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Construction of ShRNA lentiviral vector for targeted silencing of Kdm7a gene and application of shRNA lentiviral vector

A targeted silencing and lentiviral technology, applied in the fields of molecular biology and biomedicine, can solve the problems of functional research limitations, unreported and unseen research on osteogenic differentiation, etc.

Inactive Publication Date: 2018-12-11
天津医科大学代谢病医院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the study on the regulation of KDM7A on the osteogenic differentiation of MSCs has not been reported
However, using liposomes for transient transfection rather than stable transfection, the KDM7A plasmid was gradually reduced as the cells proliferated
Therefore, studies on the function of KDM7A in mouse bone marrow mesenchymal cells are limited.
So far, no KDM7A recombinant lentiviral vector that can sustainably, stably and efficiently express in mouse bone marrow mesenchymal cells has been seen

Method used

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  • Construction of ShRNA lentiviral vector for targeted silencing of Kdm7a gene and application of shRNA lentiviral vector
  • Construction of ShRNA lentiviral vector for targeted silencing of Kdm7a gene and application of shRNA lentiviral vector
  • Construction of ShRNA lentiviral vector for targeted silencing of Kdm7a gene and application of shRNA lentiviral vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of shRNA lentiviral vector targeting silencing Kdm7a gene

[0029] 1. Design the shRNA sequence targeting Kdm7a silencing. The sense strand and antisense strand oligonucleotides of the Kdm7a shRNA coding sequence were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0030] 2. Synthesis of target fragments: The sense strand and antisense strand oligonucleotides were dissolved at a concentration of 50 μM, and 5 μl each was taken in a 0.2 mL EP tube for denaturation and annealing, and the reaction was carried out in a PCR instrument under the following conditions: 95°C for 1 min, 50 °C for 2 min, then cool down at room temperature.

[0031] 3. Obtaining the linear vector fragment: the pLVX-shRNA2 plasmid was digested with restriction endonucleases BamHI and EcoRI, electrophoresed on 1.2% agarose gel, and the linear vector fragment was recovered.

[0032] 4. T4 ligase ligated the target fragment and the linear vector fragment, transformed...

Embodiment 2

[0041] Example 2 Packaging pLVX-Kdm7a shRNA recombinant lentivirus

[0042] 1. Recovery and passage of 293T cells: Recovery: Take 293T cells frozen in a liquid nitrogen tank, quickly place them in water at 37-38°C, and shake. After it was completely melted, centrifuge at 1000rpm for 10min at room temperature. Discard the frozen storage solution, resuspend the cells in DMEM medium containing 10% FBS and double antibodies, inoculate them into cell culture flasks, and store at 37°C, 5% CO 2 Incubate in a cell culture incubator. Every 3 days, replace the cell culture medium. Observe the state of the cells under a microscope, and when the confluence of the cells reaches 80% to 90%, they can be passaged.

[0043] 2. Cell inoculation: 24 hours before transfection, digest 293T cells with 0.25% trypsin, and inoculate into a new 10cm culture dish. When the confluence of the cells in the well plate reached 80%-90%, the empty lentiviral plasmid pLVX shRNA2 (Ctrl LV) and pLVX-Kdm7a shRNA...

Embodiment 3

[0061] Example 3 Effect of pLVX-Kdm7a shRNA recombinant lentivirus on osteogenic differentiation of mouse bone marrow mesenchymal cells

[0062] 1. Cell inoculation: 24 hours before transfection, the mouse bone marrow mesenchymal cells were digested with 0.25% trypsin, and inoculated into a 24-well plate. When the confluence of the cells in the well plate reached 30%-40%, the control lentivirus pLVX shRNA2 (Ctrl LV) and pLVX-Kdm7a shRNA recombinant lentivirus (Kdm7a-KD LV) were infected.

[0063] 2. The infection system and method are as follows:

[0064]

[0065] 3. Observe the state of the cells after infection, replace the medium after 24-48 hours of infection, collect the cells, extract total RNA, reverse transcribe and synthesize cDNA, and detect the expression of Kdm7a in primary mouse bone marrow mesenchymal cells after infection with lentivirus by qRT-PCR mRNA expression.

[0066] 4. Induce osteogenic differentiation and detect the mRNA expression of specific fact...

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Abstract

The invention designs a shRNA sequence aiming at histone demethylase KDM7A and constructs a lentiviral vector for coding the shRNA sequence. A 293T cell is transinfected, and a lentivirus particle fortargeted silencing of a Kdm7a gene is acquired through packaging and is used for infecting mesenchymal stem cells of a mouse. The constructed Kdm7a gene is high in interference efficiency of the lentivirus to host cells and capable of specifically, continuously and efficiently inhibiting the endogenous expresion of Kdm7a in the mesenchymal stem cells of the mouse and promoting the differentiationof the mesenchymal stem cells of the mouse to osteoblasts. As KDM7A can be taken as a latent target for treating bone defect and osteoporosis, a method provided by the invention can be used for researching and developing relevant diseases.

Description

technical field [0001] The invention relates to the fields of molecular biology and biomedicine, and mainly relates to the construction of an interference lentiviral vector targeting silencing Kdm7a and its effect on the directed differentiation of mouse bone marrow mesenchymal cells. Background technique [0002] 1. RNA interference (RNA interference, RNAi) is a regulatory method for silencing gene expression, which exists widely in organisms. Common RNA interference is mediated by small RNAs with a length of 20-30 nucleotides. Small hairpin RNA (shRNA) is a method commonly used in the field of molecular biology to inhibit the endogenous expression of genes. It is a double-stranded RNA containing a hairpin structure obtained based on an expression vector. The lentiviral vector can integrate the target gene into the host DNA, and stably express it in various types of mammalian cells, and suppress gene expression for a long time, which also lays the foundation for stable exp...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A61K48/00A61P19/08A61P19/10
CPCC12N15/86A61K48/0025A61K48/0066C12N15/66C12N2740/15043
Inventor 王宝利刘颖杨晓月
Owner 天津医科大学代谢病医院
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