Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector

A technology of PLEGFP-N1, plegfp-n1-spmyod1, applied in the directions of botanical equipment and methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc.

Inactive Publication Date: 2011-03-30
徐日福 +3
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0009] 1 It is not specifically ...

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  • Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector
  • Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector
  • Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector

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preparation example Construction

[0109] 2.6 Preparation of Competent Cells:

[0110] Apply CaCl 2 Prepare Escherichia coli competent DH5a strains by sensitization method. After overnight shaking culture at 37°C and 225rpm, the bacterial liquid was streaked on LB solid medium, cultured overnight in a 37°C incubator, and single clones were screened. Select a single colony and inoculate in 5mL LB medium, culture overnight, inoculate the culture in LB medium at 1 / 100, and cultivate until A 600 Up to 0.45-0.55, with CaCl 2 Sensitized Escherichia coli. The details are as follows: after 10 minutes of ice-bathing the bacterial solution, centrifuge at 4000 rpm for 20 minutes at 4°C to collect the bacterial cells, and then add ice-cold 0.1M CaCl 2 Sensitization solution, suspended bacteria, ice-bathed for 10 minutes, after re-collecting the bacteria, add ice-cold 0.1M CaCl 2 , resuspend the cells, add glycerol to a final concentration of 15%, aliquot 100 μL / tube, store at -70°C, and use for transformation.

[0111...

Embodiment 1

[0218] Liposome 2000-mediated expression of PLEGFP-N1-spMyoD1 vector plasmid in C2C12 cells. C2C12 cells not transfected with expression vector, cultured under the same conditions, and with the same concentration were used as negative control.

[0219] method:

[0220] Take the cultured C2C12 cells when the concentration reaches 50%, and add 2ml of antibiotic-free DMEM+10% fetal bovine serum culture medium to the cells, and the cells grow to 80%-90%, and the transfection can begin. At the same time, 30ul of 20ng / ul vector plasmid was added to Opti-MEM 220ul serum-free medium, with a total volume of 250ul, mixed gently, and left for 5 minutes. Take liposome 200010ul, add to Opti-MEM 240ul serum-free medium, total volume 250ul, mix gently. Leave for 5 minutes. Add the liposome mixture to the vector plasmid mixture and mix gently; let stand for 20 minutes. Change the medium of the cells every 20 minutes, add 1.5ml of antibiotic-free medium (DMEM+10% fetal bovine serum), and a...

Embodiment 2

[0223] Liposome 2000-mediated expression of PLEGFP-N1-spMyoD1 vector plasmid in 293T cells, 293T cells not transfected with expression vector, cultured under the same conditions, and with the same concentration were used as negative control.

[0224] method:

[0225] Take the cultured 293T cells when the concentration reaches 50%, add 2ml of antibiotic-free DMEM+10% fetal bovine serum culture medium to the cells, and the cells grow to 80%-90%, and the transfection can start. At the same time, 30ul of 20ng / ul vector plasmid was added to Opti-MEM 220ul serum-free medium, with a total volume of 250ul, mixed gently, and left for 5 minutes. Take liposome 2000 10ul, add to Opti-MEM 240ul serum-free medium, total volume 250ul, mix gently. Leave for 5 minutes. Add the liposome mixture to the carrier plasmid mixture and mix gently; let stand for 20 minutes. Change the medium of the cells every 20 minutes, add 1.5ml of antibiotic-free medium (DMEM+10% fetal bovine serum), and add a ...

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Abstract

The invention relates to construction of an efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector, belonging to the technical fields of animal gene engineering and animal molecule breeding engineering. The construction of the efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector is characterized by artificially synthesizing a skeletal muscle specific promoter sequence, using a RT-PCR method to amplify a gene overall length sequence from a skeletal muscle tissue of a pig, designing a fusion primer, using an overlapped PCR amplification method to obtain the promoter and a fusion gene sequence, cloning T/A to a pMD18-T vector, constructing the efficient expression vector of the PLEGFP-N1-spMyoD1 double promoter, and identifying with restriction enzyme and sequencing, packaging with a liposome method and amplifying, and monitoring virus titer and efficiency of infection. The invention has the beneficial effects: 1, Primer Premier 5.0, DNA Club and Oligo 6.0 are utilized to self-design the primer; 2, the restriction that an objective gene can not be subject to specific tissue expression in the past is overcome; 3, pertinency and accuracy of study on the objective gene functions are simultaneously improved; and 4, detection is fast and convenient, and has strong reliability.

Description

technical field [0001] The invention belongs to the technical fields of animal genetic engineering and animal molecular breeding engineering. Background technique [0002] With the continuous improvement of people's living standards and changes in dietary structure, the market's demand for livestock products has gradually changed from quantity to quality, and traditional conventional breeding methods have been unable to meet the needs of development. The breeding of transgenic animals has increasingly attracted great attention from many countries in the world and has invested a lot of manpower and material resources in research and development. From the successful acquisition of the first batch of transgenic pigs by Hammer et al. (1985) to the present, the research on transgenic pigs has gone through more than 20 years of history, including the genetic improvement of pig production performance and the use of pigs as donors for xenotransplantation. , have achieved a series o...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N15/66C12N5/10A01K67/027
Inventor 徐日福秦宁王志贤包艳波
Owner 徐日福
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