Breeding method for transgenic pigs expressing sIFITM3 genes
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A technology of transgenic pigs and breeding methods, which is applied in the field of biomedical technology and disease-resistant breeding, can solve the problems of low selection efficiency, long breeding cycle, and inability to meet the needs of disease-resistant varieties, and achieve the effect of easy method and simple operation
Inactive Publication Date: 2012-03-28
HUAZHONG AGRI UNIV
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[0003] Conventional breeding technology is difficult to analyze complex traits such as disease resistance, selection efficiency is
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Embodiment 1
[0034] A method for preparing porcine interferon-induced transmembrane 3 gene (interferon induced transmembrane 3, IFITM3) for preparing transgenic pigs, the steps of which are:
[0035] 1. Acquisition of porcine interferon-induced transmembrane 3 gene:
[0036] Referring to the GenBank HQ641403.1 IFITM3 gene sequence, a pair of primers were designed, RNA was extracted from porcine lymph node tissue, and sIFITM3 gene was amplified by RT-PCR. The primer sequences are as follows:
[0037] sIFITM 3F: 5'-tttgaattccaccatgaactgcgct-3'
[0038] sIFITM 3R: 5'-tttctcgagtcagatcatcgga-3'
[0039] A two-step method was used to amplify. Firstly, cDNA was obtained by reverse transcription, and then used as a template for PCR amplification. The reaction system was: 0.25 μL template plasmid, 5 μL 10×LA buffer, 1.0 μL each of pTRIM25F and pTRIM25R primers (final Concentration is 10pmol / L), LA enzyme 0.25μL, dNTPS 1.0μL, add ddH2O to 50μL. Act at 94°C for 3 minutes; then act at 94°C for 30 s...
Embodiment 2
[0043] A kind of cultivation for expressing the transgenic pig of pig IFITM3 gene, its steps are:
[0044] 1. Construction of large white pig fibroblast cell line stably expressing sIFITM3 gene:
[0045] 1.1 Vector linearization:
[0046] The vector plasmid pcDNACAsIITM3 was extracted and linearized with AccI, then precipitated with ethanol and dissolved in sterile water.
[0047] 1.2 Lipofectamine transfection:
[0048] 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) at 37°C for 20 minut...
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Abstract
The invention discloses a breeding method for transgenic pigs expressing sIFITM3 (swine interferon induced transmembrane 3) genes. The breeding method comprises the steps as follows: firstly, the construction of swine fibroblast cell lines stably expressing the sIFITM3 genes comprises the following steps: the step a refers to the construction and the linearization of pcDNACAsIITM3 vectors, wherein, primers are designed, lymph node tissue RNA (Ribose Nucleic Acid) of pigs is extracted, the sIFITM3 genes are obtained through RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification, ethanol precipitation is carried out, and sterile water is used for dissolving; the step b refers to liposome transfection, wherein, swine fibroblasts expressing sIFITM3 are constructed; the step c refers to screening and proliferation of nuclear donor cells transfected with the sIFITM3 genes; secondly, recombination blastula acquisition based on a manual nucleus transplantation method comprises the following steps: the step a refers to the preparation of recipient cells; the step b refers to enucleation and injection of the nuclear donor cells; and thirdly, the embryo transplantation and the acquisition of the transgenic pigs comprise the following steps: the step a refers to embryo transplantation, wherein, bred blastulas are transferred to a fallopian tube of a surrogate sow, and then detection is carried out; and the step b refers to transgenic individual identification. The breeding method is feasible, and is simple and convenient to operate; in addition, the pigs are the transgenic pigs expressing sIFITM3, and have the potential capability to resist animal virus such as foot-and-mouth disease virus, Japanese encephalitis virus, swine influenza virus and the like.
Description
technical field [0001] The invention relates to the fields of biomedical technology and disease-resistant breeding. It specifically relates to a method for breeding transgenic pigs resistant to animal viral diseases. The transgenic pigs expressing swine interferon induced transmembrane 3 (sIFITM3) gene are prepared by manual somatic cell nuclear transfer technology. Background technique [0002] The prevalence of animal infectious diseases is the main reason that seriously restricts the sustainable development of my country's aquaculture industry. The three basic links of its prevalence are the source of infection, the route of transmission and susceptible animals. Controlling the source of infection, cutting off the route of transmission and improving the disease resistance of susceptible animals are the basic measures for the prevention and control of zoonotic diseases, among which improving the disease resistance of susceptible animals is fundamental. The main ways to im...
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