CN118028375A - Application of ZBED6 gene in regulation and control of animal fat deposition and fat cell differentiation - Google Patents
Application of ZBED6 gene in regulation and control of animal fat deposition and fat cell differentiation Download PDFInfo
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- CN118028375A CN118028375A CN202311838043.2A CN202311838043A CN118028375A CN 118028375 A CN118028375 A CN 118028375A CN 202311838043 A CN202311838043 A CN 202311838043A CN 118028375 A CN118028375 A CN 118028375A
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
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- C—CHEMISTRY; METALLURGY
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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
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of ZBED6 gene in regulating and controlling animal fat deposition and fat cell differentiation.
Background
Type 2 diabetes has become a serious health problem worldwide. Type 2 diabetes is a chronic metabolic disorder highly associated with obesity, closely associated with fat deposition. The adipose tissue of the mammal is an important energy storage and energy metabolism endocrine organ and plays an important role in glucose metabolism, insulin sensitivity, heat generation, immunoregulation and the like. Adipose tissues of mammals can be classified into white adipose tissues and beige adipose tissues according to functions. White adipose tissue is used as main adipose tissue of organism, is mainly responsible for storing energy under the condition of sufficient nutrition, and can form fat mobilization when the organism lacks energy, and triglyceride in white adipose tissue is decomposed into free fatty acid for energy consumption of whole body. Brown adipose tissue is rich in mitochondria, and generates heat by consuming substrates such as fatty acid and glucose, and is mainly distributed around the inter-scapular, two sides of spine, perirenal, thoracic artery and inferior vena cava. The modes of brown fat heat production mainly comprise UCP 1-dependent heat production (mitochondrial uncoupling) and UCP 1-independent heat production (Ca 2+ null cycle, creatine substrate cycle and TAG-fatty acid cycle), and the brown fat can resist obesity and treat immune diseases by consuming heat. On the one hand, UCP+ cells in white fat are induced to activate into beige fat when the organism is stimulated by cold, epinephrine and the like, and the beige fat has the characteristic of brown fat heat generation, such as multi-room fat drops and UCP1 gene expression. On the other hand, under the induction of genes such as PDGFRα, SCA1, CD81, αSMA, PPARγ, PRDM16, EBF2 and the like, fat precursor cells are synthesized from the head into beige fat cells. The ability of adipocytes to respond rapidly to environmental changes is essential for maintaining the balance and health of the body's energy metabolism. There is increasing evidence that induction of beige adipocyte formation has an important contribution to maintaining physical health and treating diseases, but research on targets for regulating beige adipocyte formation is still very limited at present, and development of new molecular targets for the treatment of obesity and diabetes is urgently required. Thus, there is a need to further screen and identify molecular targets that regulate mammalian fat deposition and thermogenic adipocyte production.
Disclosure of Invention
The invention aims to provide application of ZBED6 genes in regulating and controlling fat deposition and fat cell differentiation of animals.
To achieve the object of the present invention, in a first aspect, the present invention provides any one 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 fat development and/or thermogenic animal models (animal models for studying obesity and related diseases);
4) Is used for breeding low-fat high-lean-percentage pigs.
In the invention, 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.
Further, inhibiting expression or activity of ZBED6 gene or its encoded protein at a transcriptional or translational level, or knocking out ZBED6 gene from an animal genome to reduce fat deposition in an animal, reduce lipid-forming differentiation efficiency of precursor adipocytes in an animal, and reduce thermogenic gene expression (reduce thermogenic capacity of precursor adipocytes);
Further, 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 can be at least one selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low-molecular compound, peptide, antibody, ZBED6 gene targeting vector, etc.
The ZBED6 gene targeting vector can be constructed based on genome editing technologies such as CRISPR, TALEN or ZFN.
In one embodiment of the present invention, the inhibitor is siRNA, and the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 1 or 2.
Further, 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 precursor fat cells in animals, and promote thermogenic gene expression (increase the thermogenic capacity of precursor fat cells).
Still further, 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.
