Method for delaying senescence of mesenchymal stem cells through FOXP1 gene editing and mutation

A kind of stem cell and gene editing technology, applied in the field of cell biology, can solve the problems of poor miRNA effect, off-target effect and gene mutation, risks brought by operators, etc., to improve the potential of disease treatment, promote proliferation, and improve organ repair Effect

Active Publication Date: 2021-06-11
安可来(重庆)生物医药科技有限公司
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Problems solved by technology

[0015] However, the above methods have the following problems: 1) use viral vectors, such as lentiviruses, or adenoviral vectors to transfect cells, provide donor DNA for CRISPR/Cas9, and carry out DNA recombination
The viral vectors used in these manipulations all carry the risk of random insertion into the cell genome or pose a ris

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  • Method for delaying senescence of mesenchymal stem cells through FOXP1 gene editing and mutation
  • Method for delaying senescence of mesenchymal stem cells through FOXP1 gene editing and mutation
  • Method for delaying senescence of mesenchymal stem cells through FOXP1 gene editing and mutation

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Embodiment 1

[0058] This embodiment relates to a method for delaying the aging of human umbilical cord mesenchymal stem cells (MSCs) through FOXP1 gene editing. Using the artificial chromosome episomal-Cas9n vector, two reverse sgRNAs near the FOXP1 gene mutation site were inserted in series; meanwhile, donor DNA containing about 500 bp homology arms at the left and right sides of the mutation site was prepared by PCR. Episomal-Cas9n-FOXP1sgRNA and donor DNA were co-transfected into human umbilical cord mesenchymal stem cells, the cells were diluted and cultured to form single-cell clones, and after antibiotic screening, human-derived FOXP1 (T176G, T277G, K393G) point mutations were identified Umbilical cord mesenchymal stem cells.

[0059] The method comprises the steps of:

[0060] 1) Isolation and culture of human umbilical cord mesenchymal stem cells

[0061] (1) Take out a 15cm dish (petri dish with a diameter of 15cm), cut up the fetal umbilical cord with scissors, suck up and disc...

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Abstract

The invention relates to the technical field of cell biology, in particular to a method for delaying senescence of mesenchymal stem cells through FOXP1 gene editing and mutation. The method includes the following steps: (1) separating and culturing human umbilical cord mesenchymal stem cells; (2) adding plasmids, donor DNA (deoxyribonucleic acid) and lipidosome into the culture solution obtained in the step (1) for transfection; (3) transferring the human umbilical cord mesenchymal stem cells transfected in the step (2) into a human umbilical cord mesenchymal stem cell culture medium containing penicillin and streptomycin, and performing culturing; (4) carrying out screening culture on the human umbilical cord mesenchymal stem cells cultured in the step (3); and (5) carrying out digestion passage on the human umbilical cord mesenchymal stem cells cultured in the step (4), and performing amplifying until stably transferred cell strains are obtained. Compared with the prior art, the method has the advantages that the proliferation speed of the mesenchymal stem cells can be obviously promoted by one time, and the cell senescence process can be delayed by 50%.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for delaying the aging of mesenchymal stem cells through FOXP1 gene editing and mutation. Background technique [0002] Mesenchymal stem cells (MSCs) are non-hematopoietic adult pluripotent stem cells. Friedenstein first proposed the concept of MSCs to define a group of stem cells that can form fibroblast clonal colonies (CFU-F) in vitro , but also osteoblast, chondrocyte and adipocyte differentiation (Friedenstein et al., 1966). It has a high capacity for self-renewal while possessing the ability to differentiate into osteoblasts, chondrocytes, and adipocytes (Caplan, 2017). Most studies regard osteogenic, adipogenic and chondrogenic differentiation ability as the "gold standard" for defining mesenchymal stem cells (Bianco et al., 2008b). MSC cell surface markers include CD73, CD90, and CD105, but do not express CD11b, CD14, CD34, CD45, and HLA-DR. [0003] At p...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/88C12N15/87C12N15/55C12N15/12C12N15/10C12N5/10
CPCC12N15/85C12N15/88C12N15/87C12N9/22C07K14/4702C12N15/102C12N5/0668C12N2800/107C12N2510/00C12Q2521/327C12Q2525/161
Inventor 郭熙志凌世烽
Owner 安可来(重庆)生物医药科技有限公司
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