Method for breeding transgenic pig for over expression of pig PBD-2 gene

A technology of PBD-2 and transgenic pigs, which is applied in the field of biomedical technology and disease-resistant breeding, can solve the problems of difficult analysis of disease resistance, long breeding cycle, and low selection efficiency, and achieve the goal of easy method, simple operation, and high efficiency Effect

Inactive Publication Date: 2012-06-27
HUAZHONG AGRI UNIV
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  • Claims
  • Application Information

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Problems solved by technology

However, conventional disease-resistant breeding technology is difficult to analyze complex traits such as disease resistance, selection effi

Method used

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  • Method for breeding transgenic pig for over expression of pig PBD-2 gene
  • Method for breeding transgenic pig for over expression of pig PBD-2 gene
  • Method for breeding transgenic pig for over expression of pig PBD-2 gene

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Embodiment 1

[0037] A kind of preparation method of porcie beta-defensin 2 (porcie beta-defensin 2, PBD-2) gene for transgenic pig, its steps are as follows:

[0038] 1. Acquisition of porcine beta defensin 2 gene:

[0039] According to GenBank NM_214442.1 gene sequence, design a pair of primers:

[0040] PBD2 01: CCG GAATTC ATGTGGGCCCTCTGCTTG, where the underlined part is the EcoRI restriction site;

[0041] PBD2 02: CCG CTCGAG TCAGGGTCAGCGGATGCA, where the underlined part is the XhoI restriction site.

[0042] RNA was extracted from porcine liver tissue, first reverse-transcribed into cDNA, and then PCR amplified using this as a template to obtain the target DNA fragment with a length of 234bp. The system and procedure of PCR reaction are as follows:

[0043] The 50 μL reaction system contained: 5 μL 10×PCR reaction buffer (purchased from Treasure Bioengineering Dalian Co., Ltd.), 5 μL dNTP (2.5 mM each), 5 μL specific upstream primer PBD2 01 (20 μM), 5 μL specific downstream prim...

Embodiment 2

[0048] A kind of cultivation for expressing the transgenic pig of pig PBD-2 gene, its steps are:

[0049] 1. Construction of large white pig fibroblast cell line stably overexpressing PBD-2 gene:

[0050] 1.1 Lipofectamine transfection:

[0051] According to conventional methods ("Animal Cell Culture Technology and Application", edited by Wang Jie, Beijing: Chemical Industry Press, 2004), the fibroblasts used as nuclear donor cells---large white fetal pigs were isolated and prepared. Fetuses were isolated by removing the fetal membranes, washed 3 times in phosphate buffered saline containing antibiotics, and then the limbs, head and viscera were removed. The tissue was mechanically ground with small scissors, and then digested with trypsin-EDTA (ethylenediaminetetraacetic acid) for 20 minutes at 37°C to prepare fibroblasts, which were treated with 15% (v / v) fetal calf serum (FCS, Gibco, Life Technologies Inc.), Dulbecco's modified Eagle's medium (DMEM, Gibco, Life Technologi...

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Abstract

The invention discloses a method for breeding a transgenic pig for the over expression of a pig PBD-2 gene. The method comprises the following steps: (1) building and straightening a pc DNA 3.1 (+)/pCA-PBD2 carrier in such a manner that a primer is designed, the liver tissue RNA of a pig is extracted, RT-PCR is enlarged to obtain a PBD-2 gene, alcohol is used for precipitation, and sterilization water is used for dissolution; (2) transfecting lipidosome and screening cells in such a manner that a pig fibroblast for over-expression of the PBD-2 is built; (3), obtaining a recombined blastula according to a manual clone method; (4), transplanting an embryo in such a manner that the bred blastula is transplanted into the fallopian tube of a surrogate sow; and (5), identifying the transgenic pig. The method has the advantage that the operation is feasible, simple and convenient. After being cloned, the pig PBD-2 gene is inserted into the expression vector, and a pig fibroblast over expressing the pig PBD-2 is screened out through cell transfection; the pig fibroblast is transplanted to the megagametocyte of the sow through manual cloning to obtain a reconstructed blastula; and a masculine blastula is transplanted to the fallopian tube of the recipient sow to obtain the transgenic pig for over-expression of the PBD-2 gene. The method has a wide anti-bacterial capability, and provides a favorable material for disease-resistant breeding research.

Description

technical field [0001] The invention relates to the fields of biomedical technology and disease-resistant breeding. Specifically, it relates to a method for breeding transgenic pigs resistant to animal bacterial diseases, and prepares transgenic pigs overexpressing porcie beta-defensin 2 (porcie beta-defensin 2, PBD-2) gene by manual somatic cell nuclear transfer technology. Background technique [0002] Antibacterial drugs play an important role in the clinical treatment of human and animal diseases, but due to the widespread and irrational use of antibacterial drugs, pathogenic bacteria are increasingly resistant to various antibacterial drugs used in humans and animals, and very serious bacterial resistance has emerged situation, so that some bacterial diseases can hardly be controlled by effective drugs. The emergence of bacterial drug resistance has aroused widespread concern from all walks of life around the world, and is considered by the World Health Organization to...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/873A61D19/04A01K67/027
Inventor 周锐杨希
Owner HUAZHONG AGRI UNIV
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