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Method for completely silencing target gene of embryo by improving microinjection of siRNA

A microinjection and embryo technology, applied in the direction of using microinjection method, other methods of inserting foreign genetic materials, recombinant DNA technology, etc., can solve the problems of 100% failure rate and affect the experimental effect, so as to make it easy to judge the injection result , easy to master and generalize

Inactive Publication Date: 2018-03-30
ANIMAL HUSBANDRY RES INST OF HEILONGJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that the success rate of injection cannot reach 100% during microinjection, which affects the effect of subsequent experiments.

Method used

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  • Method for completely silencing target gene of embryo by improving microinjection of siRNA
  • Method for completely silencing target gene of embryo by improving microinjection of siRNA
  • Method for completely silencing target gene of embryo by improving microinjection of siRNA

Examples

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Effect test

Embodiment 1

[0033] A method for improving microinjection of siRNA to completely silence embryonic target genes, the method comprising the steps of:

[0034] S1: Remove all granulosa cells from fertilized oocytes with hyaluronidase;

[0035] S2: After washing with PZM-5 medium for 3 times, put it into an incubator at 38.5 degrees Celsius, 100% humidity, and 5% carbon dioxide to recover for 15 minutes;

[0036] S3: Under an inverted microscope, use a microsyringe pump to inject 15 pl of the target liquid;

[0037] S4: Check the fluorescence of the injected embryos under a fluorescent microscope, and if there is a fluorescent agent, the injection is successful.

Embodiment 2

[0039] According to the improved method of microinjecting siRNA to completely silence embryonic target genes described in Example 1, the content of hyaluronidase in the decumulus fluid is 0.1%, and the content of EDTA is 0.1%.

Embodiment 3

[0041] According to an improved method of microinjecting siRNA to completely silence embryonic target genes described in Example 1 or 2, the method of removing the granulosa cells described in the oocytes was mixed by a vortex mixer for 30 seconds.

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Abstract

The invention provides a method for completely silencing a target gene of an embryo by improving microinjection of siRNA. In a developmental biology study, in order to study gene functions, the expression of genes often needs to be inhibited, and siRNA interference is a common means, so that a specific target mRNA can be silenced, the silencing efficiency of the target gent cannot reach 100%, andan expected effect is not achieved. The method for completely silencing the target gene of the embryo by improving microinjection of the siRNA comprises the following steps: S1: removing all granularcells from fertilized oocytes with hyaluronidase; S2: washing the granular cells with a PZM-5 culture medium, placing the washed granular cells into a culture incubator with the temperature of 38.5 DEG C, the humidity of 100% and the carbon dioxide of 5% for recovery for 15min; S3: injecting 15pl of a target liquid with a microinjection pump under an inverted microscope; S4: detecting fluorescencein the embryo after injection under a fluorescence microscope, and determining that the injection is successful if the embryo contains a fluorescent agent. The method is applied to the technical field of developmental biology.

Description

Technical field: [0001] The invention relates to an improved method for microinjecting siRNA to completely silence embryonic target genes. Background technique: [0002] In developmental biology research, in order to study gene function, it is often necessary to inhibit gene expression, siRNA interference is a commonly used method, which can silence specific target mRNA. According to current reports, the efficiency of target gene silencing has not been able to reach 100%, so that the expected effect cannot be achieved. The reason is that the success rate of the injection cannot reach 100% during the microinjection, which affects the follow-up experiment effect. Invention content: [0003] The purpose of the present invention is to provide an improved method for microinjection of siRNA to completely silence the target gene of the embryo, which can effectively improve the success rate of microinjection and ensure that the success rate reaches 100%, so that in further experi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89
CPCC12N15/89
Inventor 郭镇华刘娣吕蕾赛福杨云春付博汪亮刘自广马红李忠秋张冬杰
Owner ANIMAL HUSBANDRY RES INST OF HEILONGJIANG ACADEMY OF AGRI SCI
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