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Establishment and application of gene system capable of inducing high-efficiency expression in human pluripotent stem cells

A high-efficiency expression technology of human pluripotent stem cells, which is applied in the establishment and application of an inducible high-efficiency expression gene system, can solve the problems of low efficiency of exogenous genes, and achieve high survival rate and proliferation efficiency, low cost, and convenient operation Effect

Inactive Publication Date: 2020-11-27
WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the inadequacies of existing technologies such as the low efficiency of overexpressing foreign genes in pluripotent stem cells, and provide a gene system that can induce high-efficiency expression in human pluripotent stem cells. The system has the following advantages: stable system; high-efficiency expression of specific exogenous genes in human pluripotent stem cells; overexpression of exogenous genes can be induced at a specific time, etc.

Method used

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  • Establishment and application of gene system capable of inducing high-efficiency expression in human pluripotent stem cells
  • Establishment and application of gene system capable of inducing high-efficiency expression in human pluripotent stem cells
  • Establishment and application of gene system capable of inducing high-efficiency expression in human pluripotent stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Induced high expression of human VGLL4 gene in human embryonic stem cells.

[0050] (1) Construction of PB-TRE6P-P2A-EGFP vector plasmid:

[0051] Step 1: Design P2A-EGFP-specific seamless cloning primers based on the P2A-EGFP gene CDS sequence and the MCS site of the plasmid PB-TRE6P. The sequence of the P2A-EGFP forward primer is shown in SEQ ID: 1, which contains enzyme cutting sites: Hind III, BamH I, EcoR I; the sequence of the P2A-EGFP reverse primer is shown in SEQ ID: 2, which contains Restriction site Sal I.

[0052] Step 2: PCR amplification.

[0053] Using Polymerase Phusion TM High-Fidelity DNA Polymerase (Thermo Fisher, Cat#F530S) was used to amplify the P2A-EGFP gene by PCR. The amplification reaction system was 50ul. After the reaction, 10ul of the PCR product was purified to obtain the P2A-EGFP gene fragment.

[0054] Step 3: Carry out digestion and ligation of DNA fragments by conventional molecular cloning methods.

[0055] Digest the PB-TRE6P pla...

Embodiment 2

[0068] As described in Example 1, during the process of inducing the expression of the foreign gene VGLL4, DOX was not added to prevent the expression of the foreign gene VGLL4.

[0069] In another embodiment, after the overexpression of DOX is induced, DOX can be withdrawn at any time as needed.

[0070] In another embodiment, after DOX is withdrawn, if overexpression is required, DOX is added at any time to induce the expression of the exogenous gene VGLL4.

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Abstract

The invention discloses establishment of a gene system capable of inducing high-efficiency expression in human pluripotent stem cells. The system is specifically based on a PiggyBac transposition subsystem. A PiggyBac transposon plasmid containing an exogenous gene is rapidly constructed in vitro in a seamless cloning manner, and co-transfects the human pluripotent stem cells with PB200PA plasmidscontaining transposase genes, so that a human pluripotent stem cell model capable of efficiently expressing exogenous genes through DOX induction is established. The method is simple and rapid, the constructed gene system is stable, the specific exogenous gene can be efficiently expressed in the human pluripotent stem cells, the exogenous gene overexpression can be induced in specific time, and the gene system can be widely applied to related gene research of the pluripotent stem cell.

Description

technical field [0001] The invention relates to the establishment technology of an expression gene system, in particular to the establishment and application of an inducible high-efficiency expression gene system in human pluripotent stem cells. Background technique [0002] Human pluripotent stem cells have strong self-renewal and self-replication capabilities, and can differentiate into all cells in the body, forming all tissues and organs in the human body. The cultivation of human pluripotent stem cells in vitro provides a powerful impetus for research in organ regeneration, repair and disease treatment. It has great application prospects in basic research on factors affecting proliferation, differentiation, and migration regulation, clinical research on cell transplantation and gene carrier therapy, and mechanism research in the process of transplantation treatment of diseases. [0003] In recent years, due to species differences, there are great differences between an...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90
CPCC12N15/85C12N15/90C12N2800/107C12N2800/90C12N2830/002
Inventor 王永煜权颖怡
Owner WENZHOU MEDICAL UNIV
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