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miRNA for promoting maturing of bovine oocytes and application thereof

An oocyte, mature technology, applied in DNA/RNA fragments, germ cells, animal cells, etc., can solve the problem that the role of exosomal miRNA has not been studied much.

Inactive Publication Date: 2019-03-12
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on exosomal miRNA in cells mainly focuses on the participation of exosomal miRNA in the process of cell migration, differentiation, proliferation, etc., but there is not much research on the role of exosomal miRNA in bovine in vitro embryo culture

Method used

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  • miRNA for promoting maturing of bovine oocytes and application thereof
  • miRNA for promoting maturing of bovine oocytes and application thereof
  • miRNA for promoting maturing of bovine oocytes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Sequencing method to identify miRNAs that can promote the maturation of bovine oocytes

[0026] After the ovaries were collected in the slaughterhouse, they were placed in physiological saline with penicillin and streptomycin at 37°C and brought back to the laboratory within 4-6 hours. The collected ovaries were rinsed three times with preheated normal saline to remove impurities and blood, and the fat blocks, connective tissue and fallopian tubes on the surface of the ovary were removed with scissors, and the pruned ovaries were washed again with normal saline. Put the treated ovary into a beaker of normal saline with double antibody, and place it on a constant temperature platform at 37°C for later use. Then use a 10 mL syringe (with an 8-gauge needle) to aspirate the contents of follicles with a diameter of 2-8 mm on the surface of the ovary.

[0027] Divide bovine ovarian follicles into large (>8mm), medium (2-8mm), and small (<2mm) groups according to th...

Embodiment 2

[0033] Embodiment 2 detects the expression level in the bovine oocyte with real-time PCR technique

[0034] The RNA of two groups of follicles was extracted by exosome extraction kit and Trizol method, and cDNA was obtained by reverse transcription.

[0035] Using the transcribed cDNA obtained by the above reverse transcription reaction as a template, set up three duplicate wells for each template of each gene to be tested, and operate on ice.

[0036] Set up the reaction steps according to the two-step PCR. After the reaction is completed, according to the melting curve and Ct value, the relative expression value of the target gene in this template is obtained according to the formula and then analyzed.

[0037] The result is as figure 2 As shown, miRNA-378 was significantly different in different groups of cells, with the highest expression level in the small follicle group, followed by the medium follicle group, and the lowest expression level in the large follicle group...

Embodiment 3

[0038] Example 3 Application of miRNA-378 in promoting bovine oocyte maturation

[0039] Two compounds, miRNA-378agomir (analogue) and miRNA-378antagomir (inhibitor), were synthesized respectively. Among them, Agomir is a specially labeled and chemically modified double-stranded small RNA that regulates the biological functions of target genes by simulating endogenous miRNA. Antagomir is a specially labeled and chemically modified single-stranded small RNA designed according to the mature miRNA sequence, and is a highly effective inhibitor specially used to inhibit endogenous microRNA.

[0040] Add miRNA analogs to bovine oocyte in vitro maturation culture medium by liposome encapsulation (TAKALA kit): Mix 3 μL of miRNA and 3 μL of transfection with 22 μL of buffer respectively, and then mix the two mixtures Let it stand still for 20 minutes, drop into 450 μL bovine oocyte maturation medium in vitro and shake well (final concentration is 120nnol / L). The medium is preheated wi...

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Abstract

The invention discloses miRNA-378 and application thereof. The miRNA-378 is differentially expressed in the bovine oocytes of different sizes. Proofed by verification, a miRNA-378 inhibitor can be used for promoting the maturing of the bovine oocytes. Based on this, the invention also discloses a culture medium containing the miRNA-378 inhibitor for culturing the bovine oocytes and a method for correspondingly promoting the maturing of the bovine oocytes. The miRNA-378 and the application thereof disclosed by the invention have important meaning on promoting the growth and development of embryos outside the bovine body.

Description

technical field [0001] The invention belongs to the field of oocyte culture, and in particular relates to a miRNA for promoting maturation of bovine oocytes and application thereof. Background technique [0002] Exosomes were first discovered in sheep reticulocytes in 1983, and Johnstone named them "exosomes" in 1987. Exosomes are disc-shaped vesicles with a diameter of 40-100 nm, which can be secreted by various cells under normal and pathological conditions. Exosomes are mainly derived from the multivesicular bodies formed by the invagination of intracellular lysosomal particles, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane and the cell membrane. [0003] When exosomes were discovered, they were once considered a way for cells to excrete waste. With the deepening of research on exosomes, including their biological origin, distribution, material composition and transportation, and intercellular signal transduction, E...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N5/075C12Q1/6876
CPCC12N5/0609C12N15/113C12N2310/141C12N2500/40C12Q1/6876C12Q2600/178
Inventor 滑留帅吕晨晨王二耀王璟王之保柏中林辛晓玲施巧婷陈付英徐照学
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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