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Repair method of iPSCs mitochondrial DNA mutation sites based on mitoTALENs

A technology of mutation site and repair method, which is applied in the field of gene knockout, can solve the problems of tediousness, high off-target rate, cumbersome design, etc., and achieve the effects of improving efficiency, shortening experiment time, and convenient modification work

Inactive Publication Date: 2019-03-22
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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Problems solved by technology

[0018] 1. A large number of molecular cloning and sequencing operations are required, which is very cumbersome. Although many commercial companies provide assembled modules, which shortens the construction and experiment cycle, most laboratories are difficult to complete the complete operation of TALEN technology by themselves, which has caused difficulties in its promotion. obstacle;
[0019] 2. The triplex property of ZFN technology, its design is more cumbersome than TALEN, and highly dependent on the target sequence and its upstream and downstream sequences, high off-target rate and high cytotoxicity and other restrictive factors;
[0020] 3. However, CRISPR / Cas technology is context-dependent and can only be applied to targets with upstream PAM sequences;

Method used

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  • Repair method of iPSCs mitochondrial DNA mutation sites based on mitoTALENs
  • Repair method of iPSCs mitochondrial DNA mutation sites based on mitoTALENs
  • Repair method of iPSCs mitochondrial DNA mutation sites based on mitoTALENs

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Embodiment 1

[0048] A method for repairing the mitochondrial mutation site of iPSCs derived from a MELAS patient, comprising the following steps:

[0049] (1) Obtaining MiPSCs from patients with Melas syndrome

[0050] The fibroblast reprogrammed (non-transgenic) fibroblasts obtained from the skin of patients with Melas syndrome (containing more than 90% 3243A>G mutation efficiency mtDNA), MiPSCs under the condition of no serum and no feeder layer, and the m3243A>G heterogeneity rate is greater than 80 %.

[0051] Extract the total cell DNA with a DNA extraction kit, the PCR primer sequence used: F: cctcggagcagaacccaacct, R: cgaaagggttgtagtagcccgt, the PCR product of wild type cells is 634bp, when there is a mutation in mitochondrial DNA A3243G, the PCR product can be cut by the endonuclease ApaI into Two sections: 424 and 210bp, software ImageJ for subsequent analysis;

[0052] (2) Determination of the amino acid sequence of TALEN:

[0053]Mitochondrial DNA only encodes 1% of the mitoc...

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Abstract

The invention discloses a repair method of iPSCs mitochondrial DNA mutation sites based on mitoTALENs. The method disclosed by the invention has the advantages of high selectivity, high repair efficiency and low miss rate; the repair method can be successfully applied to induced pluripotent stem cells MiPSCs of specific sources of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis andstroke-like episodes) patients.

Description

technical field [0001] The invention relates to a gene knockout method, in particular to a mitoTALENs-based method for repairing mutation sites in mitochondrial DNA of iPSCs. Background technique [0002] Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is one of the most common maternally inherited mitochondrial diseases. Molecular genetic studies have associated the MELAS phenotype with more than 10 mutations in different mitochondrial tRNAs or protein-coding genes, although the majority of patients with MELAS (>80%) were found to have mitochondrial tRNAs Leu has the A3243G mutation in the UUR gene. This mutation is estimated to occur in 1 in 6000 (260 individuals). This mt3243A>G mutation results in impaired mitochondrial translation and protein synthesis, including subunits of the mitochondrial electron transport chain complex leading to impaired mitochondrial energy production (see: Whitehead, M.T., et al., Cortic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/11
CPCC12N5/0696C12N15/85C12N2510/00
Inventor 范勇杨翌
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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