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

Method for preparing iPSC (induced Pluripotent Stem Cell) by free plasmid vector screened by non-antibiotics

A non-antibiotic screening and plasmid carrier technology, applied in the biological field, can solve the problems of insufficient safety and complex preparation of reprogramming vectors, and achieve the effects of improving reprogramming efficiency, low mRNA yield, and improving efficiency

Active Publication Date: 2020-07-31
安徽中盛溯源生物科技有限公司
View PDF9 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] In order to solve the problems of insufficient safety and complex preparation of reprogramming vectors in the prior art, the present invention transforms and optimizes reprogramming plasmids, discovers a non-antibiotic-selected episomal plasmid vector, and discloses the application of the episomal plasmid vector for reprogramming blood cells to generate induced pluripotent stem cells

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
  • Method for preparing iPSC (induced Pluripotent Stem Cell) by free plasmid vector screened by non-antibiotics
  • Method for preparing iPSC (induced Pluripotent Stem Cell) by free plasmid vector screened by non-antibiotics
  • Method for preparing iPSC (induced Pluripotent Stem Cell) by free plasmid vector screened by non-antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] This example discloses the idea of ​​constructing an episomal plasmid vector for non-antibiotic screening. The construction strategy is as follows: figure 1 shown. figure 1 The bacterial sequence part of the episomal plasmid screened by non-antibiotics contains the R6K replication origin and the RNA-OUT sucrose selection marker, and the eukaryotic expression part consists of a conventional promoter, target gene and SV40poly-A. SV40 poly-A is a simian vacuolar virus PolyA tailing signal; the target gene represents the nucleotide sequence of the inserted foreign gene. The R6K origin of replication is derived from the original R6K-gamma origin of replication, and the length is greatly reduced after transformation, which retains the core sequence required for replication, removes the upstream 77 dimer repressor binding site and the downstream 77 promoter . The plasmid at the R6K replication origin needs Rep protein, so that the non-antibiotic-selected episomal plasmid can...

Embodiment 2

[0118] This example first discloses a method for reprogramming blood cells to generate pluripotent stem cells with an episomal plasmid vector containing OriP / EBNA1 non-antibiotic selection, the flow chart is as follows image 3 shown, including the following steps:

[0119] S1. Primary isolation, culture and identification of erythroid progenitor monocytes

[0120] S11. Primary isolation and culture of erythrocyte progenitor monocytes

[0121] Collect a blood sample of at least 10 μl, transfer it to a lymphocyte separation tube, centrifuge, take the mononuclear cell layer, wash it twice with DPBS centrifugation, take a sample and count it, and take 3×10 cells according to the counting result. 6 Cells were inoculated into 6-well plates in 3 wells, and erythrocyte progenitor cell expansion medium 2ml / well was added, and placed at 37°C, 5% CO 2 cultured in an incubator. Add 2ml of fresh expansion medium to each well on the 4th and 8th day of expansion respectively.

[0122] T...

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 belongs to the biotechnology field, and particularly relates to a method for preparing an iPSC (induced Pluripotent Stem Cell) by a free plasmid vector screened by non-antibiotics. The preparation method for an induced pluripotent stem cell comprises the following step of reprogramming blood cells through the free plasmid vector screened by the non-antibiotics to obtain the induced pluripotent stem cell. According to the preparation method disclosed by the invention, the free plasmid vector screened by the non-antibiotics is used for avoiding antibiotic screening but adopting saccharose screening, and the obtained pluripotent stem cell is higher in safety and is suitable for producing the clinical iPSC. In addition, the bacterial sequence of the free reprogramming plasmid screened by the non-antibiotics is reduced to 500 bp or below, so that the level and the duration of protein expression can be improved, and reprogramming efficiency is greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing iPSCs using free plasmid vectors not screened by antibiotics. Background technique [0002] In 2006, Takahashi and Yamanaka used retroviral vectors to transfer four transcription factors OSKM (octamer-binding transcription factor 4, Oct4), sex determining region Y box protein 2 (sex determining region Y box protein 2, Sox2), Kuppel-like factor 4 (kruppel-likefactor 4, Klf4) and C-myelocytosis proto-oncogene (cellular myelocytomatosiscogene, c-Myc) were transferred into mouse fibroblasts, and induced pluripotent stem cells ( induced Pluripotent Stem Cell, iPSC). In view of the fact that iPSCs are very similar to embryonic stem cells (ESCs) in terms of biological characteristics, and effectively avoid problems such as ethical restrictions and difficulties in obtaining materials, iPSCs are widely used in disease models, drug screening, developmental b...

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): C12N5/074C12N15/85
CPCC12N5/0696C12N15/85C12N2800/108C12N2820/002
Inventor 不公告发明人
Owner 安徽中盛溯源生物科技有限公司
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