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Efficient clone selection expression vector, and preparation method and use thereof

A technology of expressing vectors and vectors, applied in the field of biology, can solve problems such as no relevant reports, and achieve the effect of improving the efficiency of transfection

Active Publication Date: 2014-06-04
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the PB system has a wide range of hosts and high transfection efficiency, and has been widely used in gene therapy in mammalian cells (Molecular Therapy, 2007, 15, 139-145), there has been no relevant report on its in vitro research

Method used

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  • Efficient clone selection expression vector, and preparation method and use thereof
  • Efficient clone selection expression vector, and preparation method and use thereof
  • Efficient clone selection expression vector, and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of pcDNA3.1(+)-ITRs vector

[0024] The construction of pcDNA3.1(+)-ITRs expression vector is as follows: figure 1 As shown, use pc DNA3.1(+) as the carrier, and follow the correct direction of the DNA sequence (see figure 1 ) Insert the ITRs sequence into the vector to obtain the eukaryotic expression vector pcDNA3.1(+)-ITRs containing the ITRs sequence. The expression vector pcDNA3.1(+)-ITRs can carry the mammalian promoter (CMV), the target gene at the multiple cloning site (MCS) and the screening gene (Neomycin) together into the genome of the host cell during transposition middle.

[0025] The specific preparation method is as follows:

[0026] (1) Find out the specific sequences of its 5'ITR and 3'ITR from the commercialized pXL-BacII plasmid vector; find the codon-optimized sequence from the reported literature (Nucleic Acids Research, 2007, 35, e87). The transcriptase (Transposase) gene sequence (GeneBank sequence number: EF587698) that...

Embodiment 2

[0041] Example 2 Preparation of pcDNA4 / TO-ITRs vector

[0042] The construction method of pcDNA4 / TO-ITRs vector is as follows figure 2 As shown, use pc DNA4 / TO as the carrier, follow the correct direction of the DNA sequence (see figure 2 ) Insert the ITRs sequence into the vector to obtain the eukaryotic expression vector pcDNA4 / TO-ITRs containing the ITRs sequence. The expression vector pcDNA4 / TO-ITRs can carry the mammalian promoter (CMV), the target gene at the multiple cloning site (MCS) and the screening gene (Zeocin) together into the genome of the host cell during transposition.

[0043] The specific preparation method is as follows:

[0044] (1) to (3) are the same as in Example 1.

[0045] (4) The expression vector pcDNA4 / TO was digested with NruI, and the expression vector pTriEx2 was digested with EcoRI and HindIII.

[0046] (5) The fragment ITR5 was connected to the expression vector pcDNA4 / TO digested with NruI to obtain a new expression vector ITR5-pcDNA4 / ...

Embodiment 3

[0052] Example 3 Application of pcDNA3.1(+)-ITRs vector in Hela cells

[0053] 1. Experimental method:

[0054] (1) Resuscitate Hela (human cervical cancer cell) cells for transfection, the medium is: RM1640+10% fetal bovine serum+1% Pen Strep.

[0055] (2) One day before transfection, seed Hela cells into a six-well plate at an amount of 4×105 / well. When the cell coverage is 80%-90%, change to medium without any antibiotics.

[0056] (3) Prepare two 1.5ml EP tubes ( 3810 microcentrifuge tube), add the following two combination plasmids respectively:

[0057] Combination 1: 2 μg pcDNA3.1(+)-EGFP

[0058] Combination 2: 1.5 μg pcDNA3.1(+)-ITRs-EGFP and 0.5 μg transcriptase

[0059] Replenish to 100 μl volume with OPTI-MEM medium respectively.

[0060] (4) Carefully add 5 μl of Fugene HD transfection reagent to the above two EP tubes respectively, carefully blow and mix with a pipette tip, and let stand at room temperature for 15 minutes.

[0061] (5) Add the mixed solut...

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Abstract

The invention discloses an efficient clone selection expression vector, which is obtained by an ITRs sequence of a piggyBac transposition system inserted into a mammal expression vector. The invention also discloses a preparation method and use thereof. The efficient clone selection expression vector is built by using the ITRs sequence of the piggyBac transposition system, and meanwhile, a transcriptase gene which is optimized by a codon and can be efficiently expressed in mice is cloned to another eukaryotic expression vector. A selection marker and a target gene are integrated into a genome of a host cell in common by coexpression of an expression vector containing the ITRs sequence and the vector containing transcriptase. Thus, the transfection efficiency and the positive clone screening rate are greatly improved.

Description

technical field [0001] The invention relates to the field of biology, in particular to a high-efficiency cloning and screening expression vector constructed by using the ITRs sequence of the piggyBac transposition system, the construction method of the vector and its application. Background technique [0002] A transposon is a mobile DNA sequence in the genome that can "jump" from one location of the genome to another through a series of processes such as cutting and reintegration. Transposons account for more than 40% of human and mouse genome sequences (Nature, 2001, 409, 860–921; Nature, 2002, 420, 520–562). [0003] Since McClintock discovered the first transposon from maize (Proc. Natl. Acad. Sci. 1950, USA36, 344–345), transposable elements have become valuable genetic analysis tools for many organisms. In prokaryotes, mutation studies using transposons have uncovered genes that play important roles in pathogenicity of harmful microorganisms (Science, 1999, 286, 2165–...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/64
Inventor 楼庄伟孔云华梅佳贾园陈侃宋云鹏吕强
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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