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Eukaryotic expression vector of one specific target mitochondrion and its construction process and use

A eukaryotic expression vector, mitochondrial technology, applied in the use of vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of short expression time, potential safety hazards, low target genes, etc., to improve the efficiency of clone screening, improve Effect of Transfection on Cloning Efficiency

Inactive Publication Date: 2006-10-18
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although viral vectors have high transfection efficiency, their preparation is complicated, immunogenic, and cannot be used repeatedly in vivo, and there are potential safety hazards, so further improvement is necessary
The biggest problem with non-viral vectors is that the transfection efficiency is low, the expression time is short, and the inserted foreign gene and the selection marker gene use their own independent expression cassettes, the selection pressure is exerted on the expression of the drug-resistant gene, and the cloned gene is screened out. The expression of the target gene is often low or even undetectable
The introduction of the sequence of the internal ribosome entry site (IRES) into the targeting vector has not been seen so far, and the establishment of a mitochondrial-targeted eukaryotic expression vector that allows simultaneous expression of the inserted gene and the screening gene

Method used

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  • Eukaryotic expression vector of one specific target mitochondrion and its construction process and use
  • Eukaryotic expression vector of one specific target mitochondrion and its construction process and use
  • Eukaryotic expression vector of one specific target mitochondrion and its construction process and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of Mitochondrial Targeting Vector

[0024] 1. Materials

[0025] The pDsRED1-nl and pIRESneo vectors are products of CLONTECH. RT-PCR and ligation kits were purchased from MBI Company. pcDNA3.1 / myc-HisA was purchased from Invitrogen. The host strain DH5α, restriction endonuclease, and DNA polymerase klenow fragment were purchased from Takara Company. The pGEM-T-Easy vector is a product of Promega. Plasmid DNA purification and gel recovery kits are products of Shanghai Shenergy Gaming Co., Ltd.

[0026] 2. Methods and results

[0027] 1. Construction of pcDNAmito and pcDNAmito-RFP

[0028] (1) Amplify the COX8 signal peptide sequence by PCR

[0029] According to the signal peptide sequence of the mitochondrial protein cytochrome C oxidative subunit VIII (COX8) (Genebank GI: 1311703), the first 29 amino acids of COX8 were selected as the leader sequence, and its cDNA sequence was shown in sequence 1 in the sequence table, and the amplificatio...

Embodiment 2

[0045] Example 2 Expression of pIRES-mito and pIRES-mito-RFP in tumor cell line Hela

[0046] 1. Materials

[0047] Lipofectamine2000 liposome was purchased from Invitrgene Company, DMEM was purchased from Gibco BRL Company, rhodamine 123 and G418 were products of Sigma Company, and Radiance2000 laser confocal microscope (LSCM) was a product of American Bio-Rad Company.

[0048] 2. Method

[0049] 1. Purify pIRESmito-RFP, pcDNAmito-RFP and pDsREDnl plasmid DNA by SDS alkaline lysis-polyethylene glycol precipitation method.

[0050] 2. Transfect Hela cells

[0051] Press 6×10 4 Hela cells were seeded on a 24-well plate and used for transfection when the cells grew to 50% confluent. Add lipofectamin2000 liposomes to pIRESmito-RFP and pDsREDnl respectively at 1:3, mix well and incubate at room temperature for 30 min, then add to the culture plate, set at 37°C, 30% CO 2 After culturing in an incubator for 5 hours, the culture medium containing plasmid DNA / liposome was discard...

Embodiment 3

[0064] Example 3 Expression of pmito-IRES-RFP in tumor cell line Hela

[0065] 1. Materials

[0066] Lipofectamine2000 liposome was purchased from Invitrgene Company, DMEM was purchased from Gibco BRL Company, rhodamine 123 and G418 were products of Sigma Company, and Radiance2000 laser confocal microscope (LSCM) was a product of American Bio-Rad Company.

[0067] 2. Method

[0068] 1. Purify pmito-IRES-RFP, pmito-RFP plasmid DNA by SDS alkaline lysis-polyethylene glycol precipitation method.

[0069] 2. Transfect Hela cells

[0070] Press 6×10 4 Hela cells were seeded on a 24-well plate and used for transfection when the cells grew to 50% confluent. Add lipofectamin2000 liposomes to Pmito-IRES-RFP and pmito-RFP respectively at 1:3, mix well and incubate at room temperature for 30min, then add to the culture plate, place at 37°C, 3% CO 2 Cultivate in an incubator for 5 hours, discard the medium containing plasmid DNA / plastids, add 10% calf serum DMEM, and continue culturi...

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Abstract

The present invention discloses one kind of target mitochondrion expression vector and its preparation process and use. The vector of the present invention contains COX precursor signal peptide sequence, HCMV enhancer, promoter, polyclonal site and polyA signal and RFP red fluorescence indicator signal. The preparation process of the vector includes the preparation of pcDNAmito, the preparation of pcDNAmito-RFP, the preparation of pIRESmito and pIRESmito-RFP, the preparation of pmito-IRES-RFP and other steps. The vector of the present invention may be used in inserting exogenous gene into polyclonal site of the vector and conveying exogenous gene into mitochondrion, and may be also used in coexpressing inserted exogenous gene with COX8 and neo gene to raise the cloning screening efficiency.

Description

technical field [0001] The invention relates to a eukaryotic expression vector, in particular to a eukaryotic expression vector specifically targeting mitochondria, and also relates to its construction method and application. Background technique [0002] Human mitochondrial DNA is the only organelle with autonomous replication ability outside the mammalian nuclear genome, which produces the energy needed by the body through the process of oxidative phosphorylation. In 1988, Wallace et al. proposed for the first time that Leber hereditary optic neuropathy was caused by mutations in mitochondrial DNA (mitochondrial DNA, mtDNA). Subsequent studies have found that about a hundred diseases are related to mtDNA mutations, including Leler hereditary optic neuroophthalmopathy, mitochondrial encephalomyopathy, lactic acidosis, diabetes, neurodegenerative diseases, tumors, and human aging. So far, there are no effective measures for the treatment of mtDNA mutation-related diseases. ...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/65
Inventor 陆应麟郝好杰徐元基王妍杜芝燕
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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