Construction and application of induced pluripotent stem cell line for expressing Pdx1/insulin double reporting genes

A pluripotent stem cell, dual reporter gene technology, applied in the field of induced pluripotent stem cell line construction, can solve the problems of laborious and time-consuming

Active Publication Date: 2018-11-30
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Optimizing specific cell lines for differentiation into islet β-cells is time-consuming and labor-intensive due to significant variability in cell differentiation efficiency

Method used

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  • Construction and application of induced pluripotent stem cell line for expressing Pdx1/insulin double reporting genes
  • Construction and application of induced pluripotent stem cell line for expressing Pdx1/insulin double reporting genes
  • Construction and application of induced pluripotent stem cell line for expressing Pdx1/insulin double reporting genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1. Construction of induced pluripotent stem cell lines expressing Pdx1 / insulin dual reporter genes

[0083] 1. Construction of Pdx1 / insulin dual reporter gene vector

[0084] The Pdx1 / insulin dual-reporter gene vector was obtained on the basis of the FⅣpTiger vector (Szabat, M., Luciani, D.S., Piret, J.M.&Johnson, J.D. Maturation of adult beta-cells revealed using a Pdx1 / insulin dual-reporter lentivirus.Endocrinology 150, 1627-1635, doi: 10.1210 / en.2008-1224 (2009)). In order to add a drug screening marker to the FIVpTiger vector, the hPGK-Puromycin resistance gene was first introduced into the vector. Preliminary tests showed that the expression efficiency of Ins1 promoter of FⅣpTiger vector was low. In order to improve the expression efficiency of the Ins1 promoter, the original Ins1-EGFP (410bp Ins1 promoter) was replaced with Ins1-hrGFP (646bp Ins1 promoter) to obtain a new vector pTiger-Pdx1-mRFP / insulin-hrGFP / hPGK-Puro (also Called pTiger-Pdx1 / insulin d...

Embodiment 2

[0098] Example 2. Directed differentiation of iPSCs expressing Pdx1 / insulin dual reporter genes into pancreatic islet β cells in vitro

[0099] In order to verify the function of the Pdx1 / insulin dual reporter gene cell line in vitro for real-time monitoring of cell differentiation, a combination of growth factors and small molecule compounds was used to induce the cells to differentiate into islet β cells ( figure 2 A).

[0100] Mimicking the embryonic pancreatogenic strategy, iPSCs were induced to differentiate into insulin-secreting cells through five stages: endoderm (stage 1), gastrula (stage 2), pancreatic precursor cells (stage 3), endocrine precursor cells (stage 4) and Insulin-secreting cells (stage 5).

[0101] 1. Optimization of induction medium in the first stage

[0102] 1. Optimization of M2 medium in the first stage to induce differentiation

[0103] The first stage of the M1 medium group before optimization: iPSCs differentiated into endoderm cells (Definit...

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Abstract

The invention discloses construction and application of an induced pluripotent stem cell line for expressing Pdx1/insulin double reporting genes, and provides a method for induced directional differentiation of pluripotent stem cells into insulin beta-cells. The method comprises the following steps that (1) the pluripotent stem cells are formed into embryonic bodies; 2) the embryonic bodies are subjected to demethylation treatment, and cells subjected to demethylation treatment are obtained; and (3) the cells subjected to demethylation treatment are sequentially differentiated into endosperm cells, archenteron cells, pancreatic precursor cells, endocrine precursor cells and the insulin beta-cells. The study proves that the hiPSC line for expressing the Pdx1-mRFP/insulin-hrGFP double reporting genes has the important value in establishing and optimizing a beta-cell differentiation method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an induced pluripotent stem cell line expressing Pdx1 / insulin double reporter gene. Background technique [0002] Induced pluripotent stem cells (iPSCs) have the potential of self-renewal and multilineage differentiation in vitro. Therefore, it has broad application prospects in the fields of disease modeling, drug discovery and regenerative medicine. iPSCs can be obtained from their own body cells, therefore, autologous cell transplantation can be performed to avoid transplant rejection, and there are no ethical and moral issues at the same time. Recent studies have shown that iPSCs can differentiate into other difficult-to-obtain cell types in vitro, such as neurons, retinal and cardiac muscle cells. When these cells were transplanted into animal disease models, the symptoms of the disease were alleviated to varying degrees, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10C12N15/85
CPCC12N5/0676C12N15/85C12N2506/02C12N2506/03C12N2501/16
Inventor 王启伟叶玲玲叶华虎蓝三春
Owner ACADEMY OF MILITARY MEDICAL SCI
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