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Long-chain RNA Lnc-2193 and application thereof

A rnalnc-2193, long-chain technology, which is applied in the field of genetic engineering to achieve the effects of improving oocyte quality, promoting maturation and development, and improving porcine oocyte maturation

Active Publication Date: 2020-04-17
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the research on porcine tissue-specific lncRNA, especially porcine follicle lncRNA and its regulatory mechanism, has not been reported so far.

Method used

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  • Long-chain RNA Lnc-2193 and application thereof
  • Long-chain RNA Lnc-2193 and application thereof
  • Long-chain RNA Lnc-2193 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Single oocyte transcriptome sequencing to obtain transcripts of lncRNA2193

[0034] 1. Library establishment

[0035] Purchase fresh Landrace pig ovaries from slaughterhouses, extract follicular fluid from follicles with a diameter of 3-6 mm, select cumulus-oocyte complexes (COCs) with more than 3 layers of cumulus coating with the aid of a stereo microscope, and place Cultured in oocyte maturation medium at 39°C, 5% CO2, 100% humidity incubator. The mature medium is TCM-199 and contains 0.1% PVA, 3.05mM D-glucose, 0.91mM Na-pyruvate, 1μg / ml gentamicin, 0.57mM cysteine, 0.5μg / ml LH, 0.5μg / ml FSH, 10ng / ml EGF. COCs matured and cultured for 44 hours were placed in an EP tube containing 0.1% hyaluronidase for vortex vibration to separate the cumulus cells from the oocytes, and the mature naked eggs with the first group of cumulus cells completely removed were selected in the washing solution (DPBS containing 1% BSA) was washed four times, and then 0.5% pronase...

Embodiment 2

[0038] Example 2 Fluorescence quantitative detection kit and detection method of pig long-chain RNA Lnc-2193 gene

[0039] In this example, a pair of fluorescent quantitative PCR detection primers was designed to identify and identify the objective presence of the porcine long-chain RNA Lnc-2193 described in Example 1 in porcine oocytes. The identification method of this embodiment includes the following steps:

[0040] 1. Sample collection

[0041] Collect mature porcine cumulus-oocyte complexes (COCs) for 0, 24 and 44 hours, 0.1% hyaluronidase to peel off the cumulus cells in the COCs, pick out the live and clean cumulus cells in each group Oocytes, each group received 60 oocytes, washed 3 times in preheated DPBS, put them into EP tubes, put the EP tubes into liquid nitrogen for quick freezing, and stored them in a -80°C refrigerator .

[0042] 2. Extraction of total RNA from samples

[0043] The total RNA of oocytes was extracted using the QIAGEN RNeasy Mini Kit, and th...

Embodiment 3

[0054] Example 3 The role of lncRNA2193 in porcine oocyte maturation

[0055] 1. Microinjection of porcine oocyte siRNA

[0056] Put the GV stage oocytes into the operating solution containing 7.5 μg / ml CB and 0.3% BSA, inject under an inverted microscope using a micromanipulator, and inject 10 pL of 20 uM siRNA. The siRNAs for knocking down lncRNA2193 in this example are siRNA411 (shown in SEQ ID NO: 9) and siRNA258 (shown in SEQ ID NO: 10).

[0057] Microinjection parameters were injection pressure 250hp, maintenance pressure 100hp, and injection time 0.3-0.5s. Viable oocytes were collected after maturation and culture for 44 hours after injection to detect the level of lncRNA2193. The result is as image 3 As shown, it shows that the siRNA of the present invention can specifically and significantly knock down lncRNA2193.

[0058] 2. Detection of porcine oocyte maturation ability

[0059] After knocking down lncRNA2193, the nuclei were stained by Hoechst33342, and the s...

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Abstract

The invention provides a long-chain RNA Lnc-2193 and application thereof. The transcript sequence of the long-chain RNA Lnc-2193 gene is any one of nucleotide sequences shown in SEQ ID NO: 1-6 in a sequence table. The invention also provides a fluorescent quantitative PCR detection primer pair and a detection kit of the long-chain RNA Lnc-2193; the detection primer pair has nucleotide sequences asshown in SEQ ID NO: 7 and SEQ ID NO: 8. According to the invention, the long-chain RNA Lnc-2193 is found to have the effect of promoting maturation and development of porcine oocytes for the first time; the long-chain RNA Lnc-2193 can be used as a target for improving pig oocyte maturation and improving oocyte quality; and the in-vitro production of more and better embryos can be promoted. The long-chain RNA Lnc-2193 has the broad application prospect in the aspects of conventional pig breeding and breed conservation and propagation of local varieties.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a long-chain RNA Lnc-2193 and its application. Background technique [0002] Pig is an important economic animal and a biomedical model animal. The research on its reproductive regulation can not only provide a reference for the study of human reproductive physiology and pathological process, but also provide a theoretical basis for improving the reproductive performance of pigs. However, low fecundity is currently a difficult problem plaguing the pig industry in my country. Low fecundity of pigs is reflected in various stages, including low meiotic maturation rate of eggs, low early embryo development rate after fertilization, low pregnancy rate and low live offspring rate, etc. Among them, the high rate of loss during porcine oocyte maturation and early embryonic development has become an important issue. Only 60%-70% of porcine germinal vesicular stage (GV stage)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11C12Q1/6888C12Q1/6851
CPCC12N15/113C12N2310/10C12N2310/14C12Q1/6851C12Q1/6888C12Q2600/158C12Q2600/178C12Q2531/113C12Q2521/107C12Q2563/107
Inventor 杨彩侠杜志强王沛超
Owner YANGTZE UNIVERSITY
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