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Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model

A technology of kinase activity and construction method, which is applied in the field of transgenic fruit fly model and its construction, can solve problems such as capillary connection disorder and mouse death, and achieve the effect of fast drug screening, easy observation, and rapid drug screening

Active Publication Date: 2013-08-14
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In mice, a defect in the EphB4 gene results in impaired capillary junctions, which eventually lead to embryonic death

Method used

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  • Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model
  • Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model
  • Transgenic drosophila model for screening EphB4 kinase activity inhibitors and construction method of transgenic drosophila model

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Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Drosophila model for screening EphB4 kinase activity inhibitors and its construction.

[0035] 1: PUAST vector transformation (PUAST à PUAST-EGFP)

[0036]In order to detect whether the target gene is transferred into the Drosophila at the pupal stage of Drosophila, it is necessary to transfer PUAST (China Plasmid Vector Strain Cell Strain Gene Collection Center-Biovector Science Lab, Inc.pUAST, see the vector map figure 1 ) carrier, which has a UAS (upstream activating sequence) sequence on it. The purpose of the carrier modification is to connect EGFP to the PUAST carrier. The process is as follows:

[0037] The PUAST vector was double digested with XhoI and XbaI, and the digested product was recovered for use; the PCR primers for EGFP were designed as follows:

[0038] Xh-EGFP-F: G TAC TCG AGC TTT ATG GTG AGC AAG GGC GAG (SEQ ID NO: 7)

[0039] X-EGFP-R: AAT CTA GAA CTT GTA CAG CTC GTC CAT G (SEQ ID NO: 8)

[0040] Use the above two primers and pEGFP-N...

Embodiment 2

[0134] Embodiment 2, using transgenic fruit fly model for drug screening

[0135] The specific steps for the Drosophila model to be used for drug screening are: adding the compound NVP-BHG712 (receptor tyrosine kinase ( RTK) >> Raf >> Raf inhibitor, American Selleck Company, website: http: / / www.selleck.cn / ), observed the changes in the eyes of Drosophila, and the results showed that the eye tumors were inhibited, and the eyes changed from rough to smooth , indicating that the compound NVP-BHG712 has an inhibitory effect on tumors.

[0136] image 3 It is a graph showing the results of rapid screening of inhibitors of hEphB4 kinase activity using the Drosophila model. In the picture, A is a wild-type fruit fly (from Bloomington Drosophila Center BL#1: Canton-S), with a smooth eye; B is hEphB4 Kinase domain; GMR-Gal4 / Cyo transgenic fruit fly, eye tumor; C is hEphB4 Kinase domain; GMR-Gal4 / Cyo transgenic fruit flies were fed with the compound NVP-BHG712 (receptor tyrosine kin...

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Abstract

The invention relates to a transgenic drosophila model for screening EphB4 kinase activity inhibitors and a construction method of the transgenic drosophila model. Plasmids are injected into yw drosophila embryos, after eclosion female drosophilae are hybridized with yw wild type male drosophilae, eclosion male drosophilae are hybridized with yw wild type female drosophilae, obtained offspring red-eye male drosophilae are hybridized with GMR-Ga14 virgin drosophila to obtain I-generation drosophilae, and virgin drosophilae of the I-generation drosophilae are selected to hybridize with yw / Y; +Cyo male drosophilae, and descendant curly-wing rougheye male drosophilae are male drosophilae in the transgenic drosophila model; the obtained male drosophilae are hybridized with + / FM7;+Cyo female drosophilae, descendant bar-eye curly-wing rougheye female drosophilae are further hybridized with the male drosophilae in the model, and bar-eyeless curly-wing rougheye female drosophilae are female drosophilae in the transgenic drosophila model. The transgenic drosophila model can be used for screening targeted drugs more quickly and conveniently.

Description

technical field [0001] The invention relates to an animal model and a construction method thereof, in particular to a transgenic fruit fly model and a construction method thereof. Background technique [0002] Eph receptors constitute the largest family of receptor tyrosine kinases and, together with their membrane-bound ligand ephrin, play important roles in embryonic patterning, neural targeting, vascular development, and adult neovascularization. There is increasing evidence that the Eph receptor signaling pathway promotes the occurrence of many tumors in the human body, such as directly promoting tumor growth or indirectly promoting tumor formation through the formation of tumor blood vessels. Eph receptors and their ligands have been detected to be overexpressed in tumors such as breast cancer, prostate cancer, and colon cancer. [0003] In particular, EphB4, which is the most studied protein in the Eph family. Inhibition of EphB4 expression using small interfering RN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/033
Inventor 陈建明杨丽容韩家淮传超李冬梅韩伟杨慧李福英
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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