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Application of RBP4 protein or coding gene thereof in regulation and control of myoblast differentiation and fusion

A technology of encoding genes and myoblasts, which is applied in the field of genetic engineering, can solve problems such as the unclear function of muscle fibers, and achieve the effect of improving expression

Active Publication Date: 2020-11-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research report on the role of RBP4 in the regulation of muscle development, and its role in muscle cell differentiation and muscle fiber formation is still unclear

Method used

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  • Application of RBP4 protein or coding gene thereof in regulation and control of myoblast differentiation and fusion
  • Application of RBP4 protein or coding gene thereof in regulation and control of myoblast differentiation and fusion
  • Application of RBP4 protein or coding gene thereof in regulation and control of myoblast differentiation and fusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The construction of embodiment 1 porcine RBP4 gene overexpression vector

[0034] 1. Acquisition of CDS region sequence of pig RBP4 gene

[0035] A pair of PCR primers were designed according to the porcine RBP4 gene sequence (Gene ID: 397124), and a BamHI restriction site CGGGATCC (SEQ ID NO.3) and a NotI restriction site ATTTGCGGCCGC (SEQ ID NO. 4), the sequences of the upstream and downstream primers are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. Utilize this pair of primers to amplify the CDS region sequence RBP4-CDS (as shown in SEQ ID NO.5) of RBP4 gene with pig cDNA as template, the length is 606bp (see figure 1 The RBP4-CDS electrophoresis shown in the figure), and gel recovery was performed on the amplified product. Use 20uL reaction system for PCR amplification: 10μL 2×Power Taq PCR Master Mix, 8μL ddH2O, 0.5μL each of upstream and downstream primers, 1μL cDNA template; PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; 30 cycles: denat...

Embodiment 2

[0048] Example 2 The effect of RBP4 gene overexpression vector in the differentiation of mouse myoblasts

[0049] The overexpression vector pCDH-RBP4 was transfected into the mouse myoblast C2C12 using the transfection reagent lipo2000, and the empty vector pCDH was used as the control, and the differentiation medium (DMEM containing 2% horse serum was cultured) after 6 hours. base) to induce differentiation of cells. After 4 days of differentiation, some cells were used to extract RNA and reverse transcribed into cDNA, and the expression changes of myogenic differentiation marker genes MyHC, MyoG, and MyoD were detected by fluorescent quantitative PCR; the other part of cells were immunofluorescently stained to detect myogenic differentiation markers Changes in the gene MyHC.

[0050] Figure 5 Shown is the change of mRNA expression levels of myoblast differentiation marker genes MyHC, MyoG, and MyoD detected by fluorescent quantitative PCR. Differentiation of muscle cells...

Embodiment 3

[0052] Example 3 Effect of RBP4 Gene Overexpression Vector in Mouse Myoblast Fusion

[0053] The overexpression vector pCDH-RBP4 was transfected into the mouse myoblast C2C12 using the transfection reagent lipo2000, and the empty vector pCDH was used as the control, and the differentiation medium (DMEM containing 2% horse serum was cultured) after 6 hours. base) to induce differentiation of cells. After 4 days of differentiation, some cells were used to extract RNA and reverse transcribed into cDNA, and the mRNA expression changes of myogenic fusion marker genes β-1integrin and Myomaker were detected by fluorescent quantitative PCR; the other part of cells were immunofluorescent stained to detect myoblasts myotube fusion.

[0054] Fluorescent quantitative PCR results such as Figure 7 As shown in the mRNA expression detection chart of the myogenic fusion marker gene, the expression level of the fusion marker gene in the overexpression group was lower than that in the control...

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Abstract

The invention relates to the technical field of gene engineering, and particularly discloses an application of RBP4 protein or a coding gene thereof in regulation and control of myoblast differentiation and fusion. It is discovered that overexpression of an RBP4 gene can inhibit myocyte differentiation and fusion, and then the application of the RBP4 protein or the coding gene thereof or a biological material containing the coding gene thereof in regulation and control of myocyte differentiation and fusion and muscle development is provided. It is defined that the RBP4 gene has the effect of inhibiting myoblast differentiation and fusion, and and a foundation is laid for further research of an action mechanism of the RBP4 gene in cell differentiation and fusion and research of the application of the RBP4 gene in the aspect of livestock meat quality character improvement in the future.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of a RBP4 protein or its coding gene in regulating the differentiation and fusion of myoblasts. Background technique [0002] C2C12 myoblast cell line is a myoblast cell line isolated from mouse C3H cells. It has a strong ability to differentiate when cultured in vitro. It can promote its muscle differentiation by changing the amount of serum in the medium. It is often selected as It is a model for studying the differentiation of skeletal muscle myoblasts in vitro, and simulates the growth and development process of skeletal muscle cells under in vitro conditions to study the specific regulatory mechanism of skeletal muscle development. [0003] Retinol-binding proteins (RBPs) are members of the hydrophobic small molecule binding protein family and are important vitamin A transporters, assisting the transport of retinoic acid / retinol in cells and in se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/85C12N15/66C12N5/10A01K67/027
CPCC07K14/4702C12N15/85C12N15/8509C12N15/66C12N5/0658A01K67/0275C12N2800/107C12N2510/00A01K2207/05A01K2227/105A01K2267/03
Inventor 张博付玉张盼胡晓湘商鹏张浩
Owner CHINA AGRI UNIV
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