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A method for fluorescent tracing of cynomolgus monkey stem cells

A fluorescent tracking, cynomolgus monkey technology, applied in the field of cell transplantation, can solve the problem of not being suitable for monitoring the biological behavior of cells, and achieve the effect of high sensitivity and strong penetration.

Active Publication Date: 2022-07-26
YUNNAN KEY LAB OF PRIMATE BIOMEDICINE RES
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Problems solved by technology

[0003] Traditionally, the effect of cell transplantation is mainly evaluated through pathological tissue sections. However, this method is not suitable for monitoring the biological behavior of cells in vivo.

Method used

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  • A method for fluorescent tracing of cynomolgus monkey stem cells
  • A method for fluorescent tracing of cynomolgus monkey stem cells
  • A method for fluorescent tracing of cynomolgus monkey stem cells

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Embodiment 1

[0062] The AAVS1 donor plasmid was purchased from Addgene, the luciferase fragment was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and the restriction endonucleases SalI, MluI and T4 ligase were purchased from NEB Company; DNA gel recovery kit, endotoxin-free plasmid The large-scale kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The Lonza 4D nucleator was purchased from LONZA; the small animal in vivo imager was purchased from PerkinElmer. Fibroblasts were established through cynomolgus monkey skin, and fibroblasts were reprogrammed by reprogramming small factors to obtain cynomolgus monkey induced pluripotent stem cells; the designed primers contained luciferase fragments, and the upstream primer was luciferase-F:5 '-ACGCGTCGACGCCACCATGGAGGACGCCA-3', the downstream primer is luciferase-R:5'-CGACGCGTTCACACGGCGATCTTGCCGT-3'. PCR reaction of luciferase gene and primer pair luciferase-F and luciferase-R, PCR reaction system 50μl; reaction co...

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Abstract

The invention discloses a method for fluorescent tracing of cynomolgus monkey stem cells. The method includes: establishing a fibroid cell line through cynomolgus monkey skin, reprogramming the fibroblasts to obtain cynomolgus monkey induced pluripotent stem cells; Gene design primer pair, luciferase gene and primer pair for PCR reaction; AAVS1 vector plasmid and PCR reaction product luciferase gene fragment are digested with restriction endonuclease, and T4 ligase is ligated to the product after restriction enzyme cleavage to obtain recombination AAVS1-luciferase plasmid, the recombinant plasmid is inserted into cynomolgus monkey induced pluripotent stem cells; the cells with the luciferase-labeled gene and the luciferin substrate are intravenously injected into cynomolgus monkeys, and then placed in a small animal in vivo imager. Fluorescence imaging. The method can non-invasively and effectively monitor a series of biological changes in vivo after cell transplantation, such as viability, moving position, distribution and proliferation, and provide real-time reference data for the safety and effectiveness of large animal cell transplantation.

Description

technical field [0001] The invention belongs to the technical field of cell transplantation, and relates to a method for fluorescent tracing of cynomolgus monkey stem cells. Background technique [0002] The non-human primate model is highly similar to humans at the genetic level, and its symptoms are closer to those of patients than rodent models and other animals, which is useful for understanding the mechanism of disease and exploring the effectiveness of new treatments. Sex has great potential. Stem cell therapy has been widely recognized as a novel therapeutic approach to treat and cure complex diseases. Cynomolgus monkey pluripotent stem cells (PSCs) are similar to human pluripotent stem cells in terms of morphology, gene expression characteristics and growth properties, but have many Differences in stem cells suggest that macaques may be the best model to answer some key questions. [0003] The transplantation effect of traditional research cells is mainly evaluated...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00G01N21/64
CPCA61K49/0045G01N21/6486
Inventor 牛昱宇吴俊模王芳李梦佳
Owner YUNNAN KEY LAB OF PRIMATE BIOMEDICINE RES