A promoter and construction method for regulating porcine aebp1, transfection vector and construction method, application
A construction method and a promoter technology, applied in the field of animal genetic engineering
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Embodiment 1
[0048] Example 1 Obtaining the corresponding deletion fragment of the porcine AEBP1 gene promoter
[0049] 1. Extraction of large white pig DNA
[0050] 1.1. DNA extraction
[0051] Collect fresh blood from Large White pigs (a conventionally reported variety), add 0.5M ethylenediaminetetraacetic acid (EDTA) as an anticoagulant (blood volume: EDTA volume ratio is 5:1), and shake well.
[0052] Leukapheresis:
[0053] (1) Centrifuge at 6000rpm at 4°C for 10min, discard the supernatant serum.
[0054] (2) Add 1.5 times the volume of double distilled water and shake gently for 10 minutes.
[0055] (3) Centrifuge at 5000 rpm at 4°C for 10 minutes, and remove the upper layer.
[0056] (4) Add 0.9% NaCl solution by mass fraction to wash the precipitate, and shake gently for 10 min.
[0057] (5) Centrifuge at 5000 rpm at 4°C for 10 minutes, remove the supernatant, and obtain the precipitate, which is white blood cells.
[0058] 1.2. Total DNA extraction:
[0059] (1) Add an equ...
Embodiment 2
[0091] Example 2 Construction of transfection vector for the corresponding deletion fragment of the promoter of porcine AEBP1 gene
[0092] 1. Construction of recombinant plasmid TA-Q
[0093] The PCR products of the three pig AEBP1 gene deletion fragments obtained in Example 1 were recovered (refer to the instructions of the recovery kit of Shanghai Laifeng Company), and were respectively connected to the pMD-18T vector (refer to the instruction manual of the pMD-18T vector of TAKARA Company), and the ligated products were transformed into Escherichia coli DH5α, the steps are as follows:
[0094] (1) Pipette 5 μL of the ligation product into 100 μL of competent cells, mix well, and place on ice for 30 minutes.
[0095] (2) Place in a 42°C water bath and heat shock for 90s.
[0096] (3) Quickly take out the tube and put it on ice, and cool it for 3-4min.
[0097] (4) Add 400 μL of LB liquid medium without ampicillin (Amp) into the tube, and recover at 37°C for 1 hour.
[0...
Embodiment 3
[0111] Example 3 Liposome-mediated cell transfection
[0112] The specific steps of liposome-mediated cell transfection refer to Lipofectamine TM 2000 (Invitrogen Company) instructions, the transfection was carried out with a 24-well cell culture dish, and the cells used were porcine kidney cells PK-15. To eliminate experimental errors, three rounds of independent experiments were performed for each deletion vector (pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3), and each experiment was performed in triplicate wells. Use 1 µg deletion vector, 3 µL liposomes per well. After the cells were transfected for 48 hours, the cell lysate was collected, and the activity of the missing promoter was analyzed by a dual-luciferase detection system.
[0113] In the present invention, the steps of cultivating and transfecting pig kidney cells PK-15 and the preparation of related reagents are as follows:
[0114] (1) Preparation of related reagents
[0115] a) Cell culture medium (a medium ...
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