A ps transposon system and its mediated gene transfer method

A subsystem and transgenic technology, applied in biochemical equipment and methods, microorganisms, nucleic acid vectors, etc., can solve the problem that DNA transposons are rare in vertebrates, and achieve the effect of improving gene transfer efficiency

Active Publication Date: 2022-07-15
SHANGHAI CELL THERAPY GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The DNA transposon-mediated gene transfer system is a transgenic technology favored by scientists, but DNA transposons with autonomous transposition activity are rare in vertebrates

Method used

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  • A ps transposon system and its mediated gene transfer method
  • A ps transposon system and its mediated gene transfer method
  • A ps transposon system and its mediated gene transfer method

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Experimental program
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Effect test

Embodiment I

[0044] Embodiment 1, the construction of transposase expression vector pcDNA3.9-PSase

[0045] 1. Synthesis of transposase PSase vector.

[0046] After the PSase transposase sequence was chemically synthesized, it was cloned into the LB vector to construct the pLB-PSase vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (eg figure 1 ).

[0047] 2. Construction of transposase eukaryotic expression plasmid and in vitro transcription helper plasmid pcDNA3.9-PSase

[0048] The transposase CDS was excised from the pLB-PSase vector with the restriction enzymes BamHI and XhoI, and the 1296bp CDS sequence was recovered from agarose gel. At the same time, the pcDNA3.9 vector was digested with BamHI and XhoI, and the 3.9kb fragment was recovered by cutting the gel as the vector frame. After linking the transposase CDS and pcDNA3.9 vector frame, transform competent cells Top10, pick a single colony into liquid medium LA for culture, and extract the plasmid for ...

Embodiment II

[0050] Embodiment II, the construction of transposon expression vector

[0051] 1. Construction of transgenic donor plasmid pLB-PS vector

[0052] 1.1 Synthesis of two terminal inverted repeat elements of PS (PS 3' and 5')

[0053] The insertion age of PS transposons in the Tc1 / Marinier family was analyzed for multiple species, and molecular reconstruction was carried out on the basis of phylogenetic comparative studies, and highly conserved 5' and 3' TIRs were identified, which were 28 bp transposons, respectively. to the complementary sequence. Add the restriction endonuclease site to this sequence and send it to Huada Gene Company for synthesis. The sequence is shown in Table 1.

[0054] 1.2 Polymerase Amplification of PS Transposable Elements

[0055] The above chemically synthesized single-stranded nucleotide chain PS5't (including the inverted repeat element at the end of PS5' and an enzyme cleavage site, the enzyme cleavage site is used to insert the target gene) and...

Embodiment III

[0070] Embodiment III, PS transposon mediates target gene expression test at mouse cell level

[0071] 1. Recovery and culture of cryopreserved cells

[0072] The pPS-PGK-NEO and pcDNA3.9-PSase plasmids were extracted with OMEGA endotoxin-free plasmid extraction kit (purchased from OMEGA company), and the final concentration of the products was adjusted to 500ng / ul for cell transfection.

[0073]Take out the cryopreservation tube containing human cervical cancer cells (Hela) from liquid nitrogen (cells stored in our laboratory), and immediately put it into warm water at 37-40°C and shake quickly until the cryopreservation solution is completely thawed; after 1-2min Complete rewarming within 10 minutes; transfer the cell suspension to a sterile centrifuge tube, add 5 mL of culture medium, and blow gently; centrifuge the cell suspension at 800-1000 rpm for 5 min, discard the supernatant; add 1 mL of complete cell pellet to the centrifuge tube containing the cell pellet The medi...

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Abstract

The invention discloses a PS transposon system and a gene transfer method mediated by it, comprising a transgenic donor plasmid that can be inserted into two terminal repeat sequences of a target gene cassette and a transposase auxiliary plasmid that provides transposition activity. The target gene is introduced into the recipient genome through the PS transposon system. The gene transfer method based on the PS transposon system provided by the present invention can efficiently mediate gene transfer after verification at the cell and embryo levels, and can be applied to many biotechnological fields: 1) Using the method provided in the present invention can effectively Inserting the target gene cassette into the host genome can improve the efficiency of gene transfer; 2) Combined with gene capture technology, it can effectively carry out animal gene function research; 3) It can mediate human gene therapy, etc.

Description

technical field [0001] The invention relates to the establishment of a gene transfer method mediated by a PS transposon system, and also discloses a method for constructing a transgenic donor plasmid and a transposase eukaryotic expression and in vitro transcription auxiliary plasmid involved in the method, and the preparation method thereof. Transgenic animals, studies of gene function and applications in human gene therapy. The invention belongs to the field of animal genetic engineering. Background technique [0002] Gene transfer is an important biotechnological method at present, which can mediate the stable integration of foreign genes into the host chromosome, so it has important application value in the fields of studying gene function, preparing transgenic organisms and human gene therapy. [0003] Transposon is a DNA sequence that can jump freely on the genome. It was first discovered in the corn chromosome by Mc.Clintock in the 1940s, and later in various organis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/90C12N2800/107C12N2800/106
Inventor 宋成义王赛赛高波宗文成沈丹王亚丽产舒恒桑亚通
Owner SHANGHAI CELL THERAPY GRP CO LTD
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