The heat-generating gene of the present invention may be selected from FASN, ACACA, ACLY and the like. The reference sequence numbers of the FASN, ACACA, ACLY genes from pigs on NCBI are NM_001099930.1, NM_001114269.1 and NM_001105302.1 respectively, and the reference sequence numbers of the FASN, ACACA, ACLY genes from mice on NCBI are NM_007988.3, NM_133360.3 and NM_001199296.1 respectively.
The use of the invention encompasses non-disease diagnostic and therapeutic purposes.
The animals according to the invention are mammals, such as pigs, mice.
In a second aspect, the invention provides an siRNA targeting mouse ZBED6 gene, the nucleotide sequence of which is shown as SEQ ID NO. 1 or SEQ ID NO. 2.
In a third aspect, the present invention provides a method of promoting the differentiation efficiency and/or thermogenic function of beige fat in pigs and mice, the method comprising: the expression of the ZBED6 gene is enhanced by genetic engineering techniques.
Further, the enhanced pathway may be 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.
The object of the invention can be further achieved by the following technical measures.
The present invention provides the use of a biomaterial comprising the ZBED6 gene in any of the following aspects:
1) Regulating the formation of pig and mouse adipocytes;
2) Research on fat development and metabolism of animals;
3) Cultivating a pig variety;
4) Preparing a medicament for preventing/treating obesity and diabetes;
5) And constructing a low-fat high-lean animal model.
The biological material includes, but is not limited to, host animals, host cells, adenovirus vectors.
The invention also provides ZBED6 gene knockout pigs and application of any one of the following aspects of the ZBED6 gene knockout pigs, primary adipocytes and ZBED6 gene knockout mice:
1) Reducing fat deposition in pigs and mice;
2) Reducing the differentiation efficiency of porcine and mouse adipocytes;
3) Reducing the thermogenesis efficiency of porcine and mouse adipocytes
4) Researching the characteristics of fat traits of pigs and mice;
5) Cultivating a low-fat high-lean-percentage pig variety;
6) Provides a research platform for obesity and diabetes mechanism.
The invention also provides the use of ZBED6 gene overexpression adenoviruses or any of the following aspects of the editing system of the targeted ZBED6 gene that can promote ZBED6 gene expression:
1) Increasing fat deposition;
2) Increasing the differentiation efficiency of adipocytes;
3) The characteristics of fat development were studied.
For example, the editing system of the ZBED6 gene is an operating system for promoting the expression of the ZBED6 gene by an adenovirus-coated over-expression vector.
The invention also provides the use of a ZBED6 gene inhibitor for any of the following aspects of a gene editing system that reduces ZBED6 gene expression:
1) Reducing the differentiation efficiency of the 3T3 precursor adipocyte cell line;
2) Improving animal fat deposition;
3) Is used for researching obesity and related diseases.
For example, the editing system of ZBED6 gene is an operating system for siRNA to inhibit expression of mouse ZBED6 gene.
The invention also provides siRNA of a target mouse ZBED6 gene, which has the nucleotide sequence as follows:
SEQ ID NO:1：5′-CCAGCUUCCAUGGAAGAAU-3′
SEQ ID NO:2：5′-AUUCUUCCAUGGAAGCUGG-3′
The invention also provides an animal model for reducing fat deposition of pigs and mice, wherein ZBED6 genes of the pigs and the mice are knocked out by a gene editing technology, and primary fat cells of the pigs are separated; the expression of the mouse ZBED6 gene is reduced by knocking down and silencing the ZBED6 gene.
For example, the ZBED6 gene is knocked out by gene editing techniques, resulting in systemic deficiency of ZBED6 gene in pigs and mice; or knocking down the expression of ZBED6 gene by siRNA.
The invention also provides a method for promoting the differentiation efficiency and/or the thermogenesis function of the beige fat of pigs and mice, and the expression of the ZBED6 gene is increased by a genetic engineering technology.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention provides a method for increasing the expression of ZBED6 genes to promote the differentiation and heat production of beige fat cells of pigs and mice, provides an animal model for inhibiting the fat deposition and heat production of the pigs and the mice, and provides a research platform and theoretical basis for regulating and controlling the fat deposition and the formation of fat heat production cells of the pigs and the mice.
