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Parental plasmid for obtaining minicircle DNA, and application thereof

A plasmid and parental technology, applied in the field of parental plasmids to obtain microcircle DNA, can solve the problems of low microcircle DNA acquisition, cumbersome microcircle DNA steps, etc.

Active Publication Date: 2014-12-24
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the technical problem to be solved in the present invention is to provide a method for obtaining the parental plasmid of microcircle DNA and its transgene in view of the defects that the steps of obtaining microcircle DNA by using LR cloning enzyme are relatively cumbersome and the amount of microcircle DNA is low. and application, using the parental plasmid described in the present invention can efficiently, quickly and economically obtain microcircle DNA, and obtain transgenic cell lines efficiently and rapidly

Method used

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  • Parental plasmid for obtaining minicircle DNA, and application thereof
  • Parental plasmid for obtaining minicircle DNA, and application thereof
  • Parental plasmid for obtaining minicircle DNA, and application thereof

Examples

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Effect test

Embodiment 1

[0057] The construction of embodiment 1 parental plasmid vector

[0058] The construction strategy of pEGFP-N1-attB-attR-attL-ΦC31 plasmid described in the present invention is as follows figure 1 As shown, the attR sequence, the attL sequence and the ΦC31 integrase gene were sequentially connected to the pEGFP-N1-attB plasmid.

[0059] 1. Connect the attR fragment

[0060] Primer design: attR-up (containing a SpeI restriction enzyme site), its sequence is shown in SEQ ID NO: 1 in the sequence listing;

[0061] attR-down (containing a DraIII restriction site), its sequence is shown in SEQ ID NO: 2 in the sequence listing.

[0062] The attR double-stranded fragment was obtained by PCR amplification.

[0063] attR and attRrc single chains were synthesized by Invitrogen.

[0064] attR sequence, the sequence of which is shown in SEQ ID NO:3 in the sequence listing (125bp);

[0065] The attRrc sequence is shown in SEQ ID NO: 4 in the sequence listing (125bp).

[0066] PCR rea...

Embodiment 2

[0092] Embodiment 2LR recombination reaction

[0093] The reaction system (10 μl) consists of the following components: DNA (2k8 plasmid prepared in Example 1) 200ng, 10×TE buffer 1μl, LR clonase (LR clonase) 2μl, add ddH 2 0 to 10 μl. You can also use 1×TE buffer to dissolve the plasmid (2k8) during plasmid extraction. At this time, the reaction system (10μl) is: DNA (2k8 plasmid) 200ng, LR clonase (LR clonase) 2μl, add 1×TE buffer to 10 μl.

[0094] Reaction conditions: Incubate at 25°C for 3h to 14h in a PCR instrument. The LR cloning reaction process of the plasmid is as follows: figure 2 shown.

[0095] The LR reaction system was digested with EcoRI restriction enzyme. If the pEGFP-N1-attB-attR-attL-ΦC31 plasmid undergoes LR recombination reaction, two bands of 2.1Kb (minicircle DNA) and 5.2Kb (microplasmid) can be cut out, and if it is the original parental plasmid, it can be cut out Two bands of 1.2Kb and 6.1Kb. The enzyme digestion system is: EcoRI enzyme 1μl, 1...

Embodiment 3

[0097] Example 3 Transfection of HeLa cells

[0098] use GenJet TM Plus DNA In Vitro Tranfection Reagent (SignaGen Laboratories, Cat. No. SL100499) was used to transfect the LR reaction system obtained in Example 2 into HeLa cells in a 12-well plate.

[0099] Transfection steps:

[0100] (1) Use 750 μl of serum-free DMEM (GIBCO, Cat. No. 11995-073) to change the medium for Hela cells.

[0101] (2) Prepare two 1.5ml EP tubes, marked as tube A and tube B respectively. If it is the experimental group, add 38 μl of serum-free DMEM, 10 μl of LR recombination reaction system, and 3 μl of pPGKPhiC31obpA plasmid (200ng / μl) into tube A, and mix by pipetting 3-4 times. If it is a control group, add 38 μl of serum-free DMEM, 1.39 μl of pEGFP-N1-attB plasmid (100 ng / μl), and 3.72 μl of pPGKPhiC31obpA plasmid (200 ng / μl) in tube A, and mix by pipetting 3 to 4 times. Tube B of the experimental group was the same as that of the control group. 38 μl of serum-free DMEM and 4 μl of transfec...

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Abstract

The invention discloses a parental plasmid for obtaining minicircle DNA, and an application thereof. The parental plasmid is constructed by inserting an attR segment and an attL segment and a [Phi]C31 integrase gene expression cassette in a starting vector pEGFP-N1-attB, wherein the attR segment is inserted between cleavage sites of restriction enzymes SpeI and DraIII of the starting vector; the attL segment is inserted in the cleavage sites of restriction enzymes Af1 III and Ase I of the starting vector; and the [Phi]C31 integrase gene expression cassette is inserted in a cleavage site of the restriction enzyme Af1 III of the starting vector. The minicircle DNA for stable integration can be obtained rapidly, efficiently and economically by using the parental plasmid; subsequent transgenosis operational method is simple and practicable; and the parental plasmid has important biology significance and practical application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a parental plasmid for obtaining microcircle DNA and its application. Background technique [0002] Minicircle DNA Vector (Minicircle DNA Vector) is a supercoiled DNA molecule, which is the product of site-specific recombination of parental plasmid (Parental Plasmid, PP). Under the action of recombinase, the parental plasmid is converted into two circular DNAs, one containing a large number of bacterial backbone sequences, called miniplasmid (Miniplasmid, MP); the other is a eukaryotic expression framework containing only the target gene, namely Minicircle DNA (Minicircle, MC). Compared with traditional plasmid vectors, microcircle DNA as a vector has the advantages of good safety, high expression level of the target gene carried, and long duration of transgene expression (Darquet AM, Cameron B, Wils P, et al. A new DNA vehicle for nonviral gene delivery: supercoiled minicircle.Gen...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N1/21A61K48/00C12R1/19
Inventor 马晴雯刘浏曾凡一
Owner SHANGHAI CHILDRENS HOSPITAL
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