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Vector used for oriented screening of homologous recombination PGC, and construction method and application thereof

A vector and positive screening technology, which is applied to the vector and construction and application fields of directional screening of homologous recombination PGCs, can solve problems such as labeling PGCs, and achieve the effect of avoiding screening work.

Active Publication Date: 2013-11-20
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, direct injection of these transgenic vectors into zebrafish embryos did not mark PGC well

Method used

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  • Vector used for oriented screening of homologous recombination PGC, and construction method and application thereof
  • Vector used for oriented screening of homologous recombination PGC, and construction method and application thereof
  • Vector used for oriented screening of homologous recombination PGC, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] A carrier for directional screening of homologous recombination PGC, the preparation steps of which are:

[0042] (1) Construct the positive selection vector p(UAS:mRFP) to UAS-driven mRFP expression marker PGC.

[0043] The plasmid pBK-kalTA4 (Distel, M., Wullimann, M.F. and Koster, R.W. (2009) Optimized Gal4 genetics for permanent gene expression mapping in zebrafish. Proc Natl Acad Sci U S A.106,13365-13370.) and PCS2-mRFP (Addgene) as templates, UAS-mRFP-F and UAS-mRFP-m-R, UAS-mRFP-m-F and UAS-mRFP-R as primers, UAS and The full-length coding sequence of mRFP was connected into a DNA chain UAS:mRFP. The amplification conditions are: pre-denaturation at 95°C for 4 minutes; denaturation at 95°C for 30 seconds, renaturation at 62-58°C (0.5°C per cycle) for 30 seconds, extension at 68°C for 60 seconds, 8 cycles; Denaturation at 95°C for 30 seconds, renaturation at 63°C for 30 seconds, extension at 68°C for 60 seconds, 15 cycles; extension at 68°C for 10 minutes; s...

Embodiment 2

[0054] The verification of the positive and negative screening vector p(UAS:mRFP,UAS:puma) function, the steps are:

[0055] (1) Preparation of embryos of the activated strain Tg (kop:KalTA4): the transgenic vector pTol2 (kop:KalTA4-UTRnos1, CMV: EGFP-SV40) (sequence shown in SEQ ID NO.2) was introduced into zebrafish fertilized eggs middle. After the transgenic fish are sexually mature, they are crossed with wild-type female fish, and the crossed embryos are cultured at 28°C for 24 hours. Embryos that can emit green fluorescence are selected under a fluorescent dissecting microscope and continue to be cultured, and finally the pure line Tg(kop:KalTA4) is established. (A vector for efficiently labeling zebrafish PGC and the preparation method and application of transgenic fish, application number: 201310169181.6).

[0056] (2) The p(UAS:mRFP) and p(UAS:mRFP,UAS:puma) plasmids were respectively overexpressed in Tg(kop:KalTA4) embryos.

[0057] By microinjection instrument and...

Embodiment 3

[0061] The application of p(UAS:mRFP,UAS:puma) vector in directional screening of zebrafish homologous recombination PGC is as follows:

[0062] (1) Prepare the homologous recombination plasmid targeting PGC based on the fluorescence-apoptosis positive and negative screening system.

[0063] By molecular cloning technique (conventional methods, molecular cloning experiment guide, second edition, J. Sambrook et al., Science Press, 1993), using plasmid p(UAS:mRFP, UAS:puma) as the vector backbone, in UAS :mRFP-UTRnos1 expression cassette is inserted into the left and right arm DNA sequences of the target gene, and the UAS:puma-UTRnos1 expression cassette is located outside the left and right arm sequences, and a homologous recombination vector targeting the target gene nanog is constructed. See image 3 . Taking the target gene nanog (Accession#BC162318) as an example, the specific method is as follows:

[0064]Using wild-type zebrafish DNA as a template, the left arm DNA sequ...

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Abstract

The invention discloses a vector used for oriented screening of homologous recombination PGC, and a preparation method and an application thereof. According to the invention, expression cassette UAS: mRFP-UTRnos1 and expression cassette UAS: puma-UTRnos1 are connected in series, such that vector p (UAS: mRFP, UAS: puma) which can be used in oriented screening of homologous recombination PGC can be constructed. The sequence of the vector is represented by SEQ ID NO.1. The vector has fluorescent specific high-efficiency PGC marking function, and has pro-apoptotic gene PGC specific rejection function. P (UAS: mRFP, UAS: puma) is used as a vector frame, targeting gene homologous sequence left and right arms are respectively positioned on two sides of the expression cassette UAS: mRFP-UTRnos1, and the expression cassette UAS: puma-UTRnos1 is positioned on the outer side of the homologous sequence, such that a homologous recombination vector is prepared. The homologous recombination vector is introduced into a Tg (kop: KalTA4) embryo, and embryo expressing mRFP in PGC is selected, such that embryo subjected to homologous recombination germ cells is obtained.

Description

technical field [0001] The invention belongs to the technical field of fish bioengineering. The invention relates to a vector for directional screening of homologous recombination PGC, a preparation method of the vector, and an application of the vector in directional screening of zebrafish homologous recombination PGC. The inventive vector for directional screening of homologous recombination PGC can efficiently utilize homologous recombination technology to prepare zebrafish strains with specific genetic modifications. Background technique [0002] Homologous recombination technology is the most commonly used method to construct a gene knockout mouse model. The basic steps are: first, transfer the targeting vector into mouse embryonic stem cells; and then identify embryos with homologous recombination through a suitable screening system Stem cells; then the genetically modified embryonic stem cells are cultured into mouse individuals through chimera technology, so as to pr...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12Q1/68
Inventor 孙永华熊凤魏志强朱作言
Owner INST OF AQUATIC LIFE ACAD SINICA
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