Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes

A TMEM59L, interaction technology, applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc., can solve problems such as limited research

Inactive Publication Date: 2012-11-14
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Epigenetic mechanisms play a role in the pathogenesis of late-onset Alzheimer's disease (LOAD), but genome-wide identification of disease loci is limited

Method used

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  • Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes
  • Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes
  • Specific interaction of dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: vector construction

[0018]Use Olige 6.0 software to design primers for human DCF1, mouse DCF1, human TMEM59L, mouse TMEM59L, APP(695), GDI1, GDI2, and BACE2 genes, and introduce restriction sites and protective bases into the upstream and downstream primers, respectively. After the genes were amplified by PCR, they were connected into the corresponding vectors, as shown in Table 1.

[0019]

Embodiment 2

[0020] Example 2: Plasmid transfected cells

[0021] Transfect human and mouse DCF1 and TMEM59L plasmids into U251, HEK293T, C17.2, 4T1, U87 and other cells. The transfection process is as follows:

[0022] 1. The day before transfection, cells were trypsinized and counted, and the cells were plated in a 6-well plate so that the density on the day of transfection was 70%. Add 1.5mL of serum-containing, normal growth medium without antibiotics to each well.

[0023] 2. When transfecting, first replace the serum-containing medium in the 6-well plate with serum-free medium, 1 mL per well, and place at 37°C, 5% CO 2 in the incubator.

[0024] 3. For each well of cells, dilute 3 μg of plasmid DNA with 250 μL of serum-free medium DMEM.

[0025] 4. For each well of cells, dilute 3 μL Lipofectamine 2000 reagent with 250 μL serum-free medium DMEM, and let stand at room temperature for 5 minutes.

[0026] 5. Mix diluted plasmid DNA (step 3) and diluted Lipofectamine 2000 (st...

Embodiment 3

[0031] Example Three: Organelle Staining

[0032] After DCF1 and TMEM59L were transfected with green or red fluorescent plasmids, they were stained with corresponding organelle dyes for co-localization observation.

[0033] (1) Fluorescent labeling of the Golgi apparatus

[0034] Remove the cell culture medium, and wash the cells grown on the coverslip with an appropriate amount of solution such as D-Hanks solution. Remove the washing solution, add the prepared Golgi-Tracker Red staining solution, and incubate with the cells at 4°C for 30 minutes. Recover the Golgi-Tracker Red staining working solution, wash the cells about 3 times with the cell culture medium pre-cooled in an ice bath, replace with fresh culture medium and incubate at 37°C for 30 minutes. After washing once more with fresh culture medium, observation is usually performed with a fluorescence microscope or a confocal laser microscope. At this time, it can be observed that the Golgi apparatus shows bright a...

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Abstract

The invention relates to specific interaction of a dentritic cell factor 1 (DCF1) genes and transmembrane protein 59 like (TMEM 59 L) genes. Under the overexpression condition of the DCF1 and the TMEM 59 L, amyloid precursor protein (APP) can be highly and specifically inhibited from separating from golgi apparatus, the transportation of GDP dissociation inhibitor 1 (GDI1) and GDP dissociation inhibitor 2 (GDI2) are partially inhibited, the transportation of beta-site APP cleaving enzyme 2 (BACE2) protein is not inhibited, and a new drug target is provided for diagnosing and treating the Alzheimer disease.

Description

technical field [0001] The present invention relates to the specific interaction of DCF1 gene and TMEM59L gene. Especially the application of the interaction between DCF1 gene and TMEM59L gene in inhibiting the transportation of APP protein, GDI1 protein or GDI2 protein. Background technique [0002] Dendritic cell factor 1 (DCF1), also known as TMEM59 (transmembrane protein 59), is Type transmembrane protein, the understanding of this protein is still not very clear. In 2008, Wang, L; et al showed by cloning, expressing, and silencing the DCF1 gene that the overexpression of DCF1 kept neural stem cells in an undifferentiated state; silencing the DCF1 gene, the cells tended to differentiate into neurons and glial cells. Through the gene chip analysis of mouse neural stem cells, the regulatory network of DCF1 was revealed, and 36 genes were inferred to be mainly involved in the regulation of DCF1, especially pou6f1 may play an important role in the transcription of DCF1 an...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12Q1/02G01N33/68
Inventor 文铁桥王海烽
Owner SHANGHAI UNIV
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