A method for improving the efficiency of chemically induced reprogramming by using crotonic acid to activate zscan4 and other second-cell phase genes and lengthen telomeres

A chemically induced, crotonic acid technique for biomedical applications

Active Publication Date: 2020-06-12
NANKAI UNIV
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of potential carcinogenic risk caused by the integration of exogenous genes into genomic DNA and the reduction of exogenous transcription factors accompanied by the reduction of induction efficiency, and to provide a method of using crotonic acid to activate the expression of Zscan4 and other second-cell stage genes, Method for extending telomeres to increase the efficiency of chemically induced reprogramming

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
  • A method for improving the efficiency of chemically induced reprogramming by using crotonic acid to activate zscan4 and other second-cell phase genes and lengthen telomeres
  • A method for improving the efficiency of chemically induced reprogramming by using crotonic acid to activate zscan4 and other second-cell phase genes and lengthen telomeres
  • A method for improving the efficiency of chemically induced reprogramming by using crotonic acid to activate zscan4 and other second-cell phase genes and lengthen telomeres

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: detect the improvement of induction efficiency after adding crotonic acid in the process of chemical induction ( figure 2 ,3)

[0036] A method for improving the efficiency of chemically induced reprogramming by using crotonic acid to activate the expression of genes at the second cell stage, comprising the following steps:

[0037] Section 1. MEF Cell Isolation and Primary Culture

[0038] The 13.5-day-old pregnant mice were killed, the limbs, head and tail, and viscera were removed, the tissues were chopped, digested with 0.05mM trypsin at 37°C for 10 minutes, and then adhered to the culture. Adherent spindle cells were primary MEF cells.

[0039] 2. Adding crotonic acid to obtain chemically induced pluripotent stem cells

[0040]The primary MEF cells obtained in step 1 can be used as precursor cells for chemically induced reprogramming by culturing up to 3 passages. First, 50,000 primary MEF cells were inoculated in a six-well plate, and the next d...

Embodiment 2

[0043] Embodiment 2: Detection of the impact and mechanism of action on chemically induced reprogramming after adding crotonic acid ( Figure 4 , 5, 6, 7, 8, 9)

[0044] 1. Through the TRAP (Telomerase Activity Detection) experiment, it was confirmed that the addition of crotonic acid had no significant effect on the telomerase activity.

[0045] 2. The Western blot experiment proved that the Zscan4 protein was significantly up-regulated on the 28th day after adding crotonic acid, but had no obvious effect on other pluripotent proteins Oct4, Nanog and Lin28.

[0046] 3. Immunofluorescence staining results showed that Zscan4-positive cells increased significantly on the 28th day after adding crotonic acid, and there were significant differences in the statistical results.

[0047] 4. The results of RNA-seq sequencing also confirmed that a series of second-cell stage genes including Zscan4 were significantly activated after the addition of crotonic acid, which was consistent wi...

Embodiment 3

[0050] Example 3: Identification of CiPS induced by adding crotonic acid ( Figure 10 , 11, 12)

[0051] We performed immunofluorescence staining of pluripotency markers Nanog, Sox2 and SSEA1 on the CiPS induced by adding crotonic acid, showing that the CiPS obtained by this method has good pluripotency. The chimera experiments conducted by injection of 4-8 cells also confirmed that the CiPS obtained by this method has the in vivo developmental potential to form high chimeras, which is of great significance for future regenerative medicine and clinical applications. Compared with CiPS without adding crotonic acid, the CiPS induced by crotonic acid shared a closer gene expression profile with the normal embryonic stem cell line OG4.

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 a method for activating two-cell-stage genes such as Zscan4 by virtue of crotonic acid and lengthening telomeres so as to improve the chemically induced reprogramming efficiency. By adding 7mM crotonic acid in a second stage of a chemical inducing process, the final chemically induced reprogramming efficiency can be improved by about 3 times. Chemically induced pluripotent stem cells (CiPSCs) obtained by virtue of the method have closer gene expression profiles relative to normal embryonic stem cells. By adding crotonic acid in a middle inducing state, in terms of a mechanism, the gene expression of two-cell-stage genes such as Zscan4 can be remarkably activated, and the telomeres can be rapidly lengthened, so that DNA damage in telomere regions is reduced, and the chemically induced reprogramming efficiency is improved. The chemically induced pluripotent stem cells induced by virtue of the method have very wide application prospects and very huge application potentials in tissue engineering, regenerative medicines and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to a method for improving the efficiency of chemically induced reprogramming. Specifically, the present invention relates to the addition of the chemical substance crotonic acid in the middle phase of chemical induction, through activating the expression of a series of two-cell phase genes including Zscan4, to rapidly extend the telomere, thereby reducing the genome damage in the telomere region and improving the final Chemically induced reprogramming efficiency. Background technique [0002] In recent years, mouse and human induced pluripotent stem cells (hereinafter referred to as iPS) have been successively established. The establishment of such cells requires the forced expression of a series of exogenous transcription factor combinations, such as Oct4, Sox2, Klf4, etc. However, retroviruses or lentiviruses that mediate the expression of foreign genes have potential safety iss...

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): C12N5/0735C12N5/073
CPCC12N5/0696C12N2501/06C12N2501/115C12N2501/70C12N2501/71C12N2501/72C12N2501/727C12N2501/999C12N2506/02
Inventor 付海峰刘易飞田成磊叶孝颖绳小艳刘林
Owner NANKAI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap