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Human embryonic stem cell line capable of controlling gene expression and application thereof

A technology for embryonic stem cells and gene regulation, applied to cells modified by introducing foreign genetic material, genetic engineering, using vectors to introduce foreign genetic material, etc., to achieve high-efficiency gene knock-in and reduce difficulty

Inactive Publication Date: 2018-09-21
SOUTHERN MEDICAL UNIVERSITY
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Problems solved by technology

[0005] P300 protein has histone acetyltransferase activity, which can restructure chromatin structure, expose gene promoters and enhancers to transcription factors, and regulate gene expression. It has been reported that the gene activation effect of p300 protein is better than VP64 and other similar functional proteins, but its application in the field of gene modification is still less than that of traditional acting factors such as VP64

Method used

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  • Human embryonic stem cell line capable of controlling gene expression and application thereof
  • Human embryonic stem cell line capable of controlling gene expression and application thereof
  • Human embryonic stem cell line capable of controlling gene expression and application thereof

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Experimental program
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Effect test

Embodiment

[0046] pBLuSKP-AAVS1-AAVS1 plasmid ( figure 2 )Construct:

[0047] The base sequences OLIGO-F and OLIGO-R of gAAVS1-HindIII-gAAVS1 were designed and synthesized, as shown in SEQ ID NO:1-2.

[0048] Two partially complementary paired single-stranded DNA fragments synthesize double-stranded DNA fragments, and the specific steps are as follows:

[0049] 10ul 100uM Oligo-F and 10ul 100uM Oligo-R were pre-mixed in 1.5ml EP tubes, boiled 800ml of distilled water in a beaker, put the 1.5ml EP tubes in boiling water for 5 minutes, took out the 1.5ml EP tubes and left them at room temperature overnight, and washed them with XholI +NotI restriction endonuclease cut (see below) pBLuSKP plasmid ( figure 1 ), use 0.8% agarose gel electrophoresis to analyze the digestion product, cut the gel to recover the 2888bp band, and use NanoDrop to measure the concentration of the recovered fragment, and connect the linearized pBLuSKP plasmid and the above-mentioned double-stranded DNA fragment by...

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Abstract

The invention discloses a human embryonic stem cell line capable of controlling gene expression and application thereof. The human embryonic stem cell line capable of controlling gene expression comprises a controlled expression exogenous fusion protein system and at least one guide RNA, and the controlled expression exogenous fusion protein system comprises a tetracycline inducible expression system and fusion protein formed by Cas9 protein and p300 core domain with transcriptional activation activity; each guide RNA is a 14bp short data sequence of a targeting gene promoter region or a 20bplong data sequence of a genome region. Accordingly, expression of Cas9-p300 in human embryonic stem cells can be controlled in real time, the specific gene activation and cutting double functions of human embryonic stem cell differentiation in different stages are achieved, and application of the human embryonic stem cells in the fields of medicine science and lift science is greatly extended.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and relates to a Cas9-p300 human embryonic stem cell line with a controllable activation and cutting dual system mediated by CRISPR / Cas9, a construction method and application thereof. Background technique [0002] Human embryonic stem cells are pluripotent cells, derived from the inner cell mass of early embryos (4-5d), with the potential of self-renewal and multilineage differentiation. In 1998, American scientist James Thomson successfully cultured human embryonic stem cells in vitro for the first time, and the scientific research and clinical application of human embryonic stem cells also began. In current research, embryonic stem cells have successfully differentiated into trophoblast cells, nerve cells, liver cells, chondrocytes, hematopoietic cells, cardiomyocytes and other cells, and have also successfully established individual development models. Clinical treatments...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/63C12N15/90
CPCC12N15/113C12N15/63C12N15/907C12N2310/10
Inventor 荣知立林瑛马淑凤
Owner SOUTHERN MEDICAL UNIVERSITY
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