Preparation method of hCD46 transgenic Wuzhishan pigs
A Wuzhishan pig, transgenic technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of not establishing an accurate detection method for hCD46 transgenic Wuzhishan pig, not identifying specific insertion positions, etc. Practical value, high sensitivity, good specificity
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Embodiment 1
[0040] Example 1 Preparation of hCD46 transgenic Wuzhishan pig
[0041] 1. Preparation of Donor Cell Lines
[0042] 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.
[0043] 2. Construction of hCD46 minigene expression vector
[0044] see image 3 , to construct a hCD46 miniature gene expression vector of about 9.5kb. The constructed hCD46...
Embodiment 2
[0066] Example 2 Accurate detection of hCD46 transgenic Wuzhishan pig
[0067] 1. Obtaining the specific sequence of hCD46 transgenic Wuzhishan pig
[0068] 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 is the transformation vector sequence (hCD46 minigene expression vector sequence), and 480-994bp is the Wuzhishan pig genome sequence (see figure 2 ). That is, the sequence of SEQ ID NO: 1 is the specific sequence of the transformant of the hCD46 transgenic Wuzhishan pig prepared in the present invention, that is, the...
example 1
[0076] 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.
[0077] Enzymes and Reagents
[0078] 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.
[0079] laboratory apparatus
[0080] PCR instrument: Eppendorf Mastercycle gradient (Eppendorf Co.)
[0081] Nucleic acid electrophoresis instrument: DYY-III nucleic acid electrophoresis instrument (Beijing Liuyi Instrument Factory)
[0082] DNA electrophoresis analysis system: Kodak EDAS 290 (Kodak co.)
[0083] Other instruments include...
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