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PS transposon system and mediated gene transfer method thereof

A subsystem and transgenic technology, applied in the fields of 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 the efficiency of gene transfer

Active Publication Date: 2019-09-20
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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  • PS transposon system and mediated gene transfer method thereof
  • PS transposon system and mediated gene transfer method thereof
  • PS transposon system and mediated gene transfer method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment I

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

[0045] 1. Synthesis of transposase PSase carrier.

[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 (such as figure 1 ).

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

[0048] The transposase CDS was excised from the pLB-PSase vector with restriction endonucleases 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 3.9 kb fragment was recovered as the vector frame by gel cutting. After linking the transposase CDS and the pcDNA3.9 vector framework, transform the competent cell Top10, pick a single colony and culture it in liquid medium LA, and extr...

Embodiment II

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

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

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

[0053] The insertion age of the PS transposon in the Tc1 / Marinier family was analyzed in multiple species, and the molecular reconstruction was carried out on the basis of the comparative study of phylogenetic evolution. to the complementary sequence. The sequence was added with restriction endonuclease sites and sent 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-mentioned chemically synthesized single-stranded nucleotide chain PS5't (including the inverted repeat element at the PS5' end and a restriction site for inserting the target gene) and PS3't (including the PS3' end Inverted repeat element and restriction site, the restriction site is us...

Embodiment III

[0070] Example III, PS transposon-mediated 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 an 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 the liquid nitrogen (cells stored in our laboratory), immediately put it into warm water at 37-40°C and shake it quickly until the cryopreservation solution is completely thawed; in 1-2min Complete the rewarming within the chamber; transfer the cell suspension into a sterile centrifuge tube, add 5mL of culture medium, and blow evenly; centrifuge the cell suspension at 800-1000rpm for 5min, discard the supernatant; add 1mL of complete Gently blow the culture medium, transfer the cell suspension into a...

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Abstract

The invention discloses a PS transposon system and a mediated gene transfer method thereof, wherein the PS transposon system includes a transgenic donor plasmid capable of being inserted into a target gene cassette and having two terminal repeat sequences and a transposase assisted plasmid providing transposition activity. A target gene is introduced into a receptor genome through the PS transposon system. The gene transfer method based on the PS transposon system can efficiently mediate gene transfer through cell and embryo level verification, and can be applied in many biotechnological fields: 1) the target gene cassette can be inserted into the host genome effectively by using the method provided by the invention, and the efficiency of gene transfer is improved; 2) the study on animal gene functions can be effectively developed by combination of a gene capture technology; and 3) human gene therapy can be medicated to carry out.

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 transgene donor plasmid and a transposase eukaryotic expression and in vitro transcription auxiliary plasmid involved in the method, and its preparation Applications in transgenic animals, studying 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, which can mediate the stable integration of exogenous 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 by Mc.Clintock in the chromosome of maize in the 1940s, and later found in various organisms such as...

Claims

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Application Information

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Patent Type & Authority Applications(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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