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SiRNA for inhibiting canine p53 gene expression and canine cell model of p53 gene silence

A p53 gene and cell model technology, applied in DNA/RNA fragments, cells modified by introducing foreign genetic material, gene therapy, etc.

Inactive Publication Date: 2011-05-25
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no literature on the use of RNA interference technology to inhibit the expression of canine p53 gene

Method used

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  • SiRNA for inhibiting canine p53 gene expression and canine cell model of p53 gene silence
  • SiRNA for inhibiting canine p53 gene expression and canine cell model of p53 gene silence
  • SiRNA for inhibiting canine p53 gene expression and canine cell model of p53 gene silence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The sequence design of embodiment 1 canine p53siRNA

[0044] The gene sequence of the canine p53 gene was obtained from GenBank (accession number: AF060514.1). The siRNA sequences that may be designed to inhibit the expression of canine p53 gene are as follows:

[0045] 5'-GCCACUAGAUGGAGAAUAU-3' (SEQ ID No.1);

[0046] 3'-CGGUGAUCUACCUCUUAUA-5' (SEQ ID No. 2).

[0047] The target sequence of the canine p53 siRNA interference is located at positions 927-945 of the canine p53 gene sequence (see figure 1 ). The target sequence of the dog p53 siRNA (DNA encoding the siRNA of the present invention) is as follows:

[0048] Sense strand: 5'-GCCACTAGATGGAGAATAT-3' (SEQ ID No.3);

[0049] Antisense strand: 3'-CGGTGATCTACCTCTTATA-5' (SEQ ID No. 4).

[0050] And choose a random sequence as a control. The control sequence is as follows:

[0051] Sense strand: 5'-TTCTCCGAACGTGTCACGT-3';

[0052] Antisense strand: 3'-AAGAGGCTTGCACAGTGCA-5'.

[0053] The above-mentioned sens...

Embodiment 2

[0054] Example 2 Packaging of canine p53 siRNA lentivirus

[0055] The above synthesized DNA fragment encoding the siRNA of the present invention and the reference sequence were packaged into lentiviral vectors by Jikai Gene Chemical Co., Ltd. The DNA fragment was ligated into the pGC-LV plasmid. Positive clones were screened by PCR and identified by sequencing. Then, the resulting pGC-LV recombinant plasmid, pHelper 1.0 plasmid and pHelper 2.0 plasmid were co-transfected into 293T cells, cultured for 48 hours, packaged to produce lentivirus, and the cell supernatant was collected. The cell supernatant contains lentiviral particles packaged with the siRNA fragments of the present invention, which are concentrated to obtain a high-titer lentiviral concentrate.

[0056] Viral titers of recombinant lentiviruses were determined using 293T cells. The viral titer of the lentivirus containing the siRNA sequence of the present invention is 10 8 TU / mL, the viral titer of the lentiv...

Embodiment 3

[0057] Example 3 Screening of MDCK cell clones containing canine p53siRNA and analysis of p53 gene expression

[0058] A. On the day before the test, put 5×10 4 MDCK cells (ATCC, USA) were inoculated into three 35mmdish (cell culture dishes).

[0059] B. When the cells grow to about 30-50% cell density, aspirate the supernatant and wash twice with PBS. The MDCK cells were respectively infected with the lentivirus containing the siRNA sequence of the present invention and the lentivirus containing the control sequence, and the virus infection amount was 20 MOI. MDCK cells not infected with lentivirus were set as the control group. Add DMEM medium containing 10% (v / v) serum, at 37°C, 5% CO 2 Continue culturing in the cell culture incubator.

[0060]C. After culturing for 24 hours, remove the medium, add DMEM medium containing 10 μg / ml Puromycin (puromycin) and 10% serum, and continue culturing. Observe the growth state of the cells. When the cells in the control group of MD...

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Abstract

The invention discloses a small interfering RNA (siRNA) for inhibiting canine p53 gene expression. The sequence of the siRNA is shown as SEQ ID No: 1 or SEQ ID No: 2 in a sequence table. The invention also discloses a DNA for coding the siRNA and a recombinant vector containing the siRNA or the DNA. Preferably, the recombinant vector is a slow virus vector for infecting a canine cell to obtain a canine cell model of p53 gene silence. The canine p53 gene siRNA sequence can effectively degrade mRNA of p53 genes so as to inhibit the expression of the p53 genes. By using the siRNA sequence for interfering with the expression of the canine p53 genes, on the one hand, a treatment medicament for diseases related with the p53 genes such as cell apoptosis related diseases can be prepared; and on the other hand, the established p53 knock down cell model has great significance for researching the effect of p53 protein on innate immunity of cells and possible specific mechanisms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an siRNA for inhibiting the expression of canine p53 gene, a canine cell model of p53 gene silence and their application. Background technique [0002] Tumor suppressor protein p53 is a phosphorylated protein with a molecular weight of 53KD and has the function of transcriptional activation. p53 protein plays a very important role in cell cycle regulation, promotion of cell apoptosis, tumorigenesis and cell differentiation. In normal cells, the half-life of p53 protein is very short, and the content is very small. When cells are exposed to stresses such as ultraviolet radiation, genotoxic reagents, and virus infection, especially when the DNA of cells is damaged, the expression of p53 protein increases rapidly and is activated through phosphorylation and acetylation. The activated p53 protein enters the nucleus, transcriptionally activates and regulates the expression o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/63C12N15/867C12N5/10A61K48/00A61P35/00
Inventor 沈阳马志永邱亚峰史子学
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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