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Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid)

A mammalian electroporation technology, applied in the field of embryo transfection, can solve the problems of complicated operation, large embryo trauma, and high cost

Inactive Publication Date: 2012-01-25
NORTHWEST A & F UNIV +1
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Problems solved by technology

Microinjection can achieve accurate transfection, but it requires expensive micromanipulation instruments, and the operation is complicated, causing greater trauma to the embryo, and the number of transfections is limited

Method used

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  • Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid)
  • Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid)
  • Electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid)

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Embodiment Construction

[0021] The invention provides an electroporation method for embryo transfection with siRNA, which is especially suitable for embryo transfection of mammals, including weakening treatment of the zona pellucida of the embryo and complexation treatment of siRNA. In the following, the present invention will be further described in detail in conjunction with the specific electroporation process and transfection results, which are explanations rather than limitations of the present invention.

[0022] The siRNA transfection is based on the fact that siRNA enters the embryo through the transient micropore formed by the embryonic zona pellucida and the cell membrane under a pulsed electric field. It is a biophysical transfection method, so there is no special species requirement for the embryo to be transfected. . There may be some differences in the weakening time of the zona pellucida and the electroporation parameters when the embryos with large differences in morphology and struct...

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Abstract

The invention discloses an electroporation method for transfection of mammal embryo with siRNA (small interfering Ribose Nucleic Acid). The electroporation method comprises the following steps: (1) weakening an embryo zona pellucid; (2) preparing electroporation solution containing siRNA; and (3) conveying the embryo into the prepared electroporation solution containing siRNA, standing at the room temperature, and then conveying the embryo and the electroporation solution into an electric turn trough for electroporation. The electroporation method comprises the steps of weakening the embryo zona pellucid and complexing the siRNA, and then placing the embryo under the action of a pulsed electric field to increase the permeability and the membrane conductance of the embryo instantaneously, and thus the siRNA which is difficult to penetrate through a cell membrane under the normal physiological condition enters the cell. Statistic observation and analysis prove that the stable positive transfection efficiency of survival embryos exceeds 95%, and the practical effective utilization rate of the collected total embryo exceeds 70%. The electroporation method is a safe, simple and efficient transfection technology of mammal embryo with siRNA.

Description

technical field [0001] The invention belongs to the field of embryo transfection, and relates to RNA interference mediated by siRNA, in particular to an electroporation method for siRNA transfection of mammalian embryos. Background technique [0002] In recent years, RNA interference (RNAi) mediated by siRNA (small interfering RNA) has gradually become a powerful tool for studying gene function. Traditional methods for transducing siRNA into cells include virus-mediated method and microinjection method. The virus-mediated method can be used in difficult-to-transfect cells, but requires the cells to be in the dividing phase, and there are safety issues. Microinjection can achieve accurate transfection, but it requires expensive micromanipulation instruments, and the operation is complicated, causing greater trauma to the embryo, and the number of transfections is limited. Therefore, finding a safe, convenient and efficient embryo transfection method has become an urgent pro...

Claims

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Application Information

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IPC IPC(8): C12N15/873
Inventor 常博皞张涌彭辉苏建民
Owner NORTHWEST A & F UNIV
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