CN118028375A - Application of ZBED6 gene in regulation and control of animal fat deposition and fat cell differentiation - Google Patents

Abstract

The invention provides application of ZBED6 genes in regulating and controlling animal fat deposition and fat cell differentiation. The invention also provides a method for increasing the expression of ZBED6 genes to promote the differentiation and heat production of the beige fat cells of the pigs and the mice, and provides an animal model for inhibiting the fat deposition and the heat production of the pigs and the mice, which provides a research platform and a theoretical basis for regulating and controlling the fat deposition and the formation of the fat heat production cells of the pigs and the mice.

Description

Application of ZBED6 gene in regulating animal fat deposition and adipocyte differentiation

Technical Field

The present invention relates to the field of gene engineering technology, in particular to the application of ZBED6 gene in regulating animal fat deposition and adipocyte differentiation.

Background technique

Type 2 diabetes has become a serious global health problem. Type 2 diabetes is a chronic metabolic disorder that is highly associated with obesity and is closely related to fat deposition. Mammalian adipose tissue is an important endocrine organ for energy storage and energy metabolism, and plays an important role in glucose metabolism, insulin sensitivity, heat production, and immune regulation. Mammalian adipose tissue can be divided into white adipose tissue and beige adipose tissue according to its function. As the main adipose tissue of the body, white adipose tissue is mainly responsible for storing energy under the condition of adequate nutrition. When the body lacks energy, fat mobilization will occur, and triglycerides in white adipose tissue will be broken down into free fatty acids for energy consumption throughout the body. Brown adipose tissue is rich in mitochondria and generates heat by consuming substrates such as fatty acids and glucose. It is mainly distributed between the scapula, on both sides of the spine, around the kidneys, around the thoracic artery, and around the inferior vena cava. The main ways of brown fat heat production are UCP1-dependent heat production (mitochondrial uncoupling) and non-UCP1-dependent heat production (Ca ²⁺ ineffective cycle, creatine substrate cycle, TAG-fatty acid cycle). Brown fat achieves the effect of resisting obesity and treating immune diseases by consuming calories. When the body is stimulated by cold, adrenaline, etc., on the one hand, UCP+ cells in white fat are induced to activate beige fat, which has the characteristics of brown fat thermogenesis, such as the presence of multilocular lipid droplets and the expression of UCP1 gene. On the other hand, under the induction of genes such as PDGFRα, SCA1, CD81, αSMA, PPARγ, PRDM16, EBF2, adipocyte precursor cells will synthesize into beige adipocytes from scratch. The heterogeneity of adipocytes and their ability to respond quickly to environmental changes play an indispensable role in maintaining the body's energy metabolism balance and health. More and more evidence shows that inducing the formation of beige adipocytes makes an important contribution to maintaining physical health and treating diseases, but the current research on targets for regulating the formation of beige adipocytes is still very limited, and new molecular targets are urgently needed to develop drugs for the treatment of obesity and diabetes. Therefore, it is necessary to further screen and identify molecular targets that regulate mammalian fat deposition and thermogenic adipocyte generation.

Summary of the invention

The purpose of the present invention is to provide application of ZBED6 gene in regulating animal fat deposition and adipocyte differentiation.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides any of the following applications of the ZBED6 gene:

1) Used to regulate animal fat deposition;

2) Used to regulate animal adipocyte differentiation;

3) Used to construct animal models of fat development and/or thermogenesis (animal models for studying obesity and related diseases);

4) Used for breeding low-fat and high-lean meat pigs.

In the present invention, the reference sequence number of the ZBED6 gene derived from pig on NCBI is NM_001394675.1, and the reference sequence number of the ZBED6 gene derived from mouse on NCBI is NM_001166552.2.

Furthermore, the expression or activity of the ZBED6 gene or its encoded protein is inhibited at the transcriptional or translational level, or the ZBED6 gene is knocked out from the animal genome to reduce animal fat deposition, reduce the adipogenic differentiation efficiency of animal preadipocytes, and reduce the expression of thermogenic genes (reduce the thermogenic capacity of preadipocytes);

Furthermore, the expression or activity of the ZBED6 gene or its encoded protein is inhibited at the transcription or translation level by an inhibitor; the inhibitor can be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular weight compounds, peptides, antibodies, ZBED6 gene targeting vectors, etc.

The ZBED6 gene targeting vector can be constructed based on genome editing technologies such as CRISPR, TALEN or ZFN.

In a specific embodiment of the present invention, the inhibitor is siRNA, and its nucleotide sequence is shown in SEQ ID NO: 1 or 2.

