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lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of lncRNA marker

A technology of satellite cells and skeletal muscle, applied in the field of molecular genetics, to achieve the effect of promoting proliferation

Active Publication Date: 2021-04-23
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of muscle development, the research on the regulatory role of lncRNA is mostly focused on mice and some other model animals, and the regulatory role in the process of pig skeletal muscle development needs further research

Method used

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  • lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of lncRNA marker
  • lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of lncRNA marker
  • lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of lncRNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Acquisition of lnc-88672

[0026] Samples: The longissimus dorsi muscle tissue of 0-day-old horse body pigs was collected, wrapped in tinfoil paper, frozen in liquid nitrogen, and stored at -80°C.

[0027] Reagent: Reagent was purchased from Life Technologies; PrimeScript RT reagentKit with gDNA Eraser and DNA marker were purchased from TaKaRa; 2×Es Taq Master Mix (Dye) was purchased from Kangwei Century Biotechnology Co., Ltd.; 50×TAE buffer, nucleic acid dye, agar Sugar was purchased from Solebol.

[0028] 1) Extraction of total RNA

[0029] Take out the longissimus dorsi muscle tissue of 0-day-old horse body pigs from the -80°C refrigerator, add 1mL Trizol reagent, grind it fully with a tissue homogenizer, grind and pulverize it, let it stand for 10 minutes, and centrifuge at 4°C, 12000rpm for 10 minutes after fully lysing; Transfer the supernatant to a new 1.5mL centrifuge tube, add 200μL chloroform, shake vigorously for 15s, let stand at room temperature for 10...

Embodiment 2

[0039] Temporal expression properties of lnc-88672

[0040] Samples: The longissimus dorsi muscle tissues of 0-day-old, 28-day-old and 70-day-old horse body pigs were collected respectively, wrapped in foil paper, frozen in liquid nitrogen quickly, and stored at -80°C.

[0041] Reagent: Reagent was purchased from Life Technologies; PrimeScript RT reagentKit with gDNA Eraser and 2×SYBR Premix Ex Taq II were purchased from TaKaRa.

[0042] According to the DNA sequence (SEQ ID NO: 1) of lnc-88672, lnc-88672 quantitative primers were designed by AnnHyb software, as follows:

[0043] lnc-88672 upstream primer: 5′-ACAGCAAGCATCAGCCATCA-3′ (SEQ ID NO: 4)

[0044] Downstream primer: 5'-ACACCCTCTAGAACTCCAGGT-3' (SEQ ID NO: 5)

[0045] 18S rRNA upstream primer: 5′-ATGCCAGAGTCTCGTTCGTTAT-3′ (SEQ ID NO: 6)

[0046] Downstream primer: 5'-CGGACAGGATTGACAGATTGAT-3' (SEQ ID NO: 7)

[0047] According to the method in Example 1, total RNA was extracted and reverse transcribed from each grou...

Embodiment 3

[0051] The role of lnc-88672 in the proliferation of porcine skeletal muscle satellite cells

[0052] Cells: Porcine skeletal muscle satellite cells were isolated and cultured from the longissimus dorsi muscle tissue of 1-day-old piglets by combined digestion with 0.2% type I collagenase and 0.25% trypsin at 37°C.

[0053] Reagents: fetal bovine serum (FBS), high-glucose DMEM, and 0.25% trypsin were purchased from Gibco; type I collagenase, penicillin-streptomycin mixture, PBS buffer, and 4% paraformaldehyde were purchased from Solaibio; SDS- PAGE rapid gel preparation kit and rainbow prestained protein marker were purchased from Wuhan Boster Bioengineering Co., Ltd.; CCK-8 kit, PCNA, β-actin monoclonal antibody were purchased from Abcam; fluorescent secondary antibodies were purchased from American LI- COR company; transfection reagent riboFECTTMCP was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

[0054] For the lnc-88672 sequence, design multiple siRNA sequences ...

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Abstract

The invention belongs to the technical field of molecular genetics, and particularly relates to an lncRNA marker related to porcine skeletal muscle satellite cell proliferation and application of the lncRNA marker. The lncRNA marker is lnc-88672, and a nucleotide sequence of the lncRNA marker is SEQ ID NO.1. It is found for the first time that the expression of the lnc-88672 is related to the porcine skeletal muscle satellite cell proliferation and is continuously reduced along with the continuous increase of the day age of a pig, so that the lncRNA marker related to the porcine skeletal muscle satellite cell proliferation is provided; the porcine skeletal muscle satellite cell proliferation condition can be identified by detecting the expression of the lnc-88672; the inhibition of the expression of the lnc-88672 can promote the porcine skeletal muscle satellite cell proliferation; and a new idea and method are provided for clinical research of porcine skeletal muscle growth and development.

Description

technical field [0001] The invention belongs to the technical field of molecular genetics, and in particular relates to an lncRNA marker related to the proliferation of porcine skeletal muscle satellite cells and an application thereof. Background technique [0002] As an important part of human and animal body, skeletal muscle accounts for about 40% of its total body weight. The growth and development of skeletal muscle is inseparable from the proliferation and differentiation of myoblasts, and maintaining a dynamic balance between cell proliferation and differentiation is crucial to the growth and development of animal organisms. Skeletal muscle satellite cells are located between the sarcolemma and basement membrane. They belong to myogenic stem cells and have strong proliferation and differentiation potential. They can be used as a good model for exploring the process of porcine muscle formation in vitro, and have important guidance for exploring the process of porcine m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/113C12N5/10C12N5/077
CPCC12Q1/6888C12N5/0659C12N15/113C12Q2600/178C12Q2600/158C12Q2600/124C12N2510/00C12N2310/10
Inventor 吴怡琦曹果清秦本源杨阳杨帅蔡春波高鹏飞郭晓红李步高路畅
Owner SHANXI AGRI UNIV
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