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ShRNA sequence for specifically inhibiting GOS2 gene expression and application thereof

A gene expression and specific technology, applied in the field of shRNA sequence that specifically inhibits GOS2 gene expression, can solve the problem of short time to function

Pending Publication Date: 2022-02-08
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of siRNA in cells is transient, and the time to play a role is relatively short.
However, there are no relevant reports on the expression and effect of shRNA interference with GOS2 gene in ovarian cancer tissues.

Method used

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  • ShRNA sequence for specifically inhibiting GOS2 gene expression and application thereof
  • ShRNA sequence for specifically inhibiting GOS2 gene expression and application thereof
  • ShRNA sequence for specifically inhibiting GOS2 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] First, based on the GOS2 gene (the accession number in GENABANK is NM_015714.4), four primer pairs corresponding to shRNA were designed, which were recorded as shRNA primers. The specific sequences of the four pairs of shRNA primers are shown in Table 1. The 4 pairs of shRNA primers were annealed to form 4 short double-stranded oligonucleotide fragments to obtain shRNAs, which were named shGOS2-1, shGOS2-2, shGOS2-3, and shGOS2-4, respectively. .

[0040] shGOS2-1: 5'-GGAGCGACAGGCTCTCCAGAA-3' (SEQ ID NO: 1);

[0041] shGOS2-2: 5'-GGCCCTGTCCAACCGGCAGCA-3' (SEQ ID NO: 2);

[0042] shGOS2-3: 5'-GCCGCCAGACGTCTGCGGGAC-3' (SEQ ID NO: 3);

[0043] shGOS2-4: 5'-GCGACAGGCTCTCCAGAAGCA-3 (SEQ ID NO: 4).

[0044] Table 1. Primer pairs corresponding to shRNA sequences

[0045]

[0046] The shGOS2-1, shGOS2-2, shGOS2-3, and shGOS2-4 obtained above were connected to the double-enzyme-cut lentiviral vector Plko.1-TRC (Open Biosystem Company), respectively, and the restriction si...

Embodiment 2

[0048] Example 2: Preparation of cell lentivirus and transfection of cells with lentivirus

[0049] Preparation of Mixture A:

[0050] Solution A of the experimental group: 0.75 µg helper plasmid pLP1 + 0.35 µg helper plasmid pLP2 + 0.49 µg helper plasmid pL PSVG + 0.61 µg target plasmid Plko.1-TRC-shRNA-shGOS2 obtained in Example 1 OpenBiosystem Company, Sangon Bioengineering (Shanghai) Co., Ltd.).

[0051] Solution A of the control group: 0.75 µg helper plasmid pLP1 + 0.35 µg helper plasmid pLP2 + 0.49 µg helper plasmid pL PSVG + 0.61 µg vector plasmid Plko.1-TRC (Open Biosystem).

[0052] Add solution A to 0.125 μL of serum-free low-glucose DMEM medium (purchased from Thermo Fisher), mix gently, and incubate at room temperature for 5 min.

[0053] Preparation of mixture B: Take two 1.5mL EP tubes and add 125 μL of serum-free low-glucose DMEM medium, and then add 9 μL of EZ-Trans cell transfection reagent (purchased from Shanghai Liji Biotechnology Co., Ltd.) Mix lightly ...

Embodiment 3

[0059] Embodiment 3: Detection of the expression level of GOS2 gene in OVCAR8 cells

[0060] Collect the surviving OVCAR8 cells in the experimental group and the control group after puromycin (puromycin; PM) screening in Example 2, use the TRIzol method to extract RNA, and then operate according to the instructions of the real-time fluorescence quantitative kit (purchased from Novizyme) to detect The mRNA expression of target GOS2 in different groups of cells.

[0061] According to the experimental results, the Ct values ​​of the experimental group and the control group (the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value) were obtained using 2 -△△Ct Values ​​were used to calculate the silencing efficiency of shGOS2. Among them, △△Ct=△Ct of the experimental group-△Ct of the control group, △Ct=(Ct of the experimental group-Ct value of the internal reference of the experimental group) / (Ct of the control group-Ct of ...

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Abstract

The invention provides a shRNA sequence for specifically inhibiting GOS2 gene expression and application thereof, belonging to the technical field of ovarian cancer gene silencing treatment. The invention provides an shRNA sequence for specifically inhibiting the expression of the GOS2 gene, and experiments prove that the silent expression of the GOS2 gene has no obvious influence on the proliferation of the ovarian cancer cell line OVCAR8, but obviously inhibits the migration and infiltration behaviors of the ovarian cancer cell line OVCAR8; and thus, the shRNA sequence has good application in preparing gene medicines for inhibiting the migration and infiltration of the ovarian cancer cells.

Description

technical field [0001] The invention belongs to the technical field of ovarian cancer gene silencing therapy, and in particular relates to a shRNA sequence for specifically inhibiting GOS2 gene expression and application thereof. Background technique [0002] RNA interference technology (RNA interference, RNAi) is a process of effectively silencing or inhibiting the expression of target genes. It is a highly conserved genetic defense mechanism in the evolution of species, that is, a specific short sequence of double-stranded RNA to achieve post-transcriptional gene silencing techniques. Currently, RNAi technologies commonly used in laboratories mainly include siRNA oligonucleotide vectors and shRNA lentiviral plasmid expression vectors. siRNA is a short double-stranded RNA with a size of about 19-29 nt. There are two free bases at the 3´ end, which can activate RNA interference and specifically achieve mRNA degradation by binding to the complementary sequence of the target ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12N15/867C12N5/10A61K31/7088A61P35/04
CPCC12N15/113C12N15/86C12N5/0693C12N5/0682A61P35/04C12N2310/14C12N2320/32C12N2740/15043C12N2800/107C12N2510/00C12N2310/531
Inventor 刘晗青赵锦霞林春秀屠志刚
Owner JIANGSU UNIV
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