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
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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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