Drawings
FIG. 1 shows the fat deposition phenotype of ZBED6 knockout pigs in example 1 of the present invention. Wherein, 17F 4-generation bab Ma Zhu fat phenotypes (3 for boar wild type and ZBED6 knockout pigs; 5 for sow wild type, 6 for ZBED6 knockout pigs) were collected altogether, A: fat weight of the boars and sows is kg; b: a ratio of weight to weight of fat in the boar; c: the thickness of the fat at the back of the boar is in mm; d: abdomen fat weight of the boar is kg; e: wild boars and ZBED6 knock-out pig back fat and abdomen fat HE slices with a scale of 50 μm; f: quantitative statistics of the cross-sectional areas of dorsal and ventral fat cells. P <0.05 in the figure indicates significance.
FIG. 2 shows fat deposition phenotype of ZBED6 knockout mice in example 2 of the present invention, in which 24 male mice data were collected, 12 wild type and ZBED6 knockout mice each. A: wild type and ZBED6 knockout mice body weight were fed with high fat for 12 weeks; b: after 12 weeks of high fat feeding, wild type and ZBED6 knockout mice were given white fat weights on their abdomen; c: wild type and ZBED6 knockout mice were cut into white fat HE sections on their abdomen after 12 weeks of high fat or high sugar feeding; d: after 12 weeks of normal or high fat feeding, wild type and ZBED6 knockout mice were tested for glucose tolerance; e: insulin sensitivity assay. In the figure, P <0.05, < P <0.01, < P <0.001 means significance, and ns means no significant difference.
FIG. 3 shows the effect of the isolation of primary precursor adipocytes after ZBED6 knockdown in Bama miniature pig of example 3 on adipocyte differentiation efficiency, and the effect of SiRNA interference on 3T3 mouse precursor adipocyte differentiation efficiency after ZBED6 is used in example 4, and the effect of overexpression of mouse ZBED6 gene in example 6 on mouse primary precursor adipocyte differentiation efficiency and marker gene expression. Wherein A: expression of the fatty synthesis marker gene protein after 8 days of induced differentiation of wild type and ZBED6 knockout primary adipocytes; b: oil red O staining and quantification conditions after 8 days of induced differentiation of wild type and ZBED6 knockout primary adipocytes; c: after knocking down ZBED6, the 3T3 precursor fat cells induce and differentiate for 8 days to synthesize the mRNA expression condition of the marker gene; d: after knocking down ZBED6, inducing 3T3 precursor fat cells to differentiate for 8 days, and expressing ZBED6 and fat synthesis marker gene proteins; e: knocking down ZBED6, performing oil red O staining after induced differentiation for 8 days, and quantifying a staining result; f: after interfering with ZBED6, the cell cycle changes; g: after the ZBED6 is over-expressed, 3T3 precursor fat cells are induced to differentiate for 8 days, and the expression condition of the ZBED6 and fat synthesis marker gene mRNA is shown; h: after the ZBED6 is over-expressed, the 3T3 precursor fat cells are induced to differentiate for 8 days, and the ZBED6 and fat synthesis marker gene protein are expressed; i: after ZBED6 is over-expressed, oil red O staining is carried out after induced differentiation is carried out for 8 days, and the staining result is quantified; j: after overexpression of ZBED6, cell proliferation was examined.
Detailed Description
The invention aims to provide a novel application of ZBED6 gene, in particular to an application of the ZBED6 gene in regulating fat deposition, fat cell differentiation efficiency and heat generating function of pigs and mice.
The ZBED6 gene of the invention is well known in the art, e.g. under accession numbers 100622313 (pig), 667118 (mouse) at NCBI.
The invention adopts the following technical scheme:
In a first aspect, the present invention provides the use of a ZBED6 encoding gene, or a biomaterial comprising a gene encoding the same, in any of the following aspects:
1) Regulating fat deposition in pigs and mice;
2) Regulating the efficiency of pig and mouse precursor adipocyte differentiation;
3) Study of mammalian fat development and metabolism;
4) Cultivating a pig variety;
5) Preparing a medicament for preventing, alleviating and/or treating obesity-related diseases;
6) And constructing a fat development and/or heat generation animal model.
