (hCD46 microgene expression vector sequence), and 480-994bp was the Wuzhishan pig genome sequence (see figure 2 ). That is, the SEQ ID NO: 1 sequence is the specific sequence of the transformant of the hCD46 transgenic Wuzhishan pig prepared in the present invention, that is, the...

example 1

[0075] The 4 hCD46 transgenic Wuzhishan pigs obtained in Example 1 and 1 wild-type (negative) Wuzhishan pig were all from Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0076] Enzymes and Reagents

[0077] Molecular biology reagents, such as dNTPs, Taq DNA polymerase, and DL2000Marker were purchased from Treasure Bioengineering (Dalian) Co., Ltd. Dalian Treasure Bioengineering Co., Ltd. Other biochemical reagents are imported subpackages or domestic analytical pure. The above transformant-specific PCR primers were synthesized by Zhongmei Taihe Biotechnology (Beijing) Co., Ltd.

[0078] laboratory apparatus

[0079] PCR instrument: Eppendorf Mastercycle gradient (Eppendorf Co.)

[0080] Nucleic acid electrophoresis instrument: DYY-III nucleic acid electrophoresis instrument (Beijing Liuyi Instrument Factory)

[0081] DNA electrophoresis analysis system: Kodak EDAS 290 (Kodak co.)

[0082] Other instruments include...

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Abstract

The invention relates to a specific sequence of the hCD46 genetically-modified Wuzhishan pig and a detection method adopting the specific sequence. With the specific sequence of the hCD46 genetically-modified Wuzhishan pig provided by the invention, the specific detection for the hCD46 genetically-modified Wuzhishan pig is realized.

Description

technical field [0001] The invention relates to detecting the hCD46 transgenic Wuzhishan pig by using the specific sequence of the hCD46 transgenic Wuzhishan pig. Background technique [0002] With the rapid development of genetic engineering technology, the research and development of transgenic animals is very rapid. In the field of agriculture, it involves the improvement of the characteristics of livestock and poultry. In the field of medicine, it involves the creation and application of various animal models. Some transgenic animals have been bred into strains. [0003] Although human homologous organ transplantation has achieved great success, organ transplantation faces difficulties such as shortage of donor organs, which has become the main reason hindering the implementation of organ transplantation. In the United States, taking heart transplantation as an example, more than 95% of heart disease patients lose their lives because of the shortage of allogeneic organs ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N5/10C12Q1/68
CPCC12N15/11C12N5/0656C12N2510/00C12Q1/6888
Inventor 潘登科王勤石宁宁冯冲龙川李刚梁琳
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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