A kind of construct and its application in preparation of animal senescent cell tracing and senescent cell clearing medicine

A technology for senescent cells and constructs, applied to cells modified by introducing foreign genetic material, animal/human peptides, using vectors to introduce foreign genetic material, etc., can solve the problem of interconnected signaling pathways in the body, complicated mutual constraints, and difficult research work , not easy to anti-aging methods and other issues

Active Publication Date: 2022-06-07
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in view of the complexity of the occurrence and development of aging, the interconnection and mutual restriction of signaling pathways in the body are extremely complicated, and the inducements, types and expression markers of aging cells are very different, making this kind of research work difficult
Even if some genes are known to be related to aging, it is still not easy to successfully engineer aging-related genes to obtain useful disease models or useful anti-aging methods

Method used

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  • A kind of construct and its application in preparation of animal senescent cell tracing and senescent cell clearing medicine
  • A kind of construct and its application in preparation of animal senescent cell tracing and senescent cell clearing medicine
  • A kind of construct and its application in preparation of animal senescent cell tracing and senescent cell clearing medicine

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0085] Preparation of cell or animal models

[0086] The inventors have designed a carrier that integrates the effects of tracing, sorting and clearing senescent cells with positive p21 expression. According to the present invention, the constructs can be used for the preparation of cell or animal models.

[0087] In the present invention, there is provided a method for preparing cells by using the construct, comprising: transfecting the cell with the construct to obtain the cell with the exogenous construct integrated into the genome.

[0088] In the present invention, a method for preparing an animal model is also provided. The method comprises: introducing the construct into germ cells or embryonic stem cells, and obtaining germ cells or embryonic stem cells integrated into the construct in the genome; using the germ cells or embryonic stem cells to generate animal bodies, the Animals can trace senescent cells, or their senescent cells can be induced to undergo apoptosis;...

Embodiment 1

[0096] Example 1. Establishment of constructs

[0097] With respect to the p21 gene, the inventors have carried out a lot of research work and carried out genetic modification of animals through gene targeting. After repeated studies, the present inventors identified a target position suitable for targeting, and targeted a specific position on the third exon of the p21 gene as a target.

[0098] Strategies for constructing genetically modified animals such as figure 1 As shown, the exogenous construct was inserted into the animal's genome by targeting a plasmid for a position after base 47 in exon 3 of a wild-type animal. The exogenous constructs are as follows:

[0099] 2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt.

[0100] Based on this construction strategy, the specific construction method is as follows:

[0101] 1. Construction of targeting plasmid (homologous recombination vector plasmid)

[0102] build as figure 2 The homologous recombination vector plasmid P21-(2...

Embodiment 2

[0122] Example 2. Acquisition and identification of homologous recombination ES cells

[0123] The homologous recombination vector plasmid P21-(2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt-pA)1 was linearized.

[0124] Embryonic stem cell targeting was performed using JM8A3 ES cells (Shanghai Southern Model Organism Research Center). The aforementioned linearized vectors were introduced into embryonic stem cells by microinjection.

[0125] By targeting embryonic stem cells (ES), the inventors obtained a series of resistant ES cell clones. After the genomic DNA of the resistant ES cell clones was extracted, the homologous recombination positive clones were screened by means of long fragment PCR.

[0126] ES cell identification: use the 5' or 3' homology arm detection primer pairs in Tables 2 to 3 for PCR amplification; 5.5kb fragments should be amplified from positive clones of 5' arm homologous recombination, and negative clones should have no product; 3' Arm homologous reco...

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Abstract

The invention belongs to the field of biotechnology, and discloses a construct integrating the functions of senescent cell localization, tracing, sorting, inducing senescent cell apoptosis and / or clearing senescent cells, and cells or animals transformed by the construct Model. The construct functions by targeting the P21 gene in the genome and inserting it into its proper position. The cells or animal models obtained by the method of the present invention can be used to study aging and aging-related diseases.

Description

technical field [0001] The present invention belongs to the field of biotechnology, more particularly, the present invention relates to a construct and its application in animal senescence tracking and elimination of senescent cells, and also relates to cells or animal models transformed by the construct. Background technique [0002] Senescent cells are cells that exit the cell cycle and enter a state of non-dividing, while maintaining cellular functions to a certain extent. The basic changes of senescent cells include nuclear swelling, increased nucleocytoplasmic ratio, changes in chromatin structure, reduced or lost cell replication capacity, cell function decline, and slowed metabolism. The more typical features are positive staining for senescence-associated β-galactosidase (SA-β-Gal); Senescence-Associated SecretoryPhenotype (SASP), that is, secretion of IL-1a, IL-1b, IL- 6, TGF-β1, TNF-α, CCL5, CXCL10 and other cytokines. The detection indicators involved in cellula...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/65C12N5/10A01K67/027
CPCC12N15/85C12N15/65C07K14/47A01K67/0273A01K2207/15A01K2227/105A01K2267/035
Inventor 何志颖刘中民陈苗苗潘欣卢艳丽
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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