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Method for improving adipose-derived stem cell differentiation efficiency through gene editing

An adipose stem cell and chondrocyte technology, applied in the field of gene editing to improve the differentiation efficiency of adipose stem cells, can solve the problems of short half-life, insufficient research, and low efficiency of exogenous growth factors, and achieve good induction effect, shorten induction time, and improve Effect of Induction Efficiency and Application Value

Active Publication Date: 2020-12-25
中赛干细胞基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the low efficiency and short half-life of exogenous growth factors, some scholars have proposed a long-term stable induction method, that is, using genes to transfect the target genes of the above growth factors to obtain endogenous growth factors, which play a role through the secretory pathway
Although genetic manipulation can control the secretion of specific growth factors, because chondrogenesis is coordinated by multiple growth factors and other signaling molecules, only one gene can be targeted at a time, which limits the application potential of gene transfection. Not enough, there is a lot of room for improvement

Method used

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  • Method for improving adipose-derived stem cell differentiation efficiency through gene editing
  • Method for improving adipose-derived stem cell differentiation efficiency through gene editing
  • Method for improving adipose-derived stem cell differentiation efficiency through gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of Adipose Stem Cells

[0023] Take 10 g of the abdominal subcutaneous fat extracted by vacuum liposuction under aseptic conditions, rinse it with PBS containing 500 U / ml double antibody for 3 times, remove the blood vessels and connective tissue visible to the naked eye, and fully cut it with ophthalmic scissors. Cut it into pieces, and wash it repeatedly with PBS to remove red blood cells as much as possible. Add 2 times the volume of 0.2% type Ⅰ collagenase, shake and mix well, and digest in a constant temperature water bath at 37 °C for 60 min. Add an equal volume of low-glucose DMEM medium containing 10% FBS to terminate the digestion. Centrifuge at 1800 r / min for 10 min. Discard the upper and middle layers, add 2 ml of complete medium (low sugar DMEM + 10% FBS + 100 U / ml penicillin-streptomycin) to resuspend the cell pellet, filter through a 70 μm cell mesh, and adjust the filtered filtrate to a cell concentration of 1 ×10 8 / L into 60 mm...

Embodiment 2

[0024] Example 2 Gene editing technology targeting miR-221

[0025] According to the precursor sequence of miR221, multiple sets of gRNA sequences were designed, among which the optimal g4-RNA was screened as the experimental sequence. The specific gRNA sequence is shown in SEQ ID NO: 1, gRNA4: TGTAGATTTCTGTGTTCGTTAGG.

[0026] In Shanghai Shenggong Company, the 5' end of the DNA sequence corresponding to gRNA4 was synthesized with the forward nucleotide sequence of CACC, and the reverse nucleotide sequence of AAAC was added at the 5' end of its complementary strand, respectively. The synthesized forward and reverse nucleotide sequences are denatured and annealed to obtain double-stranded DNA fragments. The specific experimental conditions are as follows: 1ul forward nucleotide sequence (100uM); 1ul reverse nucleotide sequence (100uM); 1ul 10×T4 Ligation Buffer (NEB); 6.5ul ddH 20; 0.5ul T4PNK (NEB). Put the reaction system in a PCR instrument, the reaction conditions are: ...

Embodiment 3

[0028] Example 3 Preparation of monoclonal antibodies targeting RUNX2

[0029] 针对RUNX2的保守区,发明人筛选获得了具有高免疫原性的抗原表位,具体序列如下:DENYSAELRNASAVMKNQVARFNDLRFVGRSGRGKSFTLTITVFTNPPQVATYHRAIK VTVDGPREPRRHRQKLDDSKPSLFSDRLSDLGRIPHPSMRVGVPPQNPRPSLNSAPSP FNPQGQSQITDPRQAQSSPPWSYDQSYPSYLSQMTSPSIHSTTPLSSTRGTGLPAITD VPRRISGASELGPFSDPRQFPSISSLTESRFSNPRMHYPATFTYTPPVTSGMSLGMSA

[0030] The antigenic peptide synthesized with this sequence was used as the immunogen, mixed with an equal volume of Freund's complete adjuvant, and BALB / c mice were subcutaneously injected with 50 μg of the fusion protein at multiple points. Four weeks after the initial immunization, multi-point subcutaneous injections on the back were used to boost the immunization, and 4 weeks later, the immunization was boosted again, each time using 50 μg of immunogen per mouse. 7-10 days after the third immunization, blood was collected from the tail vein to measure the serum antibody titer by ELISA. Four days before fusion, a mouse wit...

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Abstract

The invention relates to a method for improving adipose-derived stem cell differentiation efficiency through gene editing. After the optimal gRNA sequence is obtained through screening and optimization, miR-221 is knocked out to obtain transgenic adipose-derived stem cells, a specific monoclonal antibody is obtained through RUNX2 screening, the monoclonal antibody can inhibit the activity of RUNX2, and after the monoclonal antibody is added into an induction medium, the adipose-derived stem cells can be specifically promoted to be differentiated into chondrocytes, and good induction efficiencyand application value are realized.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for improving the differentiation efficiency of adipose stem cells by gene editing. Background technique [0002] The treatment of articular cartilage lesions, especially knee lesions, has always been one of the most challenging clinical problems. Articular cartilage belongs to hyaline cartilage, which has important functions such as buffering mechanical stress and ensuring flexible movement of joints. However, the articular cartilage tissue of adults lacks blood vessels, nerves, and lymphatic reflux, and the tissue cell types are single and highly differentiated. Chondrocytes are embedded in a large amount of dense matrix, which limits their ability to proliferate and migrate. As a result, the tissue has limited ability to heal itself, and even minor cartilage damage can lead to osteoarthritis, which progresses to progressive inflammation leading to joint degeneration and loss o...

Claims

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Application Information

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IPC IPC(8): C12N5/077C07K16/18
CPCC07K16/18C07K2317/56C12N5/0655C12N2506/1384
Inventor 张海涛陈帅
Owner 中赛干细胞基因工程有限公司
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