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Application of chicken CTGF gene in inhibition of chicken preadipocyte differentiation

A preadipocyte and gene technology is applied in the application field of chicken CTGF gene in inhibiting the differentiation of chicken preadipocytes, which can solve the problems of unclear function and regulation mechanism of chicken CTGF gene, hindering research on chicken CTGF gene function and regulation mechanism, etc.

Pending Publication Date: 2022-01-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there is no method for specific overexpression of chicken CTGF gene expression in the prior art, scientific researchers have been hindered from studying the function and regulation mechanism of chicken CTGF gene, and human beings are not clear about the function and regulation mechanism of chicken CTGF gene.

Method used

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  • Application of chicken CTGF gene in inhibition of chicken preadipocyte differentiation
  • Application of chicken CTGF gene in inhibition of chicken preadipocyte differentiation
  • Application of chicken CTGF gene in inhibition of chicken preadipocyte differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cell Culture

[0050] An immortalized chicken preadipocyte cell line (ICP2) is maintained in our laboratory. Cells were cultured in DMEM / F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and maintained at 37°C in a 5% humidified CO atmosphere.

Embodiment 2

[0051] Example 2: total RNA extraction and Real-time PCR

[0052] (1) Total RNA extraction

[0053] 1) Take out the 12-well plate, discard the medium, and wash 3 times with PBS.

[0054] 2) Add 1 mL of Trizol to each well of a six-well plate, shake well, place on ice for 5 minutes, and shake on a shaking plate for 15 minutes.

[0055] 3) Pipette the cells, transfer the Trizol in the culture plate into a DEPC-treated 1.5mL EP tube, and shake for 15s.

[0056] 4) Add 0.2 mL of chloroform to every 1 mL of Trizol, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

[0057] 5) Centrifuge at 12000 rpm for 15 minutes at 4°C.

[0058] 6) Transfer the upper aqueous phase to a new DEPC-treated 1.5mL EP tube, add an equal volume of isopropanol, mix up and down, and let stand at room temperature for 10 minutes.

[0059] 7) 4°C, 12000rpm, centrifuge for 10min.

[0060] 8) Discard the supernatant, add 1 mL of freshly prepared 75% ethanol, shake and wash t...

Embodiment 3

[0082] Example 3: Oil Red O staining, extraction colorimetry and protein correction

[0083] (1) Oil red O staining, extraction and colorimetry

[0084] 1) Remove the medium in the cells, and wash 3 times with PBS. Add 1 mL of 4% paraformaldehyde fixative solution (6-well plate) to each well, and fix at 4°C for 30 min; at this time, prepare the Oil Red O working solution, that is, after mixing the Oil Red O stock solution and deionized water in a ratio of 3:2, Filter with filter paper (use now).

[0085] 2) Discard the fixative, wash with PBS gently for 3 times, and dry in an oven at 65°C.

[0086] 3) Oil red O staining working solution was used to protect from light for 15 minutes.

[0087] 4) Discard the Oil Red O staining solution, wash gently with PBS 3 times, add 1 mL of 60% isopropanol to each well, discard immediately, and wash 3 times with PBS.

[0088] 5) Add 2mL PBS to each well, take pictures and save the pictures under an inverted microscope.

[0089] 6) PBS w...

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Abstract

The invention discloses application of a chicken CTGF gene in inhibition of chicken preadipocyte differentiation. The nucleotide sequence of the chicken CTGF gene is as shown in SEQ ID NO.1, and the coded amino acid sequence of the chicken CTGF gene is as shown in SEQ ID NO.2. According to the invention, through a method for adding an exogenous CTGF recombinant protein and constructing an endogenous pCMV-CTGF overexpression vector, an oil red O technology, a Real-time PCR technology and a Western blot technology are utilized to prove that the CTGF gene can inhibit chicken preadipocytes from being differentiated into mature adipocytes from the lipid droplet deposition capability, the transcriptome level and the proteome level of the chicken adipocytes, and thus, the invention provides novel application of the CTGF gene in regulation and control of the chicken preadipocyte differentiation process, and the CTGF gene has practical value in the fields of chicken genetic breeding and animal nutrition.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an application of chicken CTGF gene in inhibiting differentiation of chicken preadipocytes. Background technique [0002] The broiler industry has a good development prospect in the world, especially in developing countries such as China. China is the third largest chicken producer in the world after the United States and Brazil. According to the statistics of the United Nations Food and Agriculture Organization (FAO), my country's chicken production in 2017 was 13.44 million tons, accounting for 12.32% of the global chicken production. In the past few decades, through continuous breeding, the growth rate of broiler chickens has been greatly improved. However, the excessive pursuit of growth speed has led to excessive fat deposition in broiler chickens. Increased abdominal fat deposition in broilers leads to a number of adverse outcomes such as reduced feed efficiency and reproducti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/475C12N15/85C12N5/10C12N15/11
CPCC07K14/475C12N15/85C12N5/0653C12N2800/106C12N2510/00
Inventor 张慧申林用李辉周佳美汪舒萍葛耀文肖凡高海河
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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