ZB (zebrafish) transposon system and gene transfer method mediated by same

A subsystem and transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem that DNA transposons are rare in vertebrates, and achieve the effect of improving the efficiency of gene transfer

Active Publication Date: 2015-11-04
SHANGHAI CELL THERAPY GRP CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0005] DNA transposon-mediated gene transfer system is a new generation of transgenic technology, but DNA transposons with autonomous transposition activity are rare in vertebrates

Method used

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  • ZB (zebrafish) transposon system and gene transfer method mediated by same
  • ZB (zebrafish) transposon system and gene transfer method mediated by same
  • ZB (zebrafish) transposon system and gene transfer method mediated by same

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Experimental program
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Effect test

Embodiment I

[0035] The construction of embodiment 1, transposase expression vector pCMV-ZB and transposase in vitro transcription vector pTNT-ZB

[0036] 1. Cloning of ZB transposase coding region (ORF)

[0037] Using zebrafish genomic DNA as a template, the transposase gene fragment was amplified according to the primers listed in Table 1 for amplifying ZB-ORF. The reaction system was 50 μl, 10 μL 5×SF Buffer, 1 μl dNTP, 2 μL 10 μM primer ZB-ORF F, 2 μl 10 μM primer ZB-ORF R (primer sequence: ZB-ORF-F: ttCTCGAGACTAGTgccaccatgatgggtaaaaacaaagaactc (SEQ ID NO.5); ZB- ORF-R: atGGTACCGGGCCCttaatactttgtagaaaagcctt (SEQ ID NO. 6)), 1 μl Phanta TM Super-Fidelity, 1 μl zebrafish genomic DNA template, add ultrapure water to 50 μl (high-fidelity enzyme purchased from Novozyme). PCR amplification program: 94°C for 40s, 55°C for 40s, 72°C for 1m30s, cycle 30 times. PCR amplification products were detected by 1% agarose gel electrophoresis (such as figure 2 ). The agarose gel of the PCR produc...

Embodiment II

[0042] Embodiment II, the construction of transposon expression vector

[0043] 1. Construction of transgene donor plasmid pZB-Msc vector

[0044] 1.1 Construction of pT3-PST vector

[0045] PB [2] , SB [1] and Tol2 [3] Plasmid vector as template, clone 5' and 3' transposable elements, primer sequence:

[0046] SBF: ATGgatccattaaatggcgcgccgtttaaaccagttgaagtcggaagttta (SEQ ID NO. 7);

[0047] SBR: GAGgatccattaaatggccggccttaattaacagttgaagtcggaagttta (SEQ ID NO. 8);

[0048] 5'PBF: atggcgcgccttaaccctagaaagatagtctg (SEQ ID NO. 9);

[0049] 5' PBR: acgtttaaac tgatatctataacaagaaaatata (SEQ ID NO. 10);

[0050] 3' PBF: gcttaattaagtttaaactaaaagttttgttactttatagaag (SEQ ID NO. 11);

[0051] 3' PBR: atggccggccttaaccctagaaagataatcatattg (SEQ ID NO. 12);

[0052] 5' Tol2F: CGAAGCTTAGGCCTCAGAGGTGTAAAGTACTTGAGTA (SEQ ID NO. 13);

[0053] 5' Tol2R: GCTTGAATTCCCCGGGGCTAGCAAGTGATCTCCAAAAAATAAGTAC (SEQ ID NO. 14);

[0054] 3' Tol2: GATGGCCATCGCGAGCTAGCAATACTCAAGTACAATTTTAATGGAG (SEQ I...

Embodiment III

[0087] Example III, ZB transposon-mediated target gene expression test at mouse cell level

[0088] 1. Recovery and culture of cryopreserved cells

[0089] The pZB-PGK-NEO and pCMV-ZB plasmids were extracted with the OMEGA endotoxin-free plasmid extraction kit (purchased from OMEGA Company), and the final concentration of the product was adjusted to 500 ng / μL for cell transfection.

[0090] Take out the cryopreservation vials containing mouse embryonic fibroblast MEF (purchased from the Cell Bank of Chinese Academy of Sciences), mouse C2C12 cells and pig PK15 cells from liquid nitrogen, put them into warm water at 37-40°C and shake them quickly until frozen. Completely melt the stock solution; complete rewarming within 1-2min; transfer the cell suspension into a sterile centrifuge tube, add 5mL culture medium, and blow gently; centrifuge the cell suspension at 800-1000rpm for 5min, discard the supernatant; Add 1mL of complete medium to the centrifuge tube containing the cell ...

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Abstract

The invention belongs to the field of animal genetic engineering, and relates to a ZB (zebrafish) transposon system of and a gene transfer method mediated by the ZB transposon system. The ZB transposon system is derived from zebra fish, and comprises two transgenic donor plasmids with terminal repetition sequences and capable of being inserted into a target gene box, and transposase auxiliary plasmids for providing activity of transposition; the transgenic donor plasmids comprise terminal repetition sequences and Msc1 insertion cloning sites at two sides of ZB transposon. The invention further discloses a gene transfer method based on the ZB transposon system, which is characterized that target genes are guided into a receptor genome through the ZB transposon system. The system and the method can be applied to multiple field of biotechnology: (1) the method can be used for effectively inserting the target gene box into a host cell genome, so that the gene transfer efficiency is improved; (2) by combining with the gene trapping technology, the researches on functions of animal genes can be effectively developed; (3) human gene therapy can be realized through mediation.

Description

technical field [0001] The invention relates to the establishment of a transgenic method mediated by the zebrafish ZB transposon system, and also discloses a method for constructing a transgene donor plasmid and a transposase eukaryotic expression and in vitro transcription auxiliary plasmid involved in the method, and Application in preparation of transgenic animals, research on gene function and human gene therapy. The invention belongs to the field of animal genetic engineering. Background technique [0002] Gene transfer is an important biotechnology method at present, and has important application value in the fields of studying gene function, preparing transgenic organisms and gene therapy. [0003] Transposons are a type of mobile DNA sequences that can freely jump (replicate or translocate) on the genome. They were first discovered by Mc. found in species. Scientists have developed and perfected the method of transposon-mediated cytoplasmic microinjection by takin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/54
Inventor 高波沈丹宋成义钱跃薛松磊王赛赛
Owner SHANGHAI CELL THERAPY GRP CO LTD
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