A method to delay the aging of mesenchymal stem cells through foxp1 gene editing and mutation

A stem cell and gene editing technology, applied in the field of cell biology, can solve problems such as off-target effects and gene variation, poor miRNA effect, and risks brought by operators, so as to promote proliferation, improve the potential of treating diseases, and improve organ repair Effect

Active Publication Date: 2022-03-15
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 risk to the operator during the experiment
2) Traditional gene editing and mutation, mediated by wild-type Cas9 gene, are prone to off-target effects and gene mutations, which affect cell safety
3) Using microRNA for cell transfection and treatment, 1-2 miRNAs alone are not effective, and combined use can easily lead to off-target effects

Method used

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  • A method to delay the aging of mesenchymal stem cells through foxp1 gene editing and mutation
  • A method to delay the aging of mesenchymal stem cells through foxp1 gene editing and mutation
  • A method to delay the aging 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 the aging of mesenchymal stem cells through FOXP1 gene editing and mutation. The method comprises the following steps: (1) isolating and culturing human umbilical cord mesenchymal stem cells; (2) adding plasmids, donor DNA, and liposomes to the culture medium obtained in step (1) for transfection; (3) adding The human umbilical cord mesenchymal stem cells after the transfection in step (2) are transferred to the human umbilical cord mesenchymal stem cell medium containing penicillin and streptomycin for culture; (4) the human umbilical cord after step (3) is cultured The mesenchymal stem cells are screened and cultured; (5) the human umbilical cord mesenchymal stem cells cultured in step (4) are digested and passaged, and then amplified until a stable cell line is obtained. Compared with the prior art, the invention can obviously promote the proliferation speed of the mesenchymal stem cells by one time, and delay the aging process of the mesenchymal stem cells with an efficiency of 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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Patent Type & Authority Patents(China)
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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