Plasmid vector for building overexpression stable system on basis of transposase and application thereof

A plasmid vector and overexpression technology, applied in the field of genetic engineering, can solve problems such as time-consuming, time-consuming, and affecting the experimental progress of scientific researchers

Inactive Publication Date: 2017-11-28
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the construction of stable cell lines is mainly achieved through virus packaging and infection of target cells. This method takes a long time, usually about two weeks
In addition, since the size of the inserted target gene fragment is limited by the size of the viral plasmid itself, the size of the target gene fragment usually cannot exceed 3Kb, and the operator has potential safety hazards of virus infection
At the same time, overexpression plasmid vectors are usually constructed by inserting into multiple cloning sites. This method is not only inefficient, but also takes a long time, which greatly affects the experimental progress of researchers.

Method used

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  • Plasmid vector for building overexpression stable system on basis of transposase and application thereof
  • Plasmid vector for building overexpression stable system on basis of transposase and application thereof
  • Plasmid vector for building overexpression stable system on basis of transposase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0074] This example is used to study the construction scheme of the pSM1.1 series of vectors. Since the construction methods of the pSM1.1 series of vectors are basically the same, now select pSM1.1-HA-Puro-GFP, pSM1.1-HA-Puro, pSM1.1 -Flag-Puro-GFP these three plasmid vector construction schemes represent the construction method of pSM1.1 series vectors.

[0075] 1.1 pSM1.1-HA-Puro-GFP plasmid construction

[0076] Step 1: Using pLenti PGK Puro DEST plasmid as template, F1 and R1 as primers, PCR amplification of the Gateway cassette-terminator fragment of the plasmid, using pLenti PGK Puro DEST plasmid as template, F2 and R2 as primers, PCR amplification The fusion fragment of the PGK Promoter and Puro resistance gene of the augmentation plasmid is gelled to recover the above PCR product fragment, and then a round of recombination fusion PCR is performed using the Gateway cassette-terminator fragment and the fusion fragment of the PGK Promoter and Puro resistance gene as a templat...

Embodiment 2

[0086] pSM1.1-HA-Puro-GFP is the original plasmid successfully constructed by the inventor. The other series of vector plasmids are all based on the original plasmid. By only changing the tag sequence and screening gene GFP, other plasmid elements remain unchanged, so as to be modified to contain different tag sequences. A series of plasmid vectors containing the screening gene GFP. This example is used to study this original plasmid pSM1.1-HA-Puro-GFP to express the foreign gene embodiment.

[0087] 1.1 pSM1.1-HA-FBXW7-Puro-GFP plasmid construction

[0088] Using HeLa cell cDNA as a template, F11 and R11 as primers, PCR amplified the FBXW7 gene (Gene Bank accession number: NM_001349798.1), and running gel confirmed that the FBXW7 gene was specifically amplified ( Figure 5 A). The PCR product is recovered. The recovered PCR product is recombined into the pDONR221 entry vector through the BP reaction, and transformed into the super competent bacteria OmniMAX2-T1, painted Kana + Pl...

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Abstract

The invention discloses a series of plasmid vector for building an overexpression stable system on the basis of transposase and application thereof. The plasmid vector is a double-chain annular plasmid containing an IRDR-L-IRDR-R kit; the IRDR-L-IRDR-R kit comprises an IRDR-L sequence, a promotor, a tag sequence, an attR1 sequence, a reverse selective tag gene sequence ccdB, an attR2sequence, a screening gene sequence and an IRDR-R sequence; the tag is an HA tag, an Myc tag, a Flag tag or a V5 tag. The plasmid vector has a Sleeping Beauty transposon tail end reverse repeated sequence and a Gateway Cassette sequence; the overexpression stable system can be fast and efficiently built on the basis of the transposase method; various different tag sequences Tag and screening genes GFP exist, so that the conventional requirement of expressing the target gene in eukaryotic cells can be met.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to the construction of plasmid vectors using Gateway cloning technology. The series of plasmid vectors carry different tag sequences and the screening gene GFP, and integrate the target gene into the cell through the transposon system. Genome, realizing gene transfer. In particular, it relates to a plasmid vector for establishing an overexpression stable line based on transposase and its application Background technique [0002] Overexpression stable cell line refers to the integration of foreign DNA into the host cell genome, allowing the host cell to express the target gene for a long time. Compared with the transient strain, the stable cell line can greatly facilitate experimental research and reduce experimental costs. Therefore, the construction of stable gene overexpression strains is widely used in the study of gene function and mechanism. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N5/10
CPCC07K2319/00C07K2319/41C07K2319/42C07K2319/43C07K2319/60C12N15/85C12N2800/90C12N2830/34
Inventor 尹东胡开顺陈恒星李瑜
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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