Furthermore, the expression or activity of the ZBED6 gene or its encoded protein is enhanced at the transcriptional or translational level to increase animal fat deposition, increase the adipogenic differentiation efficiency of animal precursor adipocytes, and promote the expression of thermogenic genes (increase the thermogenic capacity of precursor adipocytes).

Furthermore, the enhanced approach is selected from the following 1) to 5), or an optional combination:

1) Enhanced by introducing a plasmid carrying the gene;

2) Enhanced by increasing the copy number of the gene on the chromosome;

3) Enhanced by changing the promoter sequence of the gene on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene;

5) Enhancement by introduction of enhancers.

The thermogenic gene of the present invention can be selected from FASN, ACACA, ACLY, etc. The reference sequence numbers of FASN, ACACA, and ACLY genes from pigs on NCBI are NM_001099930.1, NM_001114269.1, and NM_001105302.1, respectively, and the reference sequence numbers of FASN, ACACA, and ACLY genes from mice on NCBI are NM_007988.3, NM_133360.3, and NM_001199296.1, respectively.

The applications described in the present invention include non-disease diagnosis and treatment purposes.

The animal described in the present invention is a mammal, such as a pig or a mouse.

In a second aspect, the present invention provides a siRNA targeting the mouse ZBED6 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In a third aspect, the present invention provides a method for promoting the differentiation efficiency and/or heat production function of beige fat in pigs and mice, the method comprising: enhancing the expression of the ZBED6 gene by genetic engineering technology.

Furthermore, the enhanced approach may be selected from the following 1) to 5), or an optional combination:

1) Enhanced by introducing a plasmid carrying the gene;

2) Enhanced by increasing the copy number of the gene on the chromosome;

3) Enhanced by changing the promoter sequence of the gene on the chromosome;

4) Enhanced by operably linking a strong promoter to the gene;

5) Enhancement by introduction of enhancers.

The purpose of the present invention can be further achieved by adopting the following technical measures.

The present invention provides the use of a biomaterial containing a ZBED6 gene in any of the following aspects:

1) Regulate the formation of fat cells in pigs and mice;

2) Study animal fat development and metabolism;

3) Breeding of pig breeds;

4) Preparation of drugs for the prevention/treatment of obesity and diabetes;

5) Construct a low-fat and high-lean meat ratio animal model.

The biological materials include but are not limited to host animals, host cells, and adenovirus vectors.

The present invention also provides any of the following applications of ZBED6 gene knockout pigs and primary adipocytes thereof and ZBED6 gene knockout mice:

1) Reduce fat deposition in pigs and mice;

2) Reduced differentiation efficiency of pig and mouse adipocytes;

3) Reduce the thermogenic efficiency of fat cells in pigs and mice

4) Study the characteristics of fat traits in pigs and mice;

5) Cultivate low-fat and high-lean meat pig breeds;

6) Provide a platform for studying the mechanisms of obesity and diabetes.

The present invention also provides any of the following applications of a ZBED6 gene overexpression adenovirus or an editing system targeting the ZBED6 gene that can enhance ZBED6 gene expression:

1) Increased fat deposition;

2) Increase the differentiation efficiency of adipocytes;

3) Study the characteristics of fat development.

For example, the editing system of the ZBED6 gene is an operating system for promoting the expression of the ZBED6 gene by an overexpression vector encapsulated by an adenovirus.

The present invention also provides the use of any of the following aspects of a gene editing system in which a ZBED6 gene inhibitor can reduce ZBED6 gene expression:

1) Reduce the differentiation efficiency of 3T3 preadipocyte cell line;

2) Improve animal fat deposition;

3) Used for the study of obesity and related diseases.

For example, the editing system of the ZBED6 gene is an operating system for siRNA to inhibit the expression of the mouse ZBED6 gene.

The present invention also provides a siRNA targeting the mouse ZBED6 gene, the nucleotide sequence of which is as follows:

SEQ ID NO: 1: 5′-CCAGCUUCCAUGGAAGAAU-3′

SEQ ID NO:2:5′-AUUCUUCCAUGGAAGCUGG-3′

The present invention also provides an animal model for reducing fat deposition in pigs and mice, wherein the ZBED6 gene of pigs and mice is knocked out by gene editing technology, and primary pig fat cells are isolated; and the expression of the mouse ZBED6 gene is reduced by knocking down and silencing the ZBED6 gene.

For example, the ZBED6 gene can be knocked out through gene editing technology, causing pigs and mice to lack the ZBED6 gene systemically; or, siRNA can be used to knock down the expression of the ZBED6 gene.

The present invention also provides a method for promoting the differentiation efficiency and/or heat production function of beige fat in pigs and mice, which increases the expression of ZBED6 gene through genetic engineering technology.

