Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction and application of induced pluripotent stem cell line expressing pdx1/insulin dual reporter gene

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

Active Publication Date: 2022-06-07
ACADEMY OF MILITARY MEDICAL SCI
View PDF2 Cites 0 Cited by
  • Summary
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction and application of induced pluripotent stem cell line expressing pdx1/insulin dual reporter gene
  • Construction and application of induced pluripotent stem cell line expressing pdx1/insulin dual reporter gene
  • Construction and application of induced pluripotent stem cell line expressing pdx1/insulin dual reporter gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Construction of an induced pluripotent stem cell line expressing Pdx1 / insulin dual reporter genes

[0082] 1. Construction of Pdx1 / insulin dual reporter gene vector

[0083] The Pdx1 / insulin dual reporter gene vector was obtained by remodeling 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 selection marker to the FIVpTiger vector, the hPGK-Puromycin resistance gene was first introduced into the vector. Preliminary tests showed that the expression efficiency of the Ins1 promoter of the FIVpTiger vector was low. In order to improve the expression efficiency of 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 Call...

Embodiment 2

[0097] Example 2. Directed differentiation of iPSCs expressing Pdx1 / insulin dual reporter gene into pancreatic β cells in vitro

[0098] In order to verify the function of the Pdx1 / insulin dual reporter gene cell line to monitor cell differentiation in vitro in real time, a combination of growth factors and small molecule compounds was used to induce cell differentiation into islet β cells ( figure 2 A).

[0099] Mimic embryonic pancreatogenesis strategy to induce iPSCs to differentiate into insulin-secreting cells through 5 stages: endoderm (stage 1), gastrulation (stage 2), pancreatic precursor cells (stage 3), endocrine precursor cells (stage 4) and Insulin secreting cells (stage 5).

[0100] 1. Optimization of the first stage induction medium

[0101] 1. Stage 1 M2 medium optimized to induce differentiation

[0102] M1 medium group before optimization Stage 1: iPSCs differentiate into endoderm cells (Definitive endoderm, DE): When the iPSCs expressing the Pdx1 / insulin ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses the construction and application of an induced pluripotent stem cell line expressing Pdx1 / insulin double reporter gene. The invention provides a method for directional induction and differentiation of pluripotent stem cells into pancreatic islet β cells, comprising the following steps: 1) forming embryonic bodies from pluripotent stem cells; 2) demethylating the embryonic bodies to obtain demethylated 3) differentiate the demethylated cells into endoderm cells, gastrula cells, pancreatic precursor cells, endocrine precursor cells and islet β cells in sequence. This study demonstrates the value of hiPSC lines expressing Pdx1‑mRFP / insulin‑hrGFP dual reporter genes in establishing and optimizing β-cell differentiation methods.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the construction and application of an induced pluripotent stem cell line expressing Pdx1 / insulin double reporter genes. Background technique [0002] Induced pluripotent stem cells (iPSCs) have self-renewal and multi-directional differentiation potential in vitro. Therefore, it has broad application prospects in the fields of disease modeling, drug development and regenerative medicine. iPSCs can be obtained from autologous cells, therefore, autologous cell transplantation can be performed to avoid transplant rejection, and there are no ethical issues. Recent studies have shown that iPSCs can differentiate in vitro into cell types that are difficult to obtain by other methods, such as neurons, retinas, and cardiomyocytes. When these cells were transplanted into animal disease models, the symptoms of the disease were alleviated to varying degrees, such as improving motor function i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com