The biological material includes, but is not limited to, host cells, bama pig or C57BL/6J mice.
The ZBED6 coding gene or the biological material containing the coding gene can also be applied to research on pathogenesis of diabetes and obesity, cultivation of low-fat and high-lean-meat-percentage pig varieties, screening and identification of genes related to fat deposition of pigs and mice, and screening and identification of beige fat genes of the pigs and the mice.
The invention discovers that fat deposition is obviously reduced and brown fat is whitened by knocking out ZBED6 genes in vivo in mice of the bars Ma Zhu and the C57BL/6J, and the knocking-out ZBED6 can obviously reduce the adipogenic differentiation efficiency of SVF cells and reduce the thermogenic gene expression by separating wild type and ZBED 6-/-primary SVF cells for in vitro culture. The expression of ZBED6 gene can promote the adipogenic differentiation of SVF cells of pigs and mice into beige adipocytes and increase the expression of thermogenic genes, and further the application of ZBED6 gene in the adipogenic differentiation of SVF cells of pigs and mice is proposed.
It was found that mutation of IGF2 gene (NCBI accession No. nm_ 213883.2) disrupts binding to an unknown nuclear factor, causing up-regulation of IGF2 expression in skeletal muscle by a factor of 3, ultimately resulting in an increase in skeletal muscle mass and an increase in lean muscle mass of 3-4%. This binding of IGF2 occurs only after birth and does not bind to IGF2 hypermethylated regions during fetal periods. In 2009, this unknown nuclear factor was found to be a transcription factor, designated ZBED6, by regulating IGF2 expression and thus muscle growth. ZBED6 evolved from a DNA transposon, which was at the same position in ZC3H11A intron 1 of all placental mammals. ZBED6 has a basic function in the evolution process, has 2 DNA binding regions, and shows nearly 100% amino acid identity in 26 placental mammals. In addition, the ZBED6 can be knocked out in pigs and mice to obviously promote the development of tissues and organs such as skeletal muscles, spleens and the like, and the ZBED6 has important functions.
The ChIP-seq of porcine skeletal muscle and C2C12 cells reveals ZBED6 regulated target genes, all of which have the motify sequence GCTCG to which ZBED6 binds, most of which occur near the transcription initiation site. The pig ZBED6 gene is positioned on chromosome 9, the coding region is 2946nt, and 981 amino acids are coded. The mouse ZBED6 gene is positioned on chromosome 1, the coding region is 2943nt, and 980 amino acids are coded. The current research on ZBED6 is mainly focused on skeletal muscle development and atrophy, islet beta cell function, immune response and the like. There are few reports on how ZBED6 regulates fat deposition and formation of beige adipocytes.
The invention uses gene editing means to knock out ZBED6 gene, eliminates the expression of ZBED6 gene from the transcription and translation level, verifies that it can inhibit fat deposition and the generation of beige fat cells from fat tissue, primary cell and marker gene mRNA and protein level.
The invention constructs a ZBED6 gene over-expression adenovirus vector by utilizing a genetic engineering means, over-expresses the ZBED6 gene, promotes or improves the expression of the ZBED6 gene on the transcriptional and translational level, and verifies that the ZBED6 gene can promote the generation of beige adipocytes from the primary cell, the marker gene mRNA and the protein level.
In the 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 invention provides the use of porcine and mouse ZBED6 overexpressing adenovirus vectors or any of the following aspects that can promote ZBED6 gene expression:
1) Increasing the differentiation efficiency of porcine and mouse precursor adipocytes;
2) Increasing the thermogenic capacity of porcine and mouse precursor adipocytes;
3) Researching the characteristics of fat traits of pigs and mice;
4) Reducing fat deposition in mammals;
5) And (5) cultivating a low-fat high-lean-percentage high-quality pig variety.
Wherein the ZBED6 overexpressing adenovirus vector is capable of increasing expression of the porcine and mouse ZBED6 gene from both transcriptional and translational levels.