By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

The present invention provides a method for increasing the expression of ZBED6 gene to promote the differentiation and heat production of beige adipocytes in pigs and mice, and provides an animal model for inhibiting fat deposition and heat production in pigs and mice, thereby providing a research platform and theoretical basis for regulating fat deposition and the formation of fat thermogenic cells in pigs and mice.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the fat deposition phenotype of ZBED6 knockout pigs in Example 1 of the present invention. Among them, a total of 17 F4 generation Bama pigs were collected (3 wild-type boars and 3 ZBED6 knockout pigs; 5 wild-type sows and 6 ZBED6 knockout pigs), A: fat weight of boars and sows, in kg; B: ratio of fat weight to body weight of boars and sows; C: back fat thickness of boars and sows, in mm; D: abdominal fat weight of boars and sows, in kg; E: HE sections of back fat and abdominal fat of wild-type boars and ZBED6 knockout pigs, with a scale of 50 μm; F: quantitative statistics of cross-sectional areas of back fat and abdominal fat cells. *P<0.05 in the figure indicates significance.

Figure 2 shows the fat deposition phenotype of ZBED6 knockout mice in Example 2 of the present invention, wherein a total of 24 male mice data were collected, including 12 wild-type and 12 ZBED6 knockout mice. A: Body weight of wild-type and ZBED6 knockout mice under high-fat feeding for 12 weeks; B: Abdominal white fat weight of wild-type and ZBED6 knockout mice after 12 weeks of high-fat feeding; C: HE sections of abdominal white fat of wild-type and ZBED6 knockout mice after 12 weeks of high-fat or high-sugar feeding; D: Glucose tolerance test of wild-type and ZBED6 knockout mice after 12 weeks of normal feeding or high-fat feeding; E: Insulin sensitivity test. In the figure, *P<0.05, **P<0.01, ***P<0.001 indicate significance, and ns indicates no significant difference.

Figure 3 shows the effect of knocking out ZBED6 in Bama Xiang pigs in Example 3 of the present invention on the differentiation efficiency of adipocytes, as well as the effect of using SiRNA to interfere with ZBED6 on the differentiation efficiency of 3T3 mouse preadipocytes in Example 4, and the differentiation efficiency and marker gene expression of mouse primary preadipocytes after overexpressing the mouse ZBED6 gene in Example 6 of the present invention. A: protein expression of fat synthesis marker genes in wild-type and ZBED6 knockout primary adipocytes after 8 days of induction and differentiation; B: Oil red O staining and quantification of wild-type and ZBED6 knockout primary adipocytes after 8 days of induction and differentiation; C: mRNA expression of fat synthesis marker genes in 3T3 preadipocytes after 8 days of induction and differentiation after knocking down ZBED6; D: protein expression of ZBED6 and fat synthesis marker genes in 3T3 preadipocytes after 8 days of induction and differentiation after knocking down ZBED6; E: protein expression of ZBED6 and fat synthesis marker genes in 3T3 preadipocytes after 8 days of induction and differentiation after knocking down ZBED6. Oil red O staining was performed and the staining results were quantified after 8 days; F: Changes in cell cycle after interference with ZBED6; G: After overexpression of ZBED6, the mRNA expression of ZBED6 and fat synthesis marker genes in 3T3 preadipocytes induced for 8 days after differentiation; H: After overexpression of ZBED6, the protein expression of ZBED6 and fat synthesis marker genes in 3T3 preadipocytes induced for 8 days after differentiation; I: After overexpression of ZBED6, Oil red O staining was performed and the staining results were quantified after 8 days of differentiation; J: Detection of cell proliferation after overexpression of ZBED6.

Detailed ways

The present invention aims to provide a new use of the ZBED6 gene, specifically to provide its use in regulating fat deposition in pigs and mice as well as adipocyte differentiation efficiency and heat production function.

The ZBED6 gene of the present invention is well known in the art, such as its number on NCBI is 100622313 (pig) and 667118 (mouse).

The present invention adopts the following technical solution:

In a first aspect, the present invention provides a ZBED6 encoding gene, or a biomaterial containing the ZBED6 encoding gene, for use in any of the following aspects:

1) Regulate fat deposition in pigs and mice;

2) Regulate the efficiency of porcine and mouse preadipocyte differentiation;

3) Study mammalian fat development and metabolism;

4) Breeding of pig breeds;

5) Preparation of drugs for preventing, alleviating and/or treating obesity-related diseases;

6) Construct animal models of fat development and/or thermogenesis.

The biological materials include but are not limited to host cells, Bama Xiang pigs or C57BL/6J mice.

