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Method for transforming exogenous mitochondrion into mammal cells

A mammalian and mitochondrial technology, applied in the field of biogenetic engineering, can solve the problems of unclear mechanism, affecting the function of target RNA, unrealized expression and functional identification of artificially synthesized mitochondrial DNA, and achieves the effect of high purity

Active Publication Date: 2015-05-20
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One solution is to express the target gene in the nuclear genome, add the positioning sequence and import it into the mitochondria. There are some problems in this solution: First, most of the mammalian mitochondrial genome encodes functional RNA, and the RNA encoded by the nuclear genome currently enters the mitochondria The mechanism is not very clear, it is known that a positioning RNA sequence needs to be added, but whether this sequence will affect the function of the target RNA is still unknown
[0004] In 2010, Craig J.Venter's laboratory successfully used synthetic biology technology to obtain the complete sequence of artificially synthesized mouse mitochondrial DNA, but did not make any changes to it, nor did it realize the synthesis of artificially synthesized mitochondrial DNA in mitochondria and in mammalian cells. Expression and functional characterization

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  • Method for transforming exogenous mitochondrion into mammal cells
  • Method for transforming exogenous mitochondrion into mammal cells
  • Method for transforming exogenous mitochondrion into mammal cells

Examples

Experimental program
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Effect test

Embodiment 1

[0049] This example is to prove that exogenous mitochondria can enter mammalian cells through endocytosis. By stably expressing the mitochondrial-localized fluorescent protein EYFP in macrophages, the endogenous mitochondria of macrophages are labeled with EYFP, and the mitochondrial-localized fluorescent protein DsRed2 is stably expressed in NIH3T3 cells, and the mitochondria of NIH3T3 cells are labeled with DsRed2 , Isolate NIH3T3 mitochondria with DsRed2 labeling, add them to the culture system of macrophages, and observe them under a confocal microscope after 12 hours. It can be observed that DsRed2-labeled mitochondria enter into macrophages, which can present the same appearance as that in macrophages. Source consistent morphology of EYFP-tagged mitochondria. Specifically:

[0050] 1. Mouse macrophage cell line RAW264.7 cells and NIH3T3 cell culture medium (high glucose DMEM (purchased from Hyclone, product number: SH30022.01B); 10% fetal bovine serum (purchased from GI...

Embodiment 2

[0061] In this example, the circular mitochondrial DNA of the designed sequence is obtained by gene introduction into mouse mitochondrial DNA, that is, the insertion of GFP and Linker sequences, primer synthesis, and DNA splicing, that is, the circular mitochondrial DNA containing the GFP-COX-I fusion gene .

[0062] 1. Design a new artificial circular mitochondrial DNA:

[0063] This example is to obtain circular mitochondrial DNA by gene introduction into mouse mitochondrial DNA, that is, inserting GFP and Linker sequences, specifically, based on the known mitochondrial DNA sequence of wild-type C57 BL / 6J mice (sequence source : NCBI GenBank: EF108336) and inserted the GFP gene and Linker sequence (specifically as shown in Seq.ID No.1) at the 5328 site, and designed a circular mitochondria containing a GFP-COX-I fusion gene of about 5Kb DNA.

[0064] 2. According to the specific composition of the designed circular mitochondrial DNA, a plurality of 50bp-60bp DNA fragments ...

Embodiment 3

[0070] This example describes the process of assembling the circular mitochondrial DNA obtained in Example 2 with the mitochondrial shell of NIH3T3 Rho0 cells in vitro to make artificially synthesized mitochondria, and the process of extracting and identifying RNA and DNA of artificially synthesized mitochondria . figure 2 Shown is the detection of the correct transcript in the synthetic mitochondria ( figure 2 A) and DNA replication ( figure 2 B).

[0071] 1. Cultivation of Rho0 cells without mitochondrial DNA:

[0072] 1. Add 1.5 μg / mL ditercalinium or 250 ng / mL ethidium bromide, 50 μg / mL uridine (Sigma) and 110 μg / mL sodium pyruvate to the NIH3T3 cell culture medium;

[0073] 2. Continuously cultivate for one month according to the conventional method;

[0074] 3. Culture with NIH3T3 Rho0 medium (high glucose DMEM, 10% fetal bovine serum, 50 μg / mL uridine, 110 μg / mL sodium pyruvate, penicillin and streptomycin double antibodies);

[0075] 4. Collect the cells to obt...

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Abstract

Disclosed are synthetic mitochondria obtained by introducing exogenous DNA into mitochondria or mitochondrial shells. Cells containing exogenous mitochondria are then obtained by introducing the synthetic mitochondria into mammalian cells via endocytosis, thereby allowing the exogenous mitochondria to perform effectively within the cells. After being introduced, synthetic mitochondrial DNA genes can be expressed stably, and passaged effectively. The method for introducing exogenous mitochondria into cells can serve as a new mitochondrial molecular cloning method, performing gene knockout, gene knock-in, gene rearrangement etc. within mitochondria, thereby enabling any molecular cloning of mammalian mitochondrial DNA to be engineered, which has significant implications for the treatment of diseases caused by mitochondrial DNA mutations.

Description

technical field [0001] The invention relates to the field of biological genetic engineering, in particular to a method for introducing exogenous mitochondria into mammalian cells. Background technique [0002] Mitochondria are the most important organelles of eukaryotes, which are responsible for more than 90% of the energy supply of cells. Mitochondria carry an independent genome, mitochondrial DNA, which has an independent gene transcription and protein translation structure different from the nuclear genome. Mammalian cell mitochondrial DNA codes 22 tRNAs, 2 rRNAs, and 13 polypeptides were detected. These encoded polypeptides are very critical subunits in various protein complexes for mitochondrial aerobic respiration. A large number of studies have shown that mutations or reduced expression of these polypeptides can significantly inhibit cellular aerobic respiration. [0003] With the rapid development of modern molecular biology, humans have been able to carry out vari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N5/071
CPCC12N2510/00C12N15/87C12N5/0645
Inventor 刘兴国裴端卿刘景磊刘雪宾
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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