In a third aspect, the use of ZBED6 in adipocytes can be verified in vitro by knocking out ZBED6 gene and isolating primary SVF cells in pigs and mice by gene editing techniques:
1) Reducing the differentiation efficiency of porcine and mouse precursor adipocytes;
2) Reducing the thermogenic capacity of porcine and mouse precursor adipocytes;
3) Improving fat deposition in mammals;
4) Is used for researching genetic mechanism of obesity and type 2 diabetes.
Wherein the primary SVF cells are primary cells from which the ZBED6 gene is knocked out, and are capable of eliminating the expression of the ZBED6 gene from the transcriptional and translational levels.
In a fourth aspect, the present invention provides an animal model capable of reducing fat deposition and/or thermogenesis in pigs and mice by knocking out the expression of ZBED6 gene by gene editing techniques.
In a fifth aspect, the present invention provides a method capable of increasing the differentiation efficiency and/or the thermogenic capacity of porcine and mouse precursor adipocytes by increasing the expression of porcine and mouse ZBED6 gene by means of an adenovirus overexpression vector.
In a sixth aspect, the present invention provides an siRNA targeting 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, expression of the ZBED6 gene is ablated by knockout of the ZBED6 gene. The expression of ZBED6 gene was knocked down by siRNA.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 ZBED6 knockout pig fat deposition phenotype
Phenotype data of wild type and knock-out ZBED6 gene back fat and abdominal fat of Bama miniature pigs were obtained and quantified to show the effect of ZBED6 on pig fat deposition. The observation results are shown in FIG. 1. From the figure, the ZBED6 can be knocked out by the Bama miniature pig, so that the deposition of back fat and abdominal fat can be remarkably reduced. The ZBED6 gene has important role in the pig fat deposition process.
The construction method of the ZBED6 gene knockout pig comprises the following steps:
1. The sgrnas Zg1, zg2, zg3, zg4 (constructed on pX330-Cas9 vector, pX330-Cas9 constructed by inserting Cas9 protein coding gene into pX330 vector) were transfected into pig fetal fibroblasts, respectively, 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 (Zg 3, targeting efficiency 7.5%) by a generation sanger sequencing;
3. Screening 3 positive cell clones Z17, Z23 and Z28;
4. Performing somatic cell nuclear transfer on the positive cells, constructing a reconstructed embryo and transferring the reconstructed embryo to a donor sow;
5. And (3) carrying out a generation sanger sequencing on the genetically edited piglet, and identifying that the frame shift mutation occurs at 1320-T locus, which proves that the construction of the ZBED6 gene knockout pig is successful.
EXAMPLE 2 ZBED6 knockout mouse fat deposition phenotype
Phenotype data such as body weight, abdominal fat HE section and the like of C57BL/6J mice wild type and knock-out ZBED6 gene were obtained and quantified to show the effect of ZBED6 on fat deposition in mice. The observation results are shown in FIG. 2.
This example further examined glucose tolerance and insulin sensitivity in wild type and ZBED6 knockout mice in normal feeding and high fat feeding. From the results, it was found that the C57BL/6J mice were able to significantly reduce fat weight and promote glucose tolerance and insulin sensitivity in both high-fat and high-sugar states after ZBED6 knockout. It is demonstrated that knockout of ZBED6 can significantly increase the glucose metabolism ability of mice, increase energy expenditure and thereby reduce fat deposition.
The construction method of ZBED6 gene knockout mice is as follows:
1. ZBED6-flox mice were constructed with a LoxP site inserted 230bp upstream of the ZBED6 gene ATG and a FRT-Neo-FRT-LoxP site inserted 370bp downstream of the TAA.
2. Inserting 6kb homology arms at two ends of LoxP site; wherein, the left homology arm extends upwards by 6kb from 230bp upstream of the ZBED6 gene start codon, and the right homology arm extends downwards by 6kb from 370bp downstream of the ZBED6 gene start codon.
3. ZBED6-flox mice were hybridized with PGK-Cre mice to generate ZBED6 systemic knockout mice (PGK-Cre mice are systemic knockout tool mice, given by Leif Andersson professor Leif, uppsala university, sweden, PGK-Cre mice see 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 U S A.2018 Feb 27;115(9):E2048-E2057.).