The ZBED6 encoding gene of the present invention, or the biological material containing the encoding gene thereof, can also be used in studying the pathogenesis of diabetes and obesity, breeding low-fat and high-lean meat pig breeds, screening and identifying genes related to fat deposition in pigs and mice, and screening and identifying beige fat genes in pigs and mice.

The present invention knocks out the ZBED6 gene in Bama pigs and C57BL/6J mice and finds that fat deposition is significantly reduced and brown fat is whitened. Wild-type and ZBED6-/- primary SVF cells are separated for in vitro culture and it is found that knocking out ZBED6 can significantly reduce the adipogenic differentiation efficiency of SVF cells and reduce the expression of thermogenic genes. Increasing the expression of the ZBED6 gene can promote the adipogenic differentiation of pig and mouse SVF cells into beige fat cells and increase the expression of thermogenic genes, thereby proposing the application of the ZBED6 gene in fat deposition in pigs and mice and adipogenic differentiation of SVF cells.

The study found that mutations in the IGF2 gene (NCBI number NM_213883.2) disrupted binding to an unknown nuclear factor, causing a 3-fold increase in IGF2 expression in skeletal muscle, ultimately leading to an increase in skeletal muscle mass and a 3-4% increase in lean meat. This binding of IGF2 only occurs postpartum, and does not bind to the hypermethylated region of IGF2 during the fetal period. In 2009, it was discovered that this unknown nuclear factor is a transcription factor that regulates muscle growth and development by regulating the expression of IGF2, and was named ZBED6. ZBED6 evolved from a DNA transposon and is located in the same position in intron 1 of ZC3H11A in all placental mammals. ZBED6 has a basic function in the evolutionary process. The gene has two DNA binding regions and shows nearly 100% amino acid identity in 26 placental mammals. In addition, knocking out ZBED6 in pigs and mice can significantly promote the development of tissues and organs such as skeletal muscle and spleen, proving that ZBED6 has an important function.

ChIP-seq of pig skeletal muscle and C2C12 cells revealed target genes regulated by ZBED6. These target genes all have the motify sequence GCTCG bound by ZBED6, and most of the binding sites occur near the transcription start site. The pig ZBED6 gene is located on chromosome 9, with a coding region of 2946nt, encoding 981 amino acids. The mouse ZBED6 gene is located on chromosome 1, with a coding region of 2943nt, encoding 980 amino acids. At present, the research on ZBED6 mainly focuses on skeletal muscle development and atrophy, pancreatic β-cell function, immune response, etc. There are few reports on the role of ZBED6 in regulating fat deposition and the formation of beige adipocytes.

The present invention utilizes gene editing to knock out the ZBED6 gene, thereby eliminating the expression of the ZBED6 gene at the transcription and translation levels, and verifies that it can inhibit fat deposition and the formation of beige adipocytes at the levels of adipose tissue, primary cells, and marker gene mRNA and protein.

The present invention utilizes genetic engineering means to construct a ZBED6 gene overexpression adenovirus vector, overexpresses the ZBED6 gene, promotes or improves the expression of the ZBED6 gene at the transcription and translation levels, and verifies that it can promote the generation of beige adipocytes at the primary cell and marker gene mRNA and protein levels.

In the present invention, the reference sequence number of the pig ZBED6 gene on NCBI is NM_001394675.1, and the reference sequence number of the mouse ZBED6 gene on NCBI is NM_001166552.2.

In a second aspect, the present invention provides a pig and mouse ZBED6 overexpression adenovirus vector or any of the following applications that can enhance ZBED6 gene expression:

1) Increase the differentiation efficiency of porcine and mouse preadipocytes;

2) Increase the thermogenic capacity of preadipocytes in pigs and mice;

3) Study the characteristics of fat traits in pigs and mice;

4) Reduce fat deposition in mammals;

5) Cultivate high-quality pig breeds with low fat and high lean meat ratio.

Among them, the ZBED6 overexpression adenovirus vector can increase the expression of pig and mouse ZBED6 genes at the transcription and translation levels.

Thirdly, by knocking out the ZBED6 gene in pigs and mice through gene editing technology and isolating primary SVF cells, it is possible to verify in vitro the application of ZBED6 in any of the following aspects of adipocytes:

1) Reduce the differentiation efficiency of porcine and mouse preadipocytes;

2) Reduce the thermogenic capacity of preadipocytes in pigs and mice;

3) Improve fat deposition in mammals;

4) Used to study the genetic mechanisms of obesity and type 2 diabetes.

The primary SVF cells are primary cells with the ZBED6 gene knocked out, and are substances capable of eliminating the expression of the ZBED6 gene at the transcription and translation levels.

In a fourth aspect, the present invention provides an animal model capable of reducing fat deposition and/or heat production capacity in pigs and mice, wherein the expression of the ZBED6 gene is knocked out by gene editing technology.

