Expression vector for piggyBac transposon, and transgenic pig and construction method thereof

A construction method and expression vector technology, applied in the field of genetic engineering, can solve the problems of not seeing transgenic animals, etc., and achieve the effect of convenient detection and screening of cell lines and convenient deletion

Inactive Publication Date: 2013-12-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the preparation of transgenic animals using the piggyBac (pB) transposon system combined with somatic cell cloning

Method used

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  • Expression vector for piggyBac transposon, and transgenic pig and construction method thereof
  • Expression vector for piggyBac transposon, and transgenic pig and construction method thereof
  • Expression vector for piggyBac transposon, and transgenic pig and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 PiggyBac transposon expression vector carrying reporter gene and resistance gene: construction of pPB-CMV-Neo / EGFP

[0038] Include the following steps:

[0039] 1. Amplify the fusion fragment containing the loxP-Neo / EGFP gene expression cassette

[0040] Using the plasmid pPSP-PB-neoEGFP-XynB-manA (SEQ ID NO: 5, this plasmid was transformed by the applicant through conventional means of transformation, PCR, enzyme digestion and ligation) as a template, design primers, 5' Add the enzyme cutting site SalI to amplify the fusion fragment containing the loxP-Neo / EGFP gene expression cassette; the primer sequences are as follows:

[0041] Neo / EGFP-F:

[0042] 5'-GTCGACCGTGAGGCGTGCTTGTCAATGC-3' (SEQ ID NO: 1); Neo / EGFP-R:

[0043] The reaction system of 5'-GTCGACGCTTCTGAGGCGGAAAGAACCA-3'(SEQ ID NO:2) PCR is

[0044]

[0045]

[0046] PCR reaction program: 98°C for 3min; 98°C for 30s, 65°C for 30s, 72°C for 3min; 35 cycles; 72°C for 10min.

[0047] After...

Embodiment 2

[0050] Example 2 Using the expression vector pPB-CMV-Neo / EGFP of Example 1 to construct transgenic pigs

[0051] Include the following steps:

[0052] Include the following steps:

[0053] 1. Screening for stably expressed monoclonal antibodies

[0054] The transposon plasmid pPB-CMV-Neo / EGFP (i.e. the expression vector constructed in Example 1) and the transposase plasmid mPB (gifted by the Sanger Institute) were mixed and co-transfected into a porcine fetal fibroblast cell line; Cell growth medium with 10% fetal bovine serum resuscitated porcine fetal fibroblasts into a 60mm diameter cell culture dish, and used for transfection when the cells reached 50%-80% confluence. The transfection steps are as follows:

[0055] (1) Mix the transposon plasmid pPB-CMV-Neo / EGFP and the transposase plasmid mPB at a molar ratio of 3:1 and add to 1000 μL In the centrifuge tube of I (life technology), mix well;

[0056] (2) PLUS TM Mix Reagents (life technology) before use, add 7 μL ...

Embodiment 3 Embodiment 2

[0075] Example 3 The detection of the transgenic pig of the piggyBac transposon constructed in Example 2

[0076] The recipient sows were pregnant and delivered, and the transgenic pigs were detected by green fluorescent fluorescence detection, PCR, southern blotting and quantitative PCR. Detect by the following method

[0077] 1. Green fluorescence detection

[0078] Directly irradiated with blue light for detection, transgenic positive pigs can emit green fluorescence, such as image 3 As shown, all anatomical organs of the transgenic pigs emit green fluorescence, which preliminarily proves that the transgenic plasmid has been integrated and can be expressed in the transgenic pigs.

[0079] 2. Detection of DNA level

[0080] Detection of DNA level by PCR, southern blot, IPCR and other methods, Figure 4 For transgenic pig PCR, southern blot results, from Figure 4 It can be seen that the target gene fragment can be detected in the transgenic pig, but there is no target ...

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Abstract

The invention discloses an expression vector for a piggyBac transposon and a transgenic pig and a construction method thereof, belonging to the field of gene engineering. The expression vector is constructed by connecting a fusion fragment containing a loxP-Neo / EGFP gene expression cassette to pCyL50 plasmid. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has the piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The construction method for the transgenic pig provides a novel approach for preparation of a variety of transgenic animals and a simple, convenient and highly efficient approach for fabrication of biological models of transgenic animals.

Description

technical field [0001] The invention belongs to the field of genetic engineering, more specifically, the invention relates to a piggyBac transposon expression vector carrying a reporter gene and a resistance gene: pPB-CMV-Neo / EGFP and its construction method, and piggyBac transposon transgenic pigs and its construction method. Background technique [0002] The concept of transgenics arose when researchers first produced mice carrying foreign genes by pronuclear microinjection. In the past thirty-three years, researchers have developed several methods to increase the efficiency of transgenic animal production, but the most popular among researchers is the use of linearized transgenic DNA by pronuclear microinjection. Proceed to the preparation of transgenic animals. The use of prokaryotic microinjection to produce transgenic animals has very obvious advantages: for example, the equipment it needs is relatively simple, while other methods will have special requirements for e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/63C12N15/85A01K67/027
Inventor 徐志谦吴珍芳邹娴李紫聪刘德武张献伟曾芳周荣曾海玉
Owner SOUTH CHINA AGRI UNIV
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