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PiggyBac transposon mediated transgenic vector for inducing cell immortalization, its construction method and its application

A technology of transgenic carrier and induced cells, which is applied in the field of genetic engineering, can solve the problems of non-expression expression efficiency, high mortality rate, and the technology of producing transgenic animals, so as to achieve the effect of improving transgenic efficiency and easy screening

Inactive Publication Date: 2012-03-07
NORTHWEST A & F UNIV
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AI Technical Summary

Problems solved by technology

The main problems of traditional transgenic technology are: the insertion of genes through homologous recombination can easily lead to low gene integration efficiency; most of the transferred genes form tandem polymers; the transferred genes do not express or have low expression efficiency; the resulting transgenic animals Most of them are chimeras; marker gene and non-target sequence excision technology is immature, resulting in many diseases, high mortality and infertility in animals with an intubation gene
All of these seriously restrict the application of transgenic animal technology

Method used

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  • PiggyBac transposon mediated transgenic vector for inducing cell immortalization, its construction method and its application
  • PiggyBac transposon mediated transgenic vector for inducing cell immortalization, its construction method and its application
  • PiggyBac transposon mediated transgenic vector for inducing cell immortalization, its construction method and its application

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Embodiment 1

[0045] 1. The construction of the basic transgenic vector containing positive and negative screening genes mediated by IRES:

[0046] 1. Use the vectors PEGFP-Cl, pB MOKS and pORF-HSVl tk as templates to PCR amplify SV40P-neo, IRES and tk-PolyA respectively; the PCR reaction pool system is shown in Table 1.1-Table 1.3;

[0047] Table 1.1 SV40P-neo PCR system

[0048]

[0049]

[0050] Table 1.2 IRES RCR system

[0051]

[0052] Table 1.3 tk-PolyA PCR system

[0053]

[0054] 2. Apply the overlapping PCR method to construct the gene expression cassette of LoxP-SV40P-neo-IRES-tk-PolyA-LoxP, which is used for the screening of transgenic cells and negative screening after marker gene deletion;

[0055] First, prepare the reaction pool. The PCR reaction system is shown in Table 2.1. The reaction conditions are: first, pre-denaturation at 94°C for 5 minutes, 94°C for 30 seconds, 64°C for 30 seconds, and 72°C for 3 minutes, 15 cycles, and the annealing temperature decre...

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Abstract

The invention relates to a PiggyBac transposon mediated transgenic vector for inducing cell immortalization, a construction method and an application, which belongs to the gene engineering technical field. The vector is PB-rtTA-NIT-STP, which is constructed by the following method: constructing a LoxP-SV40P nco-IRES-tk-PolyA-LoxP gene expression cassette; connecting an amplified fragment of the gene expression cassette, constructing a basic transgenic vector PB-NIT; constructing a transgenic vector PB-NIT-STP containing target gene SV40T and p 53; and constructing a screening vector PB-rtTA-NIT-STP containing Tet binding domain rtTA. The transgenic vector constructed by the invention has the advantage of effectively raising the transgene efficiency, and is convenient for screening.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a PiggyBac transposon-mediated induced cell immortalization transgene carrier, a construction method and application thereof. Background technique [0002] Animal transgenic technology is a technology for rapid genetic improvement of animals by means of modern biotechnology. Modern transgenic technology not only overcomes the limitations of traditional animal breeding technology, speeds up the process of germplasm genetic improvement, but also breaks the genetic barriers between species, expanding the gene resources available for selection in the breeding of new animal varieties. Therefore, transgenic technology represents The future development direction of modern agricultural technology. The main problems of traditional transgenic technology are: the insertion of genes through homologous recombination can easily lead to low gene integration efficiency; ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66A01K67/00
Inventor 张智英张婷婷辛颖杨涵江张志强徐坤王令
Owner NORTHWEST A & F UNIV
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