In a fifth aspect, the present invention provides a method capable of improving the differentiation efficiency and/or heat production capacity of pig and mouse preadipocytes, which increases the expression of the ZBED6 gene of pigs and mice through an adenovirus overexpression vector.

In a sixth aspect, the present invention provides a siRNA targeting the ZBED6 gene, the nucleotide sequence of which is as follows:

SEQ ID NO: 1: 5′-CCAGCUUCCAUGGAAGAAU-3′

SEQ ID NO:2:5′-AUUCUUCCAUGGAAGCUGG-3′

In the method of the present invention, the expression of ZBED6 gene is eliminated by knocking out the ZBED6 gene. The expression of ZBED6 gene is knocked down by siRNA.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

Example 1 Fat deposition phenotype in ZBED6 knockout pigs

This example obtains the phenotypic data of back fat and abdominal fat in wild-type Bama Xiang pigs and after knocking out the ZBED6 gene, and quantifies them to show the effect of ZBED6 on pig fat deposition. The observation results are shown in Figure 1. It can be seen from the figure that knocking out ZBED6 in Bama Xiang pigs can significantly reduce back fat and abdominal fat deposition. This shows that the ZBED6 gene plays an important role in the process of pig fat deposition.

The construction method of ZBED6 knockout pig is as follows:

1. Transfect sgRNA Zg1, Zg2, Zg3, and Zg4 (constructed into pX330-Cas9 vector, pX330-Cas9 is constructed by inserting Cas9 protein coding gene into PX330 vector) into porcine fetal fibroblasts by electroporation;

Zg1:5′-aagaaaagaaagaagggtttgcgaattaagGGGaaaaggc-3′

Zg2: 5′-aaaaagtttagtaaggatttgggatctgggaggcctgttg-3′

Zg3: 5′-cctagtactagagcaaagacttccattgtgtggcacttct-3′

Zg4: 3′-ttgacccccagtatacctggcgggcaatttgtaacctctg-5′

2. Screening the best gene targeting efficiency (Zg3, targeting efficiency is 7.5%) through first-generation Sanger sequencing;

3. Three positive cell clones Z17, Z23, and Z28 were screened;

4. Perform somatic cell nuclear transplantation on positive cells to construct reconstructed embryos and transplant them into donor sows;

5. The gene-edited piglets were subjected to first-generation Sanger sequencing, and a frameshift mutation was identified at the 1320-T site, indicating that the ZBED6 gene knockout pig was successfully constructed.

Example 2 Fat deposition phenotype in ZBED6 knockout mice

In this example, phenotypic data such as body weight, abdominal fat weight, and abdominal fat HE sections of wild-type C57BL/6J mice and mice with ZBED6 knockout were obtained and quantified to show the effect of ZBED6 on fat deposition in mice. The observation results are shown in FIG2 .

This example further tests the glucose tolerance and insulin sensitivity of wild-type and ZBED6 knockout mice under normal feeding conditions and high-fat feeding conditions. It can be seen that after knocking out ZBED6 in C57BL/6J mice, fat weight can be significantly reduced under high-fat and high-sugar conditions, and glucose tolerance and insulin sensitivity of mice can be improved. This shows that knocking out ZBED6 can significantly improve the glucose metabolism ability of mice, increase energy consumption and reduce fat deposition.

The method for constructing ZBED6 knockout mice is as follows:

1. Construct ZBED6-flox mice, insert a LoxP site 230 bp upstream of the ATG of the ZBED6 gene, and insert a FRT-Neo-FRT-LoxP site 370 bp downstream of the TAA.

2. Insert 6kb homology arms at both ends of the LoxP site; the left homology arm is located 230bp upstream of the start codon of the ZBED6 gene and extends 6kb upward, and the right homology arm is located 370bp downstream of the start codon of the ZBED6 gene and extends 6kb downward.

3. ZBED6-flox mice were hybridized with PGK-Cre mice to generate ZBED6 systemic knockout mice (PGK-Cre mice are systemic knockout tool mice, kindly provided by Professor Leif Andersson of Uppsala University, Sweden. PGK-Cre mice can be found in Younis S, M, Massart J, et al., The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals. Proc Natl Acad Sci US A. 2018 Feb 27; 115(9): E2048-E2057.).

Example 3 Regulation of ZBED6 gene knockout on porcine preadipocyte differentiation

In this example, preadipocytes were isolated from the inguinal subcutaneous fat and perirenal fat of wild-type Bama Xiang pigs and ZBED6 knockout Bama Xiang pigs, induced to differentiate, and the difference in differentiation into adipocytes in vitro between the wild-type and ZBED6 knockout types was observed by Oil Red O. The observation results are shown in A and B in Figure 3.

