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Efficient and safe transposable element integration system and application thereof

A technology of transposon and transposase, applied in the field of molecular biology, can solve the problems of limited loading capacity, low integration efficiency, complicated preparation process of recombinant virus particles, etc.

Active Publication Date: 2015-12-16
SHANGHAI CELL THERAPY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Although the host cells have undergone multiple passages or conditional changes, the expression level remains stable
[0004] In order to achieve stable expression of exogenous genes in host cells, commonly used vector systems include: 1. Retrovirus system: can effectively infect host cells and mediate the efficient integration of exogenous gene expression cassettes into the genome, but its loading capacity is limited , and the preparation process of recombinant virus particles is complex
2. Eukaryotic expression plasmid system: The preparation process is relatively simple, but it is inserted into the host genome through random DNA recombination, and the integration efficiency is extremely low
3. Transposon system: the plasmid system is used, the preparation process is relatively simple, and the foreign gene is integrated into the genome by transposase, and the integration efficiency is relatively low
The coding genes mentioned here are relative to non-coding genes, that is, genes that can encode and produce corresponding functional proteins; if the tumor-related genes are inserted inactivated or abnormally activated, there may be a risk of carcinogenesis

Method used

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  • Efficient and safe transposable element integration system and application thereof
  • Efficient and safe transposable element integration system and application thereof
  • Efficient and safe transposable element integration system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1: Construction of pNB vector

[0120] According to the sequence of PiggyBac transposon 5' terminal repeat (SEQ ID NO: 1), polyclonal insertion site (SEQ ID NO: 2), polyA tailing signal sequence (SEQ ID NO: 3), PiggyBac transposon 3' terminal repeat (SEQ ID NO :4), PiggyBac transposase coding sequence (SEQIDNO:5) containing c-myc nuclear localization signal, CMV promoter sequence (SEQIDNO:6), spliced ​​into a long sequence (SEQIDNO:7), wherein contains c-myc The PiggyBac transposase coding sequence of the nuclear localization signal and the reverse complement of the CMV promoter sequence sequence (reverse complement here means that the expression frame of the exogenous gene is in the opposite direction to the expression frame of the PB gene, so the PiggyBac transposase coding sequence is displayed. Sequence, reverse complementary sequence of CMV promoter sequence), entrusted Shanghai Jereh Biotechnology Co., Ltd. to synthesize, and added AscI and PacI restr...

Embodiment 2

[0121] Embodiment 2: Construction of the pNB vector containing exogenous gene expression cassette

[0122] 1. According to the sequence of the EF1α promoter, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, add XbaI and EcoRI restriction sites at both ends, load the pNB vector prepared in the previous example 1, and name it pNB328 vector.

[0123] The EF1α promoter sequence is shown in SEQ ID NO:8.

[0124] 2. According to the coding sequence of EGFP, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, and add EcoRI and SalI restriction sites at both ends, load it into pNB328 vector, and name it pNB328-EGFP vector.

[0125] The EGFP coding sequence is shown in SEQ ID NO:9.

[0126] 3. According to the Luc luciferase coding sequence, entrust Shanghai Jereh Biotechnology Co., Ltd. to synthesize it, and add EcoRI and SalI restriction sites at both ends, load it into pNB328 vector, and name it pNB328-Luc vector.

[0127] The coding sequence of Luc ...

Embodiment 3

[0130] Example 3: Expression time curve of PB after transfection of Jurkat cells with pNB328 vector analyze

[0131] Prepare 5×10 6 The low-generation Jurkat (purchased from the American Standard Biological Collection, ATCC) in good growth state was respectively mixed with 6 μg of pNB328 and PB210PA-1 (providing the expression of PB transposase) through the Lonza2b-Nucleofector instrument (performed according to the instrument operation manual). Plasmid (purchased from SystemBioscience) was transfected into the nucleus, and placed at 37°C, 5% CO 2 Incubator culture. At 6, 12, 24, 48, 96 hours, and 15 days after transfection, RNA was extracted, and the relative expression of PB transposase was detected by RT-PCR. β-actin was used as an internal reference, and the specific primers were as follows:

[0132] PB-F: such as SEQ ID NO: 12, PB-R: such as SEQ ID NO: 13;

[0133] Actin-F: such as SEQ ID NO: 14, Actin-R: such as SEQ ID NO: 15.

[0134] The results showed that in...

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Abstract

The invention belongs to the field of molecular biology, relates to an efficient and safe transposable element integration system and application thereof and further relates to a nucleic acid construction body and application thereof. The nucleic acid construction body concretely and sequentially comprises the following elements of a transposable element 5' terminal repetition sequence, a polyclone insertion site, a poly A tailing signal sequence, a transposable element 3' terminal repetition sequence, a transposase coding sequence and a promoter controlling the expression of transposase. The polyclone insertion site is used for inserting an exogenous gene coding sequence and a selective promoter for controlling the expression of exogenous genes in an operability mode. The poly A tailing signal sequence has a poly A tailing signal function in the forward and reverse directions, and the direction of an expression cassette of transposase is reverse to that of an exogenous gene expression cassette. The nucleic acid construction body can be used for efficiently and safely expressing mediated exogenous genes in host cells.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an efficient and safe transposon integration system and its application. The present invention also relates to a nucleic acid construct and its use. The nucleic acid construct can be used to mediate the efficient integration and stable expression of exogenous genes in host cells, and the integration sites are mainly concentrated in the three intergenic segments in the host cell genome, which can largely avoid Risks arising from random insertion. The present invention also relates to recombinant vectors and recombinant host cells containing the nucleic acid construct. Background technique [0002] The expression forms of exogenous genes in host cells can be divided into transient expression and stable expression, among which stable expression refers to: (1) expression after exogenous genes are transfected into eukaryotic cells and integrated into the genome. The stable expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/86C12N5/10
CPCA61K39/0011A61K2039/5156A61K2039/5158C07K14/7051C07K2319/03C12N15/52C12N15/85C12N2015/8518C12N5/16C12N15/113C12N2800/107
Inventor 钱其军金华君李林芳刘韬左明辉吴红平吴孟超
Owner SHANGHAI CELL THERAPY RES INST
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