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Muscle tissue extraction method for separating bovine muscle stem cells and application of muscle tissue extraction method

A technology of muscle tissue and extraction method, which is applied in the field of cell engineering, can solve problems that have yet to be revealed, and the openness of chromatin has yet to be revealed, and achieve the effect of reducing costs

Pending Publication Date: 2022-05-24
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the genetic landscape of SE driving bovine MuSCs myofibrillar differentiation remains to be revealed; at the same time, the mechanism by which the interactive dialogue between SE and seRNA in MuSCs participates in the regulation of muscle development remains to be studied, and whether SE plays a role in regulating chromatin opening in muscle development role has yet to be revealed

Method used

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  • Muscle tissue extraction method for separating bovine muscle stem cells and application of muscle tissue extraction method
  • Muscle tissue extraction method for separating bovine muscle stem cells and application of muscle tissue extraction method
  • Muscle tissue extraction method for separating bovine muscle stem cells and application of muscle tissue extraction method

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Embodiment 1

[0045] Example 1 Establishment of an in vitro isolation and culture system for bovine muscle stem cells

[0046] In the examples of the present invention, the formula of 50 mL of type I collagenase solution (0.2%) used is shown in Table 1.

[0047] Table 1 50mL type I collagenase solution (0.2%) formula

[0048] A low-glucose DMEM basal medium 39mL (sugar content: 1.0g / L) a neutral enzyme II 10mL (final concentration: 1U / mL) A collagenase I 1mL (final concentration: 0.1mg / mL)

[0049] In the examples of the present invention, the formula of 50 mL of pancreatin solution (0.25%) used is shown in Table 2.

[0050] Table 2 50mL pancreatin solution (0.25%) formula

[0051] Phosphate buffer 39mL Pancreatin 10mL (final concentration: 1U / mL)

[0052] In the implementation of the present invention, the formulation of 50 mL of bovine muscle stem cell primary culture medium used is shown in Table 3.

[0053] Table 3 Formulation of ...

Embodiment 2

[0086] Example 2 Construction of bovine muscle stem cell super-enhancer method

[0087] In the implementation of the present invention, chromatin co-immunoprecipitation high-throughput sequencing is performed based on muscle stem cells to obtain enhancer markers constituting different chromosomes in the whole genome, and the specific steps are as follows:

[0088] (1) Acquisition of raw data constituting enhancers

[0089] ①Use antibodies specific for the methylation of the fourth lysine residue of histone H3 (referred to as H3K4me1) and the acetylation of the 27th lysine residue of histone H3 (referred to as H3K27ac) to stain the target protein of the cell sample Co-immunoprecipitation reaction was performed to obtain genomic DNA fragments bound by H3K4me1 and H3K27ac tagged proteins; ② Purify and enrich the DNA fragments respectively; ③ Perform high-throughput sequencing on the DNA fragment products respectively; The base calling analysis is converted into a sequence file (...

Embodiment 3

[0117] Example 3 Construction of super-enhancer map of bovine muscle stem cells

[0118] In the embodiment of the present invention, to construct a super-enhancer map of bovine muscle stem cells, the specific steps are:

[0119] (1) Identification of super-enhancers

[0120] ① Based on the ROSE algorithm, the H3K27ac signal value of each constituting enhancer was calculated for the enhancer binding sites identified by H3K27ac in each enhancer region on the genome;

[0121] ② Calculate the Med1 signal of each enhancer region according to the H3K27ac signal value that constitutes the enhancer;

[0122] ③Order the Med1 signal in the enhancer region, represented by the coordinate axis. For example, the Med1 enrichment on the y-axis is arranged along the x-axis, and a graph is drawn to obtain a graph. The signal value obtained at the tangent point of the tangent line with a slope of 1 on the curve is used to distinguish between super-strong enhancers and ordinary enhancers. Thresh...

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Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to an extraction method for separating muscle tissues of bovine muscle stem cells and application of the extraction method. The method comprises the following steps: (1) wiping the surface of cattle skin with alcohol, collecting muscular tissues of longissimus on the back of the cattle, and immediately rinsing with a phosphate buffer solution; (2) removing fascia and white film on the surface; and (3) rinsing with triple distilled water, and then rinsing with a phosphate buffer solution, alcohol and a phosphate buffer solution in sequence. The extraction method is different from the conventional method for treating by singly using a phosphate buffer solution, and the cost is reduced by combined treatment of three washing solutions. The method comprises the following steps: extracting muscle tissue, separating muscle stem cells from the extracted muscle tissue, using the muscle stem cells to construct a super enhancer, and optimizing and forming a method for analyzing the bovine muscle super enhancer by using an H3K4me1 and H3K27ac double-labeling method on the basis of chromosome co-immunoprecipitation high-throughput data basic analysis, which is different from a single labeling method in the prior art.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a muscle tissue extraction method for separating bovine muscle stem cells and its application. Background technique [0002] Muscle development is an important factor affecting the growth rate, meat yield, meat quality and other important economic traits of livestock, and muscle development depends on the proliferation and myofibroblast differentiation of muscle stem cells (MuSCs). MuSCs are mesenchymal stem cells derived from the mesoderm, and their functions affect the number of myoblasts, myotube fusion and regulate muscle fiber hypertrophy during myogenesis. It can be seen that MuSCs play an increasingly important role in emerging research fields such as precise improvement of livestock in animal husbandry, stem cell breeding, and in vitro and in vivo cultured meat production. At the same time, the muscle fiber differentiation of MuSCs is also closely re...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N15/113
CPCC12N5/0662C12N15/113C12N2509/00C12N2509/10
Inventor 张瑞门杨素芳韦英明梁菁媛邓彦飞郑自华王乐怡安强
Owner GUANGXI UNIV
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