This example further detected the protein expression of de novo fat synthesis genes in wild-type and ZBED6 knockout pig preadipocytes after 8 days of differentiation, and FASN, ACACA, were significantly downregulated. The results of Oil Red O staining showed that knocking out ZBED6 could significantly inhibit the differentiation efficiency of primary pig preadipocytes. This indicates that knocking out ZBED6 can inhibit de novo fat synthesis in pigs and thus inhibit the differentiation of primary pig adipocytes.

The specific process of isolating and culturing wild-type and ZBED6 knockout preadipocytes of Bama Xiang pigs is as follows:

1) One-month-old wild-type Bama miniature pig and one ZBED6 knockout Bama miniature pig were killed and disinfected with alcohol cotton. The inguinal white fat and perirenal white fat were taken out on a sterile workbench and soaked in pre-cooled PBS containing 2% penicillin/streptomycin, and then transferred to an ultra-clean workbench;

2) Wash the adipose tissue three times with precooled PBS containing 2% penicillin/streptomycin, mince the adipose tissue with scissors, add 2 volumes of DMEM/F12 medium containing 2 mg/ml type I collagenase, and place in a 37°C constant temperature shaker for digestion for 60 minutes;

3) Add an equal volume of DMEM/F12 complete medium to the digested tissue to terminate digestion, filter the tissue suspension with a 70 μm cell filter, and centrifuge at 1500 rpm for 10 min at room temperature;

4) Aspirate and discard the supernatant, add 3-5 ml of red blood cell lysis buffer to resuspend the cell pellet, let stand at 4°C for 10 min, and centrifuge at 1500 rpm for 10 min;

5) Aspirate the supernatant, add 1-2 ml DMEM/F12 complete medium to resuspend the cell pellet, inoculate it into a T75 cell culture flask, make up to 15 ml of medium, shake well, and culture in a cell culture incubator at 37°C with 5% _CO2 . Change the medium every 2 days and subculture after confluence.

The induction and differentiation process of porcine preadipocytes is as follows:

1) After the porcine preadipocytes have grown to full size, they are plated. The porcine preadipocytes in one T75 culture flask can be plated on three 12-well cell culture plates;

2) After the cells have grown confluent, differentiation induction begins after two days of contact inhibition (differentiation day 0);

3) After two days of contact inhibition, the culture medium was replaced with differentiation induction medium. The culture medium was replaced every two days.

4) Differentiation medium ratio: DMEM high glucose medium, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.1 μM dexamethasone, 0.5 μM insulin, 2 nM triiodothyronine (T3), 30 μM indomethacin, 17 μM pantothenic acid, 33 μM biotin, 1 μM rosiglitazone, and 2% FBS.

Example 4 Regulation of ZBED6 gene knockdown on mouse preadipocyte differentiation

In this example, siRNA was used to interfere with ZBED6 in 3T3 mouse preadipocytes to induce differentiation, and the difference in differentiation of 3T3 mouse preadipocytes was observed by Oil Red O. The observation results are shown in C, D, E, and F in FIG3 .

This example further detected the effect of knocking down ZBED6 on the proliferation and differentiation of 3T3 mouse preadipocytes. The results showed that knocking down ZBED6 could significantly inhibit the proliferation and differentiation of 3T3 mouse preadipocytes. The effect of knocking down ZBED6 on the expression of de novo fat synthesis genes in 3T3 mouse preadipocytes was further detected. The results showed that knocking down ZBED6 could significantly inhibit the expression of de novo fat synthesis genes. This shows that knocking down the expression of ZBED6 can inhibit fat synthesis.

The specific experimental process is as follows: After the cell confluence in the T75 cell culture flask reaches 90%, the cells are inoculated into a 12-well cell culture plate. When the cell confluence reaches 40%, siRNA transfection is performed, of which 6 wells are transfected with siRNA-NC using RNAiMAX, and the other 6 wells are transfected with siRNA-ZBED6. The siRNA used for transfection is two siRNAs with two bases T connected after the sequence SEQ ID NO: 1 or SEQ ID NO: 2 (TT overhangs are added to the 3′ end of SEQ ID NO: 1 or SEQ ID NO: 2 to form sticky ends for easy melting);

The transfection method is:

1. Inoculate cells into 12-well plates and transfect when the cell confluence reaches 40%;

2. Add 300 μL OPTI-MEM and 60 μM siRNA to tube ①, and add 300 μL OPTI-MEM and 15 μL RNAiMAX to tube ②. This is the amount for 6 wells of a 12-well plate.

