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shRNA sequence for suppressing mouse MACF1 gene expression and application thereof

A gene expression and sequence technology, applied in the field of shRNA sequence, can solve the problem of low efficiency and achieve the effect of improving efficiency

Active Publication Date: 2015-04-22
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the low efficiency of the existing shRNA sequence in inhibiting the expression of the mouse MACF1 gene, the present invention provides a shRNA sequence for inhibiting the expression of the mouse MACF1 gene and its application

Method used

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  • shRNA sequence for suppressing mouse MACF1 gene expression and application thereof
  • shRNA sequence for suppressing mouse MACF1 gene expression and application thereof
  • shRNA sequence for suppressing mouse MACF1 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Design and synthesis of shRNA sequences.

[0032] (1) Query the mRNA information of the mouse MACF1 gene through the GeneBank database (http: / / www.ncbi.nlm.nih.gov / genbank), and design 4 MACF1-shRNAs targeting the mouse MACF1 gene according to the shRNA design principle sequence, such as figure 1As shown, an irrelevant control shRNA sequence was designed at the same time as the control group (Control). The above designed shRNA sequences were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd. The four shRNAs target the 627, 1461, 3681 and 4019 positions of the mouse MACF1 gene (NM_001199136.1), respectively, and are 21 bases long.

[0033] Wherein, the action site sequence of MACF1-shRNA-627 involved in the present invention is:

[0034] 5'-GCTAGTGAATATCCGCAATGA-3';

[0035] The action site sequence of MACF1-shRNA-1461 is:

[0036] 5'-GCAGATTGCAAACAAGATACA-3';

[0037] The action site sequence of MACF1-shRNA-3681 is:

[0038] 5'-GCTGTTGG...

Embodiment 2

[0069] Example 2. Efficiency of Recombinant Lentiviral Vectors Infecting Mouse Preosteoblasts.

[0070] The lentiviral particles expressing the above four kinds of shRNA and Control-shRNA were transfected into mouse preosteoblast MC3T3-E1, and the infection efficiency of different shRNA sequences was detected by fluorescence microscopy.

[0071] (1) Experiment grouping: the experiment was divided into control group (Control), MACF1-shRNA-627 infection group, MACF1-shRNA-1461 infection group, MACF1-shRNA-3681 infection group, MACF1-shRNA-4019 infection group, a total of 5 groups , the number of cells and culture conditions were the same for all groups.

[0072] (2) Cell infection experiment method: mouse preosteoblast MC3T3-E1 was mixed with 1×10 6 Cells / well were seeded in a 6-well plate and mixed at 37°C in 5% CO 2 Cultivate for 24 hours; take 200 μl of lentivirus stock solution, dilute it 5 times with α-MEM medium containing 10% FBS, and add polybrene (Polybrene) with a f...

Embodiment 3

[0074] Example 3. Inhibition of MACF1 gene expression in mouse preosteoblasts by recombinant lentiviral vectors.

[0075] Real time PCR was used to detect the effects of four shRNAs on the expression level of MACF1 gene in mouse preosteoblasts.

[0076](1) Experiment grouping: the experiment was divided into control group (Control), MACF1-shRNA-627 infection group, MACF1-shRNA-1461 infection group, MACF1-shRNA-3681 infection group, MACF1-shRNA-4019 infection group, a total of 5 groups , the number of cells and culture conditions were the same for all groups.

[0077] (2) RNA extraction and real time PCR detection: After the cells were infected for 96 hours, the cells were lysed with Trizol, and the total RNA of each group of cells was extracted by the chloroform-isopropanol method, and the extracted mRNA was reverse-transcribed into cDNA. For: 42°C 30min; 85°C 10min. The cDNA of each group of cells was used as a template, ACTB was used as an internal reference, and Real tim...

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Abstract

The invention discloses a shRNA sequence for suppressing mouse MACF1 gene expression and application thereof to solve the technical problem of low efficiency of mouse MACF1 gene expression suppression of the conventional shRNA sequence. The technical scheme is that the shRNA sequence for suppressing mouse MACF1 gene expression is cloned to a pGLV3 lentivirus vector to obtain a recombinant lentivirus vector containing the shRNA sequence, and the recombinant lentivirus vector is used for transfecting a mouse cell to achieve the purpose of suppressing MACF1 gene expression in the cell. The shRNA sequence can remarkably suppress the mRAN expression and protein-level expression of the MACF1 gene in a mouse preosteoblast, and suppresses the functions of proliferation, migration and differentiation of the mouse preosteoblast by suppressing the expression of the MACF1 in the mouse preosteoblast. The shRNA sequence reaches a protein-level suppression ratio above 90% and the gene-level suppression ratio of 81%.

Description

technical field [0001] The invention relates to an shRNA sequence, in particular to an shRNA sequence for inhibiting mouse MACF1 gene expression. It also relates to the application of this shRNA sequence for suppressing mouse MACF1 gene expression. Background technique [0002] Microtubule actin crosslinking factor 1 (MACF1), also known as ACF7 (Actin crosslinking factor 7), is a new cytoskeleton crosslinking molecule, located on 1p31-32, containing 5380 amino acids , by regulating microfilaments and microtubule skeleton dynamics, it plays an important role in regulating various biological functions such as cell migration, cell polarization, cell adhesion, cell signal transduction, and protein transport. Due to the wide distribution of MACF1 and its important role in various biological functions, it is of great significance to study its function in different tissues. [0003] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degra...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10A01K67/027
Inventor 骞爱荣商澎胡丽芳李润芝苏佩红印崇闫琨陈志浩戚依朵孙瑜隆
Owner NORTHWESTERN POLYTECHNICAL UNIV
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