Example 3 knock-out of the ZBED6 Gene Regulation of porcine precursor adipocyte differentiation
In this example, precursor adipocytes were isolated from wild type pamphlet and ZBED6 knock-out pamphlet inguinal subcutaneous fat and perirenal fat, induced to differentiate, and differential conditions of the wild type and ZBED6 knock-out differentiation in vitro into adipocytes were observed by oil red O. The observations are shown in fig. 3 as a and B.
The present example further examined the protein expression level of the fat secondary gene after 8 days of differentiation of wild type and ZBED6 knockout pig precursor adipocytes, and the FASN, ACACA, were all significantly down-regulated. The oil red O staining result shows that the knockout ZBED6 can remarkably inhibit the differentiation efficiency of the primary precursor fat cells of pigs. It was demonstrated that knockout of ZBED6 can inhibit de novo synthesis of pig fat and thus inhibit primary adipocyte differentiation in pigs.
The specific process of separating and culturing the wild type Bama miniature pig from ZBED6 knockout precursor adipocytes is as follows:
1) Taking 1 month old wild Bama miniature pigs and ZBED6 knockout Bama miniature pigs, sterilizing the whole body by using alcohol cotton after killing, taking out inguinal white fat and perirenal white fat on a sterile workbench, soaking in PBS (phosphate buffer solution) containing 2% penicillin/streptomycin precooling, and transferring to an ultra-clean workbench;
2) Washing adipose tissue 3 times with PBS (phosphate buffer solution) precooled by 2% penicillin/streptomycin, cutting the adipose tissue into meat paste by scissors, adding 2 times of volume of DMEM/F12 culture medium containing 2mg/ml type I collagenase, and placing in a shaking table at 37 ℃ for digestion for 60 minutes;
3) Adding an equal volume of DMEM/F12 complete medium into the digested tissue to stop digestion, filtering the tissue suspension by using a 70 mu m cell filter, and centrifuging at 1500r/min for 10min at room temperature;
4) Sucking and removing supernatant, adding 3-5ml erythrocyte lysate to resuspend cell sediment, standing at 4 ℃ for 10min, and centrifuging at 1500r/min for 10min;
5) The supernatant is sucked and removed, 1-2ml of DMEM/F12 complete medium is added to resuspend the cell sediment, the cell sediment is inoculated in a T75 cell culture flask, the medium is supplemented to 15ml, after shaking evenly, the cell sediment is placed in a cell culture box containing 5% CO 2 at 37 ℃ for culture, the cell sediment is replaced once every 2d, and the cell sediment is passaged after being grown up.
The induced differentiation process of pig precursor adipocytes is as follows:
1) Plating is carried out after the pig precursor fat cells are full of growth, and 3 12-hole cell culture plates can be paved on the pig precursor fat cells in one T75 culture flask;
2) Induction of differentiation was initiated after two days of contact inhibition after cell confluence (differentiation for 0 day);
3) After two days of contact inhibition, the culture medium is replaced by an induced differentiation culture medium, and the culture medium is replaced every two days.
4) The ratio of the differentiation medium is as follows: DMEM high sugar medium, 0.5mM 3-isobutyl-1-methylxanthine (IBMX), 0.1. Mu.M dexamethasone, 0.5. Mu.M insulin, 2nM triiodothyronine (T3), 30. Mu.M indomethacin (indomethacin), 17. Mu.M pantothenic acid, 33. Mu.M biotin, 1. Mu.M rosiglitazone, 2% FBS.
Example 4 knock-down of ZBED6 Gene Regulation of mouse precursor adipocyte differentiation
In this example, ZBED6 was induced to differentiate using siRNA in 3T3 mouse precursor adipocytes, and the difference in 3T3 mouse precursor adipocyte differentiation was observed by oil red O. The observations are shown at C, D, E, F in FIG. 3.