3. Transfer the mixed solution in tube ① to tube ②, mix lightly and let stand for 10 minutes; add the mixed solution evenly to the cell culture plate. After the cells are full, contact inhibition for two days (differentiation day 0) is replaced with beige fat induction differentiation medium (89% DMEM/F12 medium, 10% fetal bovine serum, 1μM dexamethasone, 1μM rosiglitazone, 850nM insulin, 0.5mM IBMX, 1% penicillin/streptomycin), and transfection is performed at the same time as the medium is changed (transfection is performed every 72 hours thereafter); after 48 hours of differentiation, it is replaced with maintenance medium (89% DMEM/F12 medium, 10% fetal bovine serum, 1μM rosiglitazone, 850nM insulin, 1% penicillin/streptomycin), and the maintenance medium is replaced every 48 hours thereafter, for a total of 8 days of differentiation.

Specifically, 48 hours after transfection of siRNA-ZBED6, the present example detected the mRNA expression level of the ZBED6 gene (the internal reference was 18S), and the detection results were shown in A of FIG. 3 , indicating that the use of siRNA-ZBED6 can significantly (*P<0.05) reduce the mRNA expression level of the ZBED6 gene; the protein expression level of the ZBED6 gene was detected (the internal reference was Tubulin), and the results were shown in B of FIG. 3 , indicating that the use of siRNA-ZBED6 can extremely significantly (***P<0.01) reduce the protein expression level of the ZBED6 gene.

After transfection of siRNA, when the cell density reached about 90%, the present embodiment detected the effect of knocking down the ZBED6 gene on cell proliferation through EDU cell proliferation detection experiment, cell cycle detection, and cell scratch test. The experimental results are shown in C/D/E in FIG3 , indicating that knocking down the expression of the ZBED6 gene can significantly (***P<0.01) inhibit cell proliferation.

After 8 days of induction of differentiation, this example uses Oil Red O staining to verify the cell differentiation efficiency after knocking down the ZBED6 gene during the induction and differentiation of 3T3 mouse preadipocytes. The Oil Red O staining results are shown in G and H in Figure 3 (G is the Oil Red O staining result, and H is the Oil Red O staining quantitative result), which shows that after knocking down the ZBED6 gene, the differentiation efficiency of 3T3 mouse preadipocytes is significantly reduced.

From the above results, it can be seen that the proliferation and differentiation efficiency of 3T3 mouse preadipocytes were significantly reduced after knocking down the expression level of the ZBED6 gene (the knockdown efficiency reached more than 50%).

Example 5 Effect of overexpression of mouse ZBED6 gene on differentiation efficiency and thermogenic gene expression of mouse preadipocytes

In this example, ZBED6 was overexpressed in 3T3 mouse preadipocytes using a ZBED6 adenovirus overexpression vector, differentiation was induced, and the differentiation of 3T3 mouse preadipocytes was observed using Oil Red O. The observation results are shown in G, H, I, and J in FIG3 .

This example further detected the effect of overexpression of ZBED6 on the proliferation and differentiation of 3T3 mouse preadipocytes, and the results showed that overexpression of ZBED6 can significantly promote the proliferation and differentiation of 3T3 mouse preadipocytes. The effect of overexpression of ZBED6 on the expression of de novo fat synthesis genes in 3T3 mouse preadipocytes was further detected, and the results showed that overexpression of ZBED6 can significantly inhibit the expression of de novo fat synthesis genes. It shows that increasing the expression of ZBED6 can promote fat synthesis.

The specific experimental process is as follows: After the cell confluence in the T75 cell culture flask reaches 90%, it is inoculated into a 12-well cell culture plate. When the cell confluence reaches 40%, adenovirus infection is performed. Six wells use the ZBED6 adenovirus overexpression vector, and the other six wells use the adenovirus overexpression negative vector. The CDS region used to construct the vector is NM_001166552.2.

The construction method of ZBED6 adenovirus overexpression vector is as follows:

1. Construct the CDS region of ZBED6 gene onto the tool vector PSB50;

2. Transfer the CDS region of the ZBED6 gene to the PSE6041 vector through homologous recombination for subsequent adenovirus packaging;

3. Use the auxiliary vector pBHG loxΔE1,3Cre to package adenovirus using the AdMax adenovirus packaging system;

4. Transfect the vector into HEK293 cells for virus amplification, collect the cells and purify the adenovirus by ultracentrifugation;

5. The titer of purified adenovirus was tested and the virus titer was 1.14×10 ¹¹ IFU/mL.

All the vectors used above were purchased from Shanghai Shenggong Biological Co., Ltd.

