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Transposon carrier and system for preparing immortalized cell and usage method thereof

An immortalization and carrier technology, applied in the field of genetic engineering, can solve the problems of pollution, difficult DNA integration, cumbersome and time-consuming preparation process, etc., and achieve high efficiency.

Active Publication Date: 2015-10-28
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have some disadvantages. For example, the electroporation method is easy to cause pollution during the operation process and cause great damage to the cells; the DNA introduced into the cell by the transfection reagent method, electroporation method or adenovirus method is difficult to integrate into the DNA of the cell itself. ;Although lentivirus or retrovirus can mediate the insertion of exogenous DNA into the cell genome, the length of exogenous DNA is limited; in addition, each new piece of DNA needs to construct and package a new lentivirus, and the preparation process is cumbersome and time-consuming Laborious; and the amount of virus entering each cell is limited; in the process of preparing and operating lentivirus, operators need to bear safety risks

Method used

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  • Transposon carrier and system for preparing immortalized cell and usage method thereof
  • Transposon carrier and system for preparing immortalized cell and usage method thereof
  • Transposon carrier and system for preparing immortalized cell and usage method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of vectors pMPH86 and pMPN86

[0082] The workflow for constructing pMPH86 / pMPN86 is as follows Image 6 shown. Specific steps are as follows:

[0083] 1. Preparation of basic plasmid pMP. In the piggyBac plasmid pMPB2 (the plasmid comes from the Molecular Oncology Laboratory, University of Chicago, USA, its map is shown in figure 1 ) on the basis of double enzyme digestion (Spe Ⅰ and Not Ⅰ), blunt end ligation after blunt end, and the plasmid pMP was obtained after screening and identification. pMPH86 / pMPN86 was constructed on the basis of pMP.

[0084] 2. Synthesize FRT-1 DNA single-strands with restriction sites (Kpn Ⅰ and BamH Ⅰ) at both ends, and anneal (95°C, 3 minutes; 25°C, 25 minutes) to form sticky-end double-stranded DNA inserts Fragment, namely Kpn Ⅰ-FRT1-BamH Ⅰ.

[0085] Sense strand: cGAAGTTCCTATTCCGAAGTTCCTATTTCTCTAGAAAGTATAGGAACTTCg

[0086] Antisense strand: tccGAAGTTCCTATAACTTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCggtac

[00...

Embodiment 2

[0104] Example 2 Construction of immortalized mouse embryonic fibroblast cell line using piggyBac immortalized vector

[0105] In order to illustrate the effectiveness of the aforementioned vector tools, this example provides an example of using pMPH86 to create an immortalized cell line of mouse embryonic fibroblasts. A protocol for immortalizing cell lines should be found in Figure 4 the conventional program.

[0106] 1. After the CD1 mice were conceived (the first day of conception was defined as the detection of the vaginal plug), they were separated into cages and raised separately, and were euthanized 12.5-13.5 days later (the neck was broken after 5 minutes in the carbon dioxide box).

[0107] 2. Separate the embryos, rinse with 10 ml of sterile PBS, remove the internal head and neck, limbs, tail and all viscera; fully cut into pieces, and repeatedly sucked 5 times through a No. 18 syringe (BD Company, USA), and added 0.25% trypsin . Incubate at 37°C for 15 minutes;...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a transposon carrier and system for preparing an immortalized cell and a usage method thereof. The transposon carrier and system for preparing the immortalized cell and the usage method thereof aim at solving the technical problem of providing a new choice for preparing the immortalized cell. According to the technical scheme, the piggyBac transposon carrier for preparing the immortalized cell contains an SV40TAg, the two ends of the SV40TAg are provided with FRT sites, and the two ends of a fragment (hygromycin-SV40TAg or neomycin-SV40TAg) which is actually inserted into a genome contain piggyBac transposase splice sites. The invention further provides a host cell containing the carrier and the system for preparing the immortalized cell, and the system comprises the carrier and a piggyBac transposase expression carrier. By adopting the carrier, an immortalized cell system can be established more effectively and quickly.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a transposon carrier, a system and an application method for preparing immortalized cells. Background technique [0002] There are many ways to introduce DNA into cells, including the use of some transfection reagents such as calcium phosphate, polyethylene glycol, liposomes, etc., as well as strategies such as electroporation, or using viruses as media. However, these methods have some disadvantages. For example, the electroporation method is easy to cause pollution during the operation process and cause great damage to the cells; the DNA introduced into the cell by the transfection reagent method, electroporation method or adenovirus method is difficult to integrate into the DNA of the cell itself. ;Although lentivirus or retrovirus can mediate the insertion of exogenous DNA into the cell genome, the length of exogenous DNA is limited; in addition, each ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/85C12N5/10
Inventor 王宁何通川张文文梁后杰
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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