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General type PiggyBac transposon transgenosis carrier and preparation method thereof

A technology of transgene carrier and transposon, which is applied in the field of general-purpose PiggyBac transposon transgene carrier and its preparation, which can solve problems such as difficulty in determining the position of the transgene, limited length of exogenous DNA fragments, and influence on the stable expression of the transgene. Effect of Carrier Molecular Weight

Inactive Publication Date: 2014-12-10
NORTHWEST A & F UNIV +1
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Problems solved by technology

This method has many disadvantages: (1) The integration efficiency is low, so transgenic animals cannot be bred efficiently by this method in most mammals; (2) In most cases, the linearized DNA will form multiple copies of tandem repeats, namely Concatamers, which are difficult to simulate the normal physiological conditions of endogenous genes; (3) concatamers are prone to recombination between different transgene copies during passage, resulting in deletion (deletion), or due to methylation modification affecting transgene (4) Since it is difficult to determine the position in the genome where the transgene is inserted by using this method, it is also impossible to evaluate the chromosomal environment near the insertion site
[0004] Compared with simple linearized DNA, viral vectors can integrate transgenes into the genome more efficiently, but their main disadvantages are: (1) The load capacity is small, that is, the length of exogenous DNA fragments that can be carried is limited; (2) The host The virus will also be methylated to silence the expression of the transgene; (3) The preparation of the virus is cumbersome; (4) Although most of the viruses used for transgenesis are replication-defective viruses, safety is still a problem that needs to be vigilant

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  • General type PiggyBac transposon transgenosis carrier and preparation method thereof
  • General type PiggyBac transposon transgenosis carrier and preparation method thereof
  • General type PiggyBac transposon transgenosis carrier and preparation method thereof

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Embodiment Construction

[0052] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

[0053] 1. Materials and reagents

[0054] Vector: pMCIneo (stratagen), pMD18-T simple (TAKARA), pEGFP-N1 and pEGFP-C1 were purchased from Clotech,

[0055] The phspBac vector (Handler and Harrell, 2001a) was kindly provided by Professor Handler, South Atlantic Center for Agricultural Insect Diseases, USDA.

[0056] Strain: DH5α (Tiangen)

[0057] Reagents: various endonucleases (NEB, MBI), T4DNA ligase (TAKARA), CIAP (TAKARA), Klenow (TAKARA), plasmid mini-extraction kit (Shanghai Sangong), plasmid macro-extraction kit (Promega), Gel Extraction Kit (Axygen), HP DNA Transfection Reagent (Roche), G418 (Sigma), PCR Related Reagent (TAKARA), Chromosome Walking Kit (TAKARA), T4 Nucleotide Kinase (TAKARA), Blood, Cells , Tissue Genome Extraction Kit (Tiangen).

[0058] The primers used are as f...

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Abstract

The invention discloses a general type PiggyBac transposon transgenosis carrier and a preparation method thereof. The general type PiggyBac transposon transgenosis carrier comprises a donor carrier and an auxiliary carrier, the donor carrier comprises a 5' terminal repeat (5'TR) and a 3' terminal repeat (3'TR) of a PiggyBac transposon, two insulators are arranged between the 5'TR and the 3'TR, and a plurality of clone loci are arranged between insulator sequences; and the auxiliary carrier comprises a PiggyBac transposase encoding gene Pbase, a promoter is arranged on the upstream of the Pbase, and PloyA is arranged on the downstream of the Pbase. The method includes that exogenous genes are cloned in the clone loci of the donor carrier, and then the exogenous genes and the auxiliary carrier are mixed with transfection cells to stably screen cells for monoclone, and specificity of the exogenous genes is integrated with TTAA loci in genome through PiggyBac (PB) transposon elements.

Description

technical field [0001] The invention belongs to the technical field of transgenic vectors, and relates to a universal PiggyBac transposon transgenic vector and a preparation method thereof. Background technique [0002] Transgenesis is the method of introducing a DNA fragment carrying a gene into the genome. By observing the phenotypic changes of organisms produced by transgenesis, functional information of the relevant genes can be obtained. In addition, transgenic technology can also be used to improve the traits of organisms, which has huge economic value. [0003] The most commonly used transgenic method in mammalian cells or animals is to introduce linearized DNA carrying foreign genes into cells through liposomes, electroporation or microinjection, so that the DNA is randomly integrated into the genome. This method has many disadvantages: (1) The integration efficiency is low, so transgenic animals cannot be bred efficiently by this method in most mammals; (2) In mos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64
Inventor 张涌胡广东王静余源高元鹏
Owner NORTHWEST A & F UNIV
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