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Dynamic monitoring system for iPSC differentiation

A promoter, SV40 technology, applied in the field of cell biology, can solve problems such as difficult to track differentiation efficiency, difficult to large-scale culture, hindering iPSC application, etc.

Active Publication Date: 2021-08-17
CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are currently several major problems in the field of iPSC cell research, which seriously hinder the application of iPSC in clinical research and pharmaceutical fields. The problems include the following aspects: (1) The iPSC culture system is extremely expensive and difficult to cultivate on a large scale; (2) During the culture process of iPSC, some cells will differentiate autonomously, resulting in cell impurity; (3) Residual iPSC may cause teratomas; (4) It is difficult to track the differentiation efficiency of iPSC during the differentiation process
At present, there is no reporter gene system that can be used to study the above problems at the same time. In view of this, in order to solve the above problems in the field, the purpose of the present invention is to provide a dynamic monitoring system for iPSC differentiation, which includes a unique Gene expression system structure, vector containing said construct, iPSC / ESC cell line model containing said vector and its use

Method used

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  • Dynamic monitoring system for iPSC differentiation
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  • Dynamic monitoring system for iPSC differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1 pCDH-CMV-MCS1-EF1a-MCS2 plasmid construction

[0125] 1. Experimental materials

[0126] The plasmid vector pCDH-CMV-MCS-EF1a-Puro used in the examples of the present invention was purchased from Invitrogen.

[0127] 2. Construction of pCDH-CMV-MCS1-EF1a-MCS2 plasmid

[0128] The fragment including EF1-Puro was digested from the site on the pCDH-CMV-MCS-EF1a-Puro vector. Select NotI for the 5' end of the restriction site and SalI for the 3' end. Through gene synthesis, the EF1a-5'LTR (truncated)-MCS2 sequence was inserted into the above restriction plasmid, the EF1a sequence information is shown in SEQ ID NO: 1, and the 5'LTR (truncated) sequence information is shown in SEQ As shown in ID NO: 2, the sequence information of MCS2 is shown in SEQ ID NO: 3. MSC2 includes restriction enzyme cutting sites HindIII, KpnI, XbaI and ApaI in sequence.

[0129] 3. Experimental results

[0130] The experimental results show that the map of the constructed pCDH-CMV-MCS...

Embodiment 2

[0131] Embodiment 2 pCDH-CMV-GFP-EF1a-MCS2 plasmid construction

[0132] 1. Construction of pCDH-CMV-GFP-EF1a-MCS2 plasmid

[0133] Select restriction sites from the MCS1 sequence on the pCDH-CMV-MCS1-EF1a-MCS2 vector, and cut the vector into line segments. NheI (GCTAGC) was selected for the 5' end of the restriction site, and NotI (GCGGCCGC) was selected for the 3' end. Through gene synthesis, the GFP sequence was inserted into the restriction plasmid, wherein the GFP sequence information is shown in SEQ ID NO:4. GFP is green fluorescent protein, and the GFP sequence can be replaced with the sequence of other fluorescent proteins, such as: red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein, etc., or these fluorescent protein derivatives Therefore, replacing the GFP sequence with the sequence of other fluorescent proteins or derivatives thereof is also included in the protection scope of the pre...

Embodiment 3

[0136] Example 3 pCDH-CMV-GFP-EF1a-lifeact-mCherry plasmid construction

[0137] 1. Construction of pCDH-CMV-GFP-EF1a-lifeact-mCherry plasmid

[0138] Select restriction sites from the MCS2 sequence on the pCDH-CMV-GFP-EF1a-MCS2 vector, and cut the vector into line segments. HindⅢ (AAGCTT) was selected for the 5' end of the restriction site, and XbaI (TCTAGA) was selected for the 3' end. Through gene synthesis, the lifeact-mCherry sequence is inserted into the above restriction plasmid, wherein the lifeact sequence information is shown in SEQ ID NO:5, and the mCherry sequence information is shown in SEQ ID NO:6. mCherry is a derivative protein of red fluorescent protein, and the mCherry sequence can be replaced by other fluorescent protein sequences, such as: red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein, etc., or these fluorescent proteins The sequence of the derivative protein, in order t...

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Abstract

The invention discloses an iPSC differentiation dynamic monitoring system, a dual-fluorescence reporter gene vector system constructed by the invention comprises pCDH-CMV-GFP-EF1a-lift-mCherry plasmids, a hiPSC cell line constructed by transformation of the vector system can stably express red fluorescent protein for a long time and does not express green fluorescent protein, and once differentiation begins, the cells can stably express green fluorescent protein for a long time and also can express red fluorescent protein. The construction of the system provides a powerful research tool for the development of an iPSC culture medium, the detection of iPSC residues, the research of an iPSC stemness maintenance mechanism, the research of cytoskeleton change in an iPSC differentiation process, the tracking of iPSC cell fate and outcome, the optimization of a differentiation system, the detection of an iPSC cell bank, cell transplantation and other downstream researches.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a dynamic monitoring system for iPSC differentiation. Background technique [0002] Stem cells are a class of cells with different differentiation potentials and self-renewal in a non-differentiated state (Kim A, Shin D M, Choo M S.Stem cell therapy for interstitial cystitis / bladder painsyndrome[J].Current urology reports,2016,17 (1): 1.). Stem cell therapy refers to the process of using human autologous or allogeneic stem cells to infuse (or implant) into the human body for disease treatment. Line) establishment, differentiation induction, cryopreservation and recovery after cryopreservation. Currently, stem cells used for cell therapy mainly include adult stem cells (Adult stem cells, ASC), embryonic stem cells (Embryonic stem cells, ESC) and induced pluripotent stem cells (Induced pluripotent stem cells, iPSC). The source of ASC is difficult and the therape...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/66C12N15/113C12N5/10
CPCC12N15/86C12N15/65C12N15/66C12N15/113C12N5/0696C12N5/0606C12N2740/15043C12N2501/405C12N2501/727C12N2510/00
Inventor 吴理达顾雨春
Owner CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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