Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish

A zebrafish, transgenic technology, applied in the direction of using vectors to introduce foreign genetic material, applications, fish farming, etc., can solve the problem of failing to construct transgenic lines and other issues

Inactive Publication Date: 2013-07-31
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no transgenic line that can specifically express Gal4 in zebrafish primordial germ cells has been constructed.

Method used

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  • Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish
  • Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish
  • Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: A kind of preparation method of the transgenic fish of highly efficient labeling zebrafish primordial germ cells, its steps are:

[0057] 1. Preparation of transgenic lines based on the Gal4 / UAS system using mRFP to mark zebrafish primordial germ cells, see the strategy diagram figure 1 shown. Then design the primers needed to amplify each fragment in the transgenic vector. The primer sequences are shown in the table below:

[0058]

[0059]

[0060] 2. Construct the transgenic vector pTol2 (kop: KalTA4-UTRnos1, CMV: EGFP-S V40) of the activated strain Tg (kop: KalTA4).

[0061] (1) Construct the vector pMD-18T-UTRnos1.

[0062]Using the plasmid kop-new-F-EGFP-UTRnos1 as a template (Blaser, H., Eisenbeiss, S., Neumann, M., Reichman-Fried, M., Thisse, B., Thisse, C.and Raz, E .(2005) "Transition from non-motile be haviour to directed migration during early PGC development in zebrafish." J Cell Sci 118, 4027-4038), nos13'UTR-F and nos13'UTR-R we...

Embodiment 2

[0081] Embodiment 2: The application of a transgenic fish that efficiently marks zebrafish primordial germ cells in fish bioengineering, the process is:

[0082] 1. KalTA4mRNA is specifically expressed in primordial germ cells in embryos of activated strain Tg (kop:KalTA4).

[0083] (1) KalTA4 probe preparation.

[0084] A. Template preparation and purification.

[0085] Using plasmid pTol2 (kop: KalTA4-UTRnos1, CMV: EGFP-SV40) as template, Probe-KalTA4-F and T3-nos13'UTR-R (with T3 promoter sequence) as primers, PCR amplified template DNA, refer to Axygen's PCR clean up kit recovered the PCR products and used them as probes for synthetic probes.

[0086] B. Probe synthesis and recovery.

[0087]RNA probes were transcribed and synthesized according to Promega's T3 RNA polymerase instructions, and the probes were recovered according to the instructions of sigma spin post-reaction chean-up columns.

[0088] (2) In situ hybridization was used to detect the expression of KalTA...

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Abstract

The invention discloses a vector for efficiently labeling zebra fish PGC, and a preparation method and a use of transgenic fish. A Gal4/UAS transcription activation system is utilized and mRFP is used as a report gene to realize an inductive gene expression regulation technology in the primordial germ cells of zebra fish. The preparation method of the transgenic fish comprises the following steps: 1, respectively constructing activation transgenic line Tg (kop:KalTA4) and an effect transgenic line Tg (UAS:mRFP); and 2, hybridizing Tg (kop:KalTA4) male fish with Tg (UAS:mRFP) female fish to obtain the transgenic fish for efficiently labeling the primordial germ cells of zebra fish. The transgenic fish for efficiently labeling the primordial germ cells of zebra fish can be widely applied to the fish bioengineering.

Description

technical field [0001] The invention belongs to the technical field of fish bioengineering, and the invention relates to a carrier for efficiently marking zebrafish PGC, and also relates to a method for preparing a transgenic fish for efficiently marking zebrafish primordial germ cells, and also relates to the transgenic zebrafish in fish Uses of bioengineering techniques. Background technique [0002] As the carrier of genetic material, fish primordial germ cell (PGC) has become a new tool for fish bioengineering technology because of its totipotency (Yoshizaki, G., Takeuchi, Y. and Okutsu, T. .(2007)."Germ cell in fish: basic biology and biotechnological applications." Tanpakushitsu Kakusan Koso 52, 2067-2072.). However, the lack of a good labeling system remains a major obstacle for the manipulation of primordial germ cells. Therefore, it is necessary to apply current molecular genetics techniques to primordial germ cells in order to better analyze the molecular mech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K61/00A01K67/027
CPCY02A40/81
Inventor 孙永华熊凤魏志强朱作言
Owner INST OF AQUATIC LIFE ACAD SINICA
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