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Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide

An antisense oligonucleotide and plasmid technology, applied in the field of embryo transfection, to achieve the effect of simple operation, fast speed and low cost

Inactive Publication Date: 2012-07-11
NORTHWEST A & F UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports of using electroporation technology to introduce plasmid DNA or antisense oligonucleotides into pre-implantation embryos.

Method used

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  • Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide
  • Method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide

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Effect test

Embodiment 1

[0038] The method for plasmid DNA transfection to attach pre-implantation embryos, comprises the following steps:

[0039] 1. First, pretreat the plasmid DNA:

[0040] a. The specific plasmid DNA used is pIRES2-AcGFP1-Nuc (Clontech). The plasmid DNA can use different plasmid vectors to carry different target genes according to the needs. The plasmid vector to be transfected generally requires a fluorescent marker gene or Other marker genes for subsequent observation and screening;

[0041] The plasmid DNA is subjected to endotoxin removal treatment, which is to eliminate the effect of endotoxin in the plasmid on the growth and development of the embryo;

[0042] b. Dilute the endotoxin-free plasmid DNA in In the solution, the plasmid DNA is different, and the concentration of dilution during electroporation is also different. The diluted concentration of pIRES2-AcGFP1-Nuc plasmid DNA is controlled at 40 μg / ml; although the expression of each plasmid DNA is different, after ma...

Embodiment 2

[0058] A method for transfecting pre-implantation embryos with antisense oligonucleotides, comprising the following steps:

[0059] 1. First, antisense oligonucleotides are pretreated:

[0060] a. Take standard control antisense oligonucleotides as an example to illustrate. Antisense oligonucleotides can be designed for different target genes according to needs. Antisense oligonucleotides to be transfected generally have fluorescent groups Marking for subsequent observation and screening;

[0061] Specifically, the standard control antisense oligonucleotide (commercially available) labeled with lissamine will not affect the function of any gene, and is only a control group in normal experiments. Since the sequence of the antisense oligonucleotide is very short, only about 25bp, it is artificially synthesized. As long as the standard control antisense oligonucleotide can be successfully transfected, other antisense oligonucleotides targeting any gene can be transferred into i...

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Abstract

The invention discloses a method for transfection and implantation of pre-embryo by plasmid DNA (deoxyribonucleic acid) or antisense oligonucleotide, which comprises the following steps of: (1) preparing an electroporation solution to be transfected; (2) weakening embryo zona pellucida; and (3) implanting the embryo into the prepared electroporation solution, standing at the room temperature, and then implanting the embryo together with the electroporation solution into an electroporation slot for electroporation. Because the method of electroporation transfection is adopted, two or more kinds of plasmid DNA or antisense oligonucleotide can also be simultaneously transferred into the implanted pre-embryo together; and if the exogenous fragment carried by the constructed plasmid DNA is a key gene or an interference fragment targeting a key gene, the concrete effect of the key gene in the development process of the implanted pre-embryo of a mouse can be concretely researched.

Description

technical field [0001] The invention belongs to the field of embryo transfection, and relates to a method for transfecting pre-implantation embryos with plasmid DNA or antisense oligonucleotides. Background technique [0002] Mammalian early embryonic development is controlled by its own genetic mechanism. The basic method to study the genetic mechanism is to regulate the expression of key genes, observe the subsequent development and analyze the role of the key genes in the development process. At present, introducing exogenous genes into embryos to overexpress or inhibit gene expression is a consistent and classic method for studying gene functions. [0003] The transfer of exogenous genes into cells / embryos or integration into the genome is mainly through transfection, viral vector infection and microinjection, while the inhibition of gene expression mainly relies on gene knockdown and gene knockout techniques. However, as far as pre-implantation embryos are concerned, i...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/87
Inventor 彭辉张涌常博皞
Owner NORTHWEST A & F UNIV
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