Method for activating gene expression of endogenous Ngn3 and MAFA

A gene expression, endogenous technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problem that β cells do not have physiological functions.

Active Publication Date: 2018-12-21
ACADEMY OF MILITARY MEDICAL SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, conventional gene expression methods such as various viruses, liposomes, electroporation and mRNA are difficult to effectively activate the expression of endogenous genes [Miyazaki, S., Yamato, E. & Miyazaki, J. Regulated expression ofpdx- 1promotes in vitro differentiation of insulin-producing cells from embryonic stem cells.Diabetes 53,1030-1037(2004).], therefore, the β cells obtained in vitro do not have mature physiological functions

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  • Method for activating gene expression of endogenous Ngn3 and MAFA
  • Method for activating gene expression of endogenous Ngn3 and MAFA
  • Method for activating gene expression of endogenous Ngn3 and MAFA

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Embodiment 1

[0044] Example 1. Activation of endogenous Ngn3 and MAFA gene expression based on CRISPRa

[0045] 1. Materials and methods

[0046] (1) CRISPR vector

[0047] 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.

[0048] (2) Lentiviral packaging plasmids PMD2.G and PsPax2 (Addgene), 293T cells (Invitrogen), rat insulinoma cells INS-1 cells (AddexBio).

[0049] (3) Design and vector construction of promoter-specific binding sgRNA

[0050] 1. sgRNA design

[0051] Five sgRNAs ( figure 1 ). The target sequences of sgRNA are shown in Table 1 and Table 2. The sgRNA target sequence oligonucleotides were synthesized by BGI.

[0052] Table 1 Targeting sequence and position of sgRNA on Ngn3 promoter

[0053] sgRNA

Target sequence (5'-3')

PAM

chain

Posi...

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Abstract

The invention discloses a method for activating the gene expression of endogenous Ngn3 and MAFA genes. According to the method disclosed by the invention, the method for activating the expression of the endogenous Ngn3 and / or MAFA genes in targeting cells is established based on an SAM system of CRISPRa (Clustered Regularly Interspaced Short Palindromic Repeats activation). The SAM system comprises sgRNA (small guide Ribonucleic Acid) from upstream -400 to +1bp positions of a transcription starting point of the targeting Ngn3 and / or MAFA genes; a target sequence of the sgRNA aiming the Ngn3 isSEQ ID No. 2 and / or SEQ ID No. 3; a target sequence of the sgRNA aiming the MAFA is SEQ ID No. 6 and / or SEQ ID No. 10. The method is applied to a CRISPRa technology, and Ngn3 and MAFA expression of 293T cells can be efficiently activated through chromatin remodeling. The method disclosed by the invention plays an important role in embryonic development researches of inducing PSCs to be directionally differentiated into beta cells and pancreas through the genes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for activating the expression of endogenous Ngn3 and MAFA genes. Background technique [0002] The incidence of diabetes is increasing significantly year by year, and it is one of the major diseases affecting human health. At present, islet β cell transplantation is considered to be one of the most effective ways to treat diabetes. However, the application of large-scale islet β cell transplantation is limited by the shortage of cell sources and the lifelong use of immunosuppressants [Atkinson, M.A. & Eisenbarth, G.S. -6736(01)05415-0(2001).]. Therefore, finding β-cell replacement resources has become an urgent problem for diabetes cell therapy, which has important economic value and extensive social significance. [0003] Directed differentiation of pluripotent stem cells (PSCs) into islet β cells and non-islet β cells is an important research field for obtaining β cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N5/073
CPCC12N5/0604C12N5/0636C12N5/0696C12N15/86C12N2510/00C12N2740/15043
Inventor 王启伟
Owner ACADEMY OF MILITARY MEDICAL SCI
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