Zebra fish egg yolk protein origin 1 gene regulating and controlling sequence

A gene regulation and regulation sequence technology, applied in the field of life sciences, can solve problems such as unreported promoter DNA sequences, and achieve the effect of protecting self-safety and healthy reproduction

Inactive Publication Date: 2007-01-31
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cDNA and protein sequences of zebrafish vtg1 are known, but the promoter DNA sequence that regulates its expression has not been reported

Method used

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  • Zebra fish egg yolk protein origin 1 gene regulating and controlling sequence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Obtaining the DNA sequence of zfvtg1-1.7

[0018] 1. Breeding of Zebrafish

[0019] Wild-type zebrafish (Danio rerio) were purchased from the International Zebrafish Resource Center, the zebrafish culture system was imported from Aquatic Habitats, USA, and the feeding program was carried out according to the Zebrafish Book description.

[0020] 2. Extraction of Zebrafish Genomic DNA

[0021] Zebrafish genomic DNA was extracted using the Genomic DNA Extraction Kit from Promega, USA.

[0022] 3. Primer Design

[0023] The vtg1 gene predicted according to the Trust Sanger Institute zebrafish database (ID: ENSDARG00000016825 ) designed the primers of sequence 2 and sequence 3, and introduced HindIII and BamHI restriction sites:

[0024] P1: 5'-GCTGCAAGCTTACTAATAGGAAGCCAACGAAGGACC-3' (SEQ ID NO: 2)

[0025] P2: 5'-CTCGGATCCGCTACAGTCAAGGCAAGCACAACAGC-3' (SEQ ID NO: 3)

[0026] The primers were synthesized by Shanghai Saibaisheng Biotechnology Company.

[002...

Embodiment 2

[0049]Example 2 Identification of zfvtg1-1.7-EGFP promoter activity (consistent spatiotemporal expression of EGFP and vtg1)

[0050] 1. Construction of zfvtg1-1.7-EGFP transgenic zebrafish

[0051] 2. Refer to "Zebrafish Book" for the breeding of zebrafish, transgenic technology and fluorescence observation method.

[0052] (1) Zebrafish farming

[0053] Wild-type zebrafish (Danio rerio) were purchased from the International Zebrafish Resource Center, and the zebrafish culture system was imported from Aquatic Habitats, USA, and the feeding program was carried out according to the description in Zebrafish Book.

[0054] (2) Genetically modified

[0055] The recombinant plasmid was linearized with restriction endonuclease bgl II, and after purification, it was dissolved in phosphate buffer containing phenol red at a concentration of 50 ng / μL. When the fertilized egg develops to the stage of one to two cells, the transgenic operation is carried out under the dissecting microsc...

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Abstract

The present invention belongs to the field of life science. Said invention provides a regulatory controlling sequence expressed by zebra fish vitellogenin 1, (vtg 1) gene of sequence 1. Said invention utilizes the transgenic zebra fish experiment to show that said sequence has promoter activity in the zebra fish body interior, and can make downstream gene implement specific expression in liver under the induction of female sex hormone. By adopting the connection of zfvtg 1-1.7 sequence provided by said invention and reporter gene (fluorescin) the fluorescence can be observed in the living body interior of transgenic zebra fish so as to quickly and visually reflect expression of vtg 1.

Description

technical field [0001] The invention belongs to the field of life sciences. A certain regulatory sequence involved in the expression of zebrafish vitellogenin-1 (vitellogenin1, vtg1) gene. Using transgenic zebrafish experiments, it is proved that this sequence has promoter activity in zebrafish, and can promote the specific expression of downstream genes in the liver under the induction of estrogen. Background technique [0002] Vitellogenin (vtg) is the precursor of three types of vitellin, which is specifically present in the blood of mature oviparous female animals, and provides nutrients and functions such as amino acids, fats, carbohydrates, vitamins, phosphorus and sulfur for the developing embryo sexual substance. Zebrafish vtg is divided into seven subtypes, vtg1-vtg7, which are mainly expressed in the liver of mature female fish under physiological conditions; after being subjected to various environmental estrogens in the water body, male fish and juvenile fish w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12Q1/68
Inventor 宋后燕陈浩杨健
Owner FUDAN UNIV
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