The cell infection method is as follows:

1) When the cell density reaches 40%, replace with 5% fetal bovine serum-free penicillin/streptomycin medium (add 500 μL to 12-well plate);

2) Add adenovirus diluted in OPTI-MEM reduced serum medium (purchased from Gibco) (MOI = 100);

3) 4 hours later, add 5% fetal bovine serum-free penicillin/streptomycin medium to 1 mL;

4) After 8 hours, the culture medium was replaced with complete medium and cultured until subsequent differentiation induction.

Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

references:

[1]Markljung E, Jiang L, Jaffe JD, et al., ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth. PLoS Biol. 2009 Dec；7(12):e1000256.

[2] Wang D, Pan D, Xie B, et al., Porcine ZBED6 regulates growth of skeletal muscle and internal organs via multiple targets. PLoS Genet. 2021 Oct28; 17(10): e1009862.

[3] Younis S, M, Massart J, et al., The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals. Proc Natl Acad Sci US A. 2018 Feb 27; 115(9): E2048-E2057.

[4] Wang X, Younis S, Cen J, et al., ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation. Diabetologia. 2021 Oct; 64(10): 2292-2305.

[5] Wang W, Seale P. Control of brown and beige fat development. Nat Rev Mol Cell Biol. 2016 Nov；17(11):691-702.

[6] Cohen P, Kajimura S. The cellular and functional complexity of thermogenic fat. Nat Rev Mol Cell Biol. 2021 Jun; 22(6): 393-409.

Claims (10)

1. Any of the following applications of zbed6 gene:

   1) For modulating animal fat deposition;

   2) For regulating the differentiation of animal fat cells;

   3) For constructing a fat development and/or thermogenesis animal model;

   4) Used for breeding low-fat high-lean-percentage pigs;

   The reference sequence number of the ZBED6 gene derived from pigs on NCBI is NM_001394675.1, and the reference sequence number of the ZBED6 gene derived from mice on NCBI is NM_001166552.2.
2. 2. The use according to claim 1, wherein the expression or activity of ZBED6 gene or its encoded protein is suppressed at the transcriptional or translational level, or the ZBED6 gene is knocked out from the animal genome to reduce fat deposition in animals, to reduce the adipogenic differentiation efficiency of animal precursor adipocytes, to reduce thermogenic gene expression;

   The thermogenic gene is selected from FASN, ACACA, ACLY;

   the use is for non-disease diagnosis and treatment purposes.
3. 3. Use according to claim 2, characterized in that the expression or activity of the ZBED6 gene or its encoded protein is inhibited at the transcriptional or translational level by an inhibitor; the inhibitor is at least one selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low-molecular compounds, peptides, antibodies and ZBED6 gene targeting vectors;

   the ZBED6 gene targeting vector is constructed based on CRISPR, TALEN or ZFN genome editing technology.
4. 4. The use according to claim 3, wherein the inhibitor is an siRNA, the nucleotide sequence of which is shown in SEQ ID No. 1 or 2.
5. 5. The use according to claim 1, wherein the expression or activity of ZBED6 gene or its encoded protein is enhanced at the transcriptional or translational level to increase fat deposition in animals, increase the lipid-forming differentiation efficiency of animal precursor adipocytes, and promote thermogenic gene expression;

   The thermogenic gene is selected from FASN, ACACA, ACLY;

   the use is for non-disease diagnosis and treatment purposes.
6. 6. The use according to claim 5, wherein the enhanced pathway is selected from the following 1) to 5), or optionally in combination:

   1) Enhanced by introducing a plasmid having the gene;

   2) Enhanced by increasing the copy number of the gene on the chromosome;

   3) Enhanced by altering the promoter sequence of said gene on the chromosome;

   4) Enhanced by operably linking a strong promoter to the gene;

   5) Enhanced by the introduction of enhancers.
7. 7. The use according to any one of claims 1 to 6, wherein the animal is a mammal.
8. 8. The use according to claim 7, wherein the mammal is selected from the group consisting of pigs, mice.
9. 9. The siRNA targeting the mouse ZBED6 gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 2.
10. 10. A method of promoting the efficiency of differentiation and/or thermogenic function of a beige fat in pigs and mice, the method comprising: enhancing expression of ZBED6 gene by genetic engineering techniques;

    The reference sequence number of the ZBED6 gene from pigs on NCBI is NM_001394675.1, and the reference sequence number of the ZBED6 gene from mice on NCBI is NM_001166552.2;

    The enhanced pathway is selected from the following 1) to 5), or an optional combination:

    1) Enhanced by introducing a plasmid having the gene;

    2) Enhanced by increasing the copy number of the gene on the chromosome;

    3) Enhanced by altering the promoter sequence of said gene on the chromosome;

    4) Enhanced by operably linking a strong promoter to the gene;

    5) Enhanced by the introduction of enhancers.