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A chromatin remodeling-based approach to activate expression of the endogenous PDX1 gene

A gene expression and endogenous technology, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problem that cells do not have physiological functions

Active Publication Date: 2022-01-04
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current method of exogenous gene overexpression is difficult to activate the expression of endogenous related genes, therefore, the differentiated cells do not have mature physiological functions

Method used

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  • A chromatin remodeling-based approach to activate expression of the endogenous PDX1 gene
  • A chromatin remodeling-based approach to activate expression of the endogenous PDX1 gene
  • A chromatin remodeling-based approach to activate expression of the endogenous PDX1 gene

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Experimental program
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Embodiment 1

[0038] Example 1. Activation of endogenous Pdx1 gene expression based on chromatin remodeling

[0039] 1. Materials and methods

[0040] (1) CRISPR vector

[0041] Purchase lenti dCAS-VP64_Blast (hereinafter referred to as dCAS-VP64), lenti MS2-P65-HSF1_Hygro (hereinafter referred to as MPH) and lenti sgRNA(MS2)_zeo backbone from Addgene. The Addgene IDs of the three vectors are 61425, 61426 and 61427, respectively.

[0042] (2) Lentiviral packaging plasmids PMD2.G and PsPax2 (Addgene), 293T cells (Invitrogen), rat insulinoma cells INS-1 cells (AddexBio).

[0043] (3) Design and vector construction of promoter-specific binding sgRNA

[0044] 1. sgRNA design

[0045] Five sgRNAs ( figure 1 ). The target sequences of sgRNAs are listed in Table 1. The sgRNA target sequence oligonucleotides were synthesized by BGI.

[0046] Table 1 Targeting sequence and position of sgRNA on Pdx1 promoter

[0047] sgRNA Target sequence (5'-3') PAM chain Position from TSS...

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Abstract

The invention discloses a method for activating endogenous Pdx1 gene expression based on chromatin remodeling. The present invention provides a method for activating endogenous Pdx1 gene expression in target cells, which is a method for establishing and activating endogenous Pdx1 gene expression in target cells based on the CRISPR-on SAM system. The SAM system includes an sgRNA targeting the position from -400 to +1bp upstream of the transcription start point of the Pdx1 gene, and the target sequence of the sgRNA is SEQ ID No.3 and / or SEQ ID No.4. The present invention applies CRISPR-on technology to efficiently activate the expression of Pdx1 in 293T cells through chromatin remodeling. This study will play an important role in gene-induced differentiation of PSCs into β cells and embryonic development of pancreas.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for activating endogenous Pdx1 gene expression based on chromatin remodeling. Background technique [0002] The incidence of diabetes is increasing significantly year by year. According to the 2017 WHO report, the current global incidence of diabetes is about 8.5%, and there are about 422 million diabetic patients. Type 1 diabetes is caused by an autoimmune attack on the beta cells of the pancreas. When the number of remaining islet β cells is less than 10-20% of the total number of islet cells, clinical symptoms will appear [Gillespie, K.M.Type 1diabetes:pathogenesis and prevention. CMAJ 175,165-170, doi:10.1503 / cmaj.060244(2006). ]. Type 2 diabetes is due to decreased cell proliferation and increased apoptosis resulting in abnormal β-cell function and progressive cell reduction [Joost, H.G. Pathogenesis, risk assessment and prevention of type 2diabetes mellitus. 000137...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10C12N5/071
CPCC12N5/0676C12N15/86C12N2310/20C12N2506/45C12N2740/15043
Inventor 王启伟叶华虎
Owner ACADEMY OF MILITARY MEDICAL SCI