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

Construction method of human embryonic stem cells

A technology for human embryonic stem cells and construction methods, which can be used in microorganism-based methods, chemical instruments and methods, and botanical equipment and methods, etc., and can solve the problem of immune rejection without a good solution.

Active Publication Date: 2021-10-22
TONGJI UNIV
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite these encouraging findings, the problem of immune rejection associated with pluripotent stem cell transplantation has not yet been well resolved, and this has become one of the biggest obstacles to the clinical application of future cell replacement therapies

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 method of human embryonic stem cells
  • Construction method of human embryonic stem cells
  • Construction method of human embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Construction of hESCs cell line with biallelic B2M gene deletion by dual gRNA gene editing system

[0060] The dual gRNA gene editing strategy can efficiently and specifically knock out biallelic genes, and the cut DNA is mainly repaired by direct end joining without introducing insertion or deletion of nucleotides (Liu et al., 2016). To knock down the B2M gene in human embryonic stem cells (hESCs), two adjacent gRNAs were designed, one located upstream of exon 2 of the B2M gene and the other downstream of exon 3 ( figure 1 A and figure 1 B), the specific sequences are respectively shown in B2M gRNA 1 and B2M gRNA 2 in Table 1. The vast majority of mature B2M proteins are encoded by this targeting sequence.

[0061] Table 1

[0062]

[0063] Two gRNA vectors (Addgene #41824), CAG promoter-driven Cas9-P2A-GFP vector (Addgene #44719) and a vector for transient expression of puromycin (SEQ ID NO.14) were co-electrotransfected into H9 hESC (generated by Provided by P...

Embodiment 2

[0065] Construction of hESCs cell lines expressing HLA-G1 and HLA-G5

[0066] Two hESCs cell lines expressing HLA-G protein were designed ( figure 1 A). In B2MmHLAG hESCs, the HLA-G1 heavy chain is flexible (G 4 S) 4 The connecting peptide is fused to endogenous B2M protein. In order to use the coding HLA-G1 heavy chain reading frame and flexible (G 4 S) 4 The sequence of the connecting peptide replaces the stop codon located in exon 3 of the endogenous B2M gene, and the B2M gRNA3 (see Table 1) that spans the B2M stop codon is designed, so that the gene locus that is successfully knocked in will not be detected by B2M gRNA3 Combined, thereby improving the efficiency of establishing homozygous knock-in cell lines. Cas9 vector driven by CAG promoter (same as Example 1), recombinant donor (SEQ ID No.9), B2M gRNA3 vector (Addgene #41824) and transient expression puromycin vector (same as Example 1) were electroporated together into H9 hESCs. In this way, (G 4 S) 4 -HLA-G...

Embodiment 3

[0092] Detection of HLA I protein expression in genetically modified cell lines

[0093] To study the expression of β2m and HLA class I proteins in wild-type (WT), B2M null , B2M mHLAG and B2M m / sHLAGThe expression in hESCs cell lines was performed by Western Blot and flow cytometry (FACS) experiments. Before the flow cytometry experiment, IFN-γ was added to the hESCs cell culture medium to a final concentration of 25 ng / ml, and after 48 hours of culture, the flow cytometry experiment was performed. In case of IFN-γ stimulation, wild type, B2M mHLAG and B2M m / sHLAG After hESCs were treated with IFN-γ, the expression of β2m protein on the cell membrane surface was significantly increased ( image 3 A), demonstrating that the integration of HLA-G1 in the B2M gene does not affect the expression of endogenous B2M. However, even under IFN-γ stimulation, in B2M null The cell membrane surface expression of β2m protein could not be detected in hESCs ( image 3 A). Thus, it wa...

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 relates to the technical field of biology, in particular to a construction method of human embryonic stem cells. The invention provides the construction method of the human embryonic stem cells, and the construction method comprises the following steps: exogenous polynucleotide for coding B2M-HLA-G1 fusion protein is integrated in a genome of the human embryonic stem cells, the B2M-HLA-G1 fusion protein comprises a first B2M fragment and an HLA-G1 fragment, and the human embryonic stem cells obtained by integration do not express free B2M protein. The endogenous HLA-A, B and C proteins of the human embryonic stem cell line constructed by the construction method of the human embryonic stem cells cannot reach the cell membrane surface, so that the cell line cannot be recognized by CD8 + T cells, the immunological rejection of allogeneic cells can be effectively prevented, and the pluripotency and proliferation ability of the embryonic stem cells are not influenced, and the cell can still be used as a target cell source for future cell transplantation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing human embryonic stem cells. Background technique [0002] Pluripotent stem cells have the ability to infinitely divide and proliferate in vitro and differentiate into all cell types derived from the three germ layers under specific induction conditions. Therefore, they are given high hopes in the future development of regenerative medicine. As early as ten years ago, professors Thomson (Thomson et al., 1998) and Reubinoff (Reubinoff et al., 2000) and others took the lead in establishing and differentiating human embryonic stem cell lines. Based on their research, more scientists have carried out research on the application of pluripotent stem cells as a source of functional cells to repair damaged tissue functions and treat degenerative diseases. In animal models, differentiated embryonic stem cells have been shown to be resistant to spinal cord injury (Sou...

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
IPC IPC(8): C12N5/10C12N15/62C12N15/867C07K19/00C07K14/74C12R1/91
CPCC07K14/70539C12N15/86C07K2319/00C12N2740/15043
Inventor 章小清刘玲
Owner TONGJI UNIV
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