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Construction method of human dcf1 gene transgenic drosophila melanogaster model

A construction method and technology of puast-dcf1, applied in the field of construction of transgenic Drosophila model

Inactive Publication Date: 2012-11-07
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are relatively few reports on the function of this protein. The existing reports have shown that DCF1 is mainly located on the Golgi apparatus, and DCF1 and its homologous protein TMEM59-like can inhibit the transport of APP to the cell membrane and further cleavage, indicating that DCF1 and AD there is some connection

Method used

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  • Construction method of human dcf1 gene transgenic drosophila melanogaster model
  • Construction method of human dcf1 gene transgenic drosophila melanogaster model
  • Construction method of human dcf1 gene transgenic drosophila melanogaster model

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Construction and verification of the Drosophila transgenic vector pUAST-dcf1

[0025] 1. In order to obtain the Drosophila transgenic vector pUAST-dcf1, first use the cDNA of human cervical cancer cell HeLa cells as a template for PCR amplification, and the primers are dcf1 primers (5'-CGGAATTCATGGCGGCGCCGAAGGGG AG-3' and 5'-GCTCTAGATTAAATTTCAGAATGAGCAA-3 ') to obtain the target fragment with a size of 972bp (the full length of the open reading frame of the dcf1 gene), and then digest it with EcoRI and XbaI, and collect the fragment for later use.

[0026] 2. Then cut the empty vector plasmid pWIZ with EcoRI and XbaI, and collect the large fragment for later use.

[0027] 3. Finally, connect the two fragments to obtain the Drosophila transgene vector pUAST-dcf1.

[0028] 4. If figure 1 The Drosophila transgenic vector pUAST-dcf1 constructed by EcoRI and XbaI double digestion identification, it can be seen that the transgenic vector pUAST-dcf1 released a t...

Embodiment 2

[0029] Example 2: Microinjection transgenic experiment and verification of transgenic fruit flies

[0030] 1. In order to obtain human dcf1 transgenic fruit flies, the principle of germ cell line transformation mediated by P factor was used to construct transgenic fruit flies by microinjection genetic engineering technology.

[0031] 2. Put the transgenic vector pUAST-dcf1 and the auxiliary plasmid into the glass microelectrode with an elongated opening at a certain concentration ratio, and inject the mixture into the newly produced Drosophila within 1 hour to remove the egg shell with the help of the pneumatic mechanical injection arm the tail of the fertilized egg, see figure 2 After the injection, the eggs were cultured in a humid box at 16°C.

[0032] 3. After the injected fertilized eggs hatched into larvae and then emerged into adults, they were mated with wild-type w1118, and the offspring whose eyes were red were selected as transgenic fruit flies. The selected UA...

Embodiment 3

[0034] Embodiment 3: Drosophila model of the present invention can be used as the tool in nerve cell research

[0035] 1. Drosophila has natural negative geotaxis. When the fruit fly is placed in a vertical tube, it will instinctively crawl upwards, and the crawling speed reflects the movement ability of the fruit fly. The experiment used different genes, the same number (10), the same eclosion days, and the same sex (male) Drosophila were anesthetized with carbon dioxide and moved into the same test tube (18 cm long, 1.5 cm in diameter), and the recovery began after 30 minutes at 25 ° C. experiment.

[0036] 2. Keep the test tube with fruit flies in a vertical state. At the beginning of the experiment, tap the test tube gently to make the fruit flies gather at the bottom of the test tube and then start timing. After 10 seconds, count the number of fruit flies at the top and bottom of the test tube quickly and count them. Make a good record, let the fruit flies recover for ...

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Abstract

The present invention relates to a construction method of a human dcf1 gene transgenic drosophila melanogaster model. According to the invention, a drosophila melanogaster transgenic vector pUAST-dcf1 is constructed; a human dcf1 transgenic UAS drosophila melanogaster strain is obtained through microinjection; and human dcf1 transgenic drosophila melanogaster strains, with balance gene and genetic stability, of w, UAS-dcf1 / Cyo and Tm6B / Tm2 are obtained through hybrid and backcross of the human dcf1 transgenic UAS drosophila melanogaster strain with Double Balance drosophila melanogasters of w, B1 / Cyo and Tm6B / Tm2. The present invention utilizes microinjection gene engineering technology and drosophila melanogaster hybridization technology to construct the human dcf1 gene transgenic drosophila melanogaster model, and provides materials and new ideas for research on relative neurodegenerative diseases.

Description

technical field [0001] The invention relates to a method for constructing a transgenic fruit fly model, in particular to a method for constructing a human dcf1 gene transgenic fruit fly model. Background technique [0002] Drosophila melanogaster (Drosophila melanogaster) has a highly developed nervous system and a short life cycle. It is easy to raise and easy to operate and analyze genetics, cell biology, and molecular biology. It is a very practical model organism. All, Drosophila is widely used in the construction and research of neurodegenerative disease models. [0003] Dendritic cell factor 1 (DCF1), also known as transmembrane protein 59 (TMEM59), was first discovered in dendritic cells of the immune system and is a single transmembrane protein with a wide range of expression. . At present, there are relatively few reports on the function of this protein. The existing reports have shown that DCF1 is mainly located on the Golgi apparatus, and DCF1 and its homologous...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85A01K67/033
Inventor 文铁桥蒋萌
Owner SHANGHAI UNIV
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