AmiRNA-HIF-1alpha sequence containing recombinant plasmid
A technology of HIF-1 and amirna-hif-1, applied in the field of recombinant plasmids, can solve problems such as disputes over the independent role of HIF family members
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Embodiment 1
[0037] Example 1: pcDNA TM 6.2 Construction of recombinant plasmid of GW / EmGFP-amiRNA-HIF-1α (abbreviated as p-amiRNA-HIF-1α).
[0038] 1. The design, synthesis and functional verification process of HIF-1α siRNA is as follows: figure 1 shown, including the following steps:
[0039] 1.1 Using HIF-1α CDS as a template, design its siRNA sequence, following the principles of complete complementary pairing with the template and not interfering with the expression of other genes, and design its control sequence, which is synthesized by takala company. And carry out 2-MO modification at the 5' end.
[0040] Target siRNA sequence: 5'-UGUGAGUUCGCAUCUUGAU-3'
[0041] Control siRNA sequence: 5'-UACACCGUUAGCAGACACC-3'
[0042] 1.2 Culture in McCoy’s 5A complete medium containing 10% newborn bovine serum, 100U / ml penicillin, 100μg / ml streptomycin (37°C 5% CO 2 , saturated humidity) human colorectal carcinoma HTC-116 cell line, press 1×10 6 Seed cells in exponential growth phase in 2...
Embodiment 2
[0090] Example 2: Human colorectal cancer cells were transfected with amiRNA-HIF-1α plasmid, and cell proliferation was observed.
[0091] Cultured in McCoy’s 5A complete medium containing 10% newborn calf serum, 100 U / ml penicillin, 100 μg / ml streptomycin (37°C, 5% CO 2 , saturated humidity) human colorectal carcinoma HTC-116 cell line, press 1×10 6 Seed cells in exponential growth phase in 24-well plates at 37°C in 5% CO 2 Incubate overnight in an incubator until the cell density reaches 60%-80%.
[0092] The above conventionally cultured human colorectal cancer HCT-116 cells were randomly divided into blank group, vector plasmid group, and recombinant plasmid group, and different plasmids were added according to the following doses for transient transfection:
[0093] (1) Blank group: normal HCT-116 cells, n=3;
[0094] (2) Vector plasmid set: p-amiR, n=3;
[0095] (3) Recombinant plasmid group: p-amiRNA-HIF-1α, n=3.
[0096] In a 1.5ml centrifuge tube, prepare the com...
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