GFP-Addnd 3'UTR recombinant expression vector and constructing method and application thereof

An expression vector and a technology of the vector are applied in the GFP-Addnd 3'UTR recombinant expression vector and its construction and application fields, which can solve the problems of sharp reduction of natural resources and limited scale of sturgeon.

Inactive Publication Date: 2017-04-26
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since then, due to the impact of human activities such as overfishing and hydropower project construction, the natural resources of Dabry's sturgeon have declined sharply
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Method used

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  • GFP-Addnd 3'UTR recombinant expression vector and constructing method and application thereof
  • GFP-Addnd 3'UTR recombinant expression vector and constructing method and application thereof
  • GFP-Addnd 3'UTR recombinant expression vector and constructing method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Isolation and cloning of the dnd gene of Acipenser dabfieldii

[0043] 1. Extraction of total RNA

[0044] The 3-year-old Dabry's sturgeon was artificially bred, anesthetized and bled, and the gonads were taken out, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use. Take a spare sample, and extract total RNA according to the instructions of the SA Total RNA Isolation System (Promega, USA) kit.

[0045] 2. Synthesis of SMART cDNA

[0046] Press SMARTer TM Synthesize the first strand of SMART cDNA in the gonad of Dabry's sturgeon according to the operation manual of RACE cDNA Amplification Kit: (1) Mix 2 μL total RNA with 1 μL 3'-CDS Primer A / 5'-CDS Primer A, incubate at 72°C for 3 minutes, then incubate at 42°C Incubate for 2 minutes; (2) Add 1 μL SMARTIIA Oligo to the 5'RACE cDNA synthesis reaction tube, then mix well and centrifuge; ③Add 5×Buffer, DTT, RNase Inhibitor, dNTP, SMARTScribe to the reaction tube TM Mix r...

Embodiment 2

[0056] The construction of embodiment 2GFP-Addnd 3'UTR expression vector

[0057] 1. Obtaining the target fragment

[0058] (1) Design primers to amplify the Addnd 3'UTR fragment, introduce restriction sites EcoR I and Xho I (Table 1), and insert into pMD18-T vector. After sequencing verification, use The plasmid was extracted with the Plasmid Mini Kit (Omega), double-digested with restriction endonucleases EcoR I and Xho I (New England), recovered from the rubber tapping, and stored at -20°C for later use.

[0059] (2) Primers were also designed to amplify the fragment encoding the entire ORF reading frame of GFP, introducing restriction sites BamH I and EcoR I (Table 1), and inserting into the pMD18-T vector. After sequencing verification, use The plasmid was extracted with the Plasmid Mini Kit (Omega), double-digested with restriction endonucleases BamH I and EcoR I (New England), and after the rubber was recovered, it was stored at -20°C for later use.

[0060] 2. Re...

Embodiment 3

[0070] Example 3 Microinjection of early embryos of Acipenser dabryi

[0071] 1. Use RNase Free H 2 O Dilute the synthesized mRNA to 300 ng / μL.

[0072] 2. After the fertilized eggs of Dabry's sturgeon are debonded, remove the fertilized egg membranes with tweezers, and transfer them to an inclined groove with a depth of 3 mm and an angle of 45° prepared by 1.5% agarose.

[0073] 3. Perform microinjection at a room temperature of 17-18°C, adjust the direction of the embryo with tweezers, align the plant pole of the embryo with the micromanipulator, and inject the sample into the 1-cell embryo.

[0074] 4. The injected embryos were cultured at 17-18°C, changing 1 / 3 of the water every 12 hours, and adding antibiotics Amp and Stre, and observing the expression of green fluorescent protein at different developmental stages under a stereofluorescence microscope. At the same time, microinjection of pure water without mRNA was used as a control for comparison. The comparison resul...

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Abstract

The invention discloses a GFP-Addnd 3'UTR recombinant expression vector. The vector is an expression vector pCS<2+>, 3'UTR segment of an Acipenser dabryanus dnd gene and a green fluorescent protein coding region gene are connected to the expression vector pCS<2+>. The GFP-Addnd 3'UTR recombinant expression vector can be used for marking an Acipenser dabryanus original germ cell. The specific marking method comprises the steps of synthesizing mRNA of the GFP-Addnd 3'UTR recombinant expression vector in vitro, injecting the mRNA into an embryo in the embryo development 1-cell period of Acipenser dabryanus in a micro-injection mode, then conducting incubated cultivation, and observing expression conditions of the green fluorescent protein in different development periods in real time. Because the Addnd is a maternal gene, and is only expressed in the ovary and the spermary, after the marking method is adopted, expression of the green fluorescent protein can be detected in the development process of the zygote, and thus tracking migration development of the original germ cell and further deep scientific study are promoted.

Description

technical field [0001] The invention relates to a GFP-Addnd 3'UTR recombinant expression vector, its construction method, and its application in marking primordial germ cells of Acipenser dabryanus. The cell-specific gene dead end (dnd) can effectively mark the primordial germ cells of Dabry's sturgeon by using the 3' non-coding region (3'UTR) of the gene to fuse with a green fluorescent expression protein, and belongs to the technical field of bioengineering. Background technique [0002] Multicellular animals that reproduce both sexes have two major cell lines: the somatic cell line and the germ cell line (germplasm line). The somatic cell line maintains the growth and development of individual organisms; the germ cell line maintains the genetic information of the species from generation to generation by producing gametes of both sexes. The germ line originates from primordial germ cells (PGCs), and in fish and many other animals, PGCs separate from the somatic line early...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/66C12N15/89C12N15/12C07K14/46
CPCC07K14/461C12N15/65C12N15/66C12N15/85C12N15/89C12N2800/106C12N2840/007
Inventor 李创举杨晓鸽危起伟岳华梅叶欢
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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