The present example further examined the effect of knock-down ZBED6 on proliferation and differentiation of 3T3 mouse precursor adipocytes, and the results demonstrate that knock-down ZBED6 can significantly inhibit proliferation and differentiation of 3T3 mouse precursor adipocytes. The effect of knock-down ZBED6 on expression of 3T3 mouse precursor fat cell de novo synthesis genes was further examined, and the results indicate that knock-down ZBED6 can significantly inhibit expression of fat de novo synthesis genes. It was demonstrated that knock-down of the expression level of ZBED6 can inhibit fat synthesis.
The specific experimental process is as follows: after the cell confluence in a T75 cell culture flask reaches 90%, inoculating the cells to a 12-hole cell culture plate, and carrying out siRNA transfection when the cell confluence reaches 40%, wherein 6 holes are transfected with siRNA iMAX and the other 6 holes are transfected with siRNA-ZBED6, the transfected siRNAs are two siRNAs with two bases T respectively connected after the sequence SEQ ID NO:1 or SEQ ID NO:2 (TT overhang is respectively added at the 3' -end of SEQ ID NO:1 or SEQ ID NO:2 to form an adhesive tail end so as to facilitate melting);
The transfection method comprises the following steps:
1. inoculating cells into a 12-well plate, and transfecting when the cell confluence reaches 40%;
2. 300. Mu.L of OPTI-MEM and 60. Mu.M siRNA were added to ① tubes, 300. Mu. LOPTI-MEM and 15. Mu.L RNAiMAX were added to ② tubes, which is the amount of 6 wells of a 12-well plate;
3. The mixed solution in the tube ① is transferred to the tube ②, and the mixed solution is slightly mixed and kept stand for 10 minutes; the mixture was added to the cell culture plate uniformly. Two days after cell confluence (day 0 of differentiation) were subjected to inhibition and then to transfection while changing to beige fat-induced differentiation medium (89% DMEM/F12 medium, 10% fetal bovine serum, 1 μm dexamethasone, 1 μm rosiglitazone, 850nM insulin, 0.5mM IBMX, 1% penicillin/streptomycin); after 48 hours of differentiation the medium was changed to maintenance medium (89% DMEM/F12 medium, 10% foetal calf serum, 1 μm rosiglitazone, 850nM insulin, 1% penicillin/streptomycin) and then every 48 hours the medium was changed for a total of 8 days.
After the siRNA-ZBED6 is specifically transfected for 48 hours, the mRNA expression quantity of the ZBED6 gene is detected (the internal reference is 18S), and the detection result is shown as A in FIG. 3, which shows that the mRNA expression quantity of the ZBED6 gene can be obviously reduced by using the siRNA-ZBED6 (the P is less than 0.05); the protein expression level of ZBED6 gene was examined (reference is Tubulin), and the results are shown in B in fig. 3, which indicate that the use of siRNA-ZBED6 can significantly (P < 0.01) reduce the protein expression level of ZBED6 gene.
When the cell density reaches about 90%, the embodiment detects the influence on cell proliferation after knocking down the ZBED6 gene through EDU cell proliferation detection experiment, cell cycle detection and cell scratch experiment, and the experimental result is shown as C/D/E in fig. 3, which shows that the expression quantity of the knockdown ZBED6 gene can remarkably (P < 0.01) inhibit cell proliferation.
After 8 days of induced differentiation, the present example uses oil red O staining to verify the cell differentiation efficiency after knocking down ZBED6 gene in the 3T3 mouse precursor adipocyte induced differentiation, and the oil red O staining results are shown in G and H (G is oil red O staining results, H is oil red O staining quantitative results) in fig. 3, which show that the 3T3 mouse precursor adipocyte differentiation efficiency is significantly reduced after knocking down ZBED6 gene.
From the above results, it was found that the proliferation and differentiation efficiency of 3T3 mouse precursor adipocytes was significantly reduced (the knock-down efficiency was 50% or more) after knocking down the expression level of ZBED6 gene.
EXAMPLE 5 Effect of over-expressed mouse ZBED6 Gene on differentiation efficiency of mouse precursor adipocytes and thermogenic Gene expression
In this example, ZBED6 was overexpressed in 3T3 mouse precursor adipocytes using a ZBED6 adenovirus overexpression vector, induced to differentiate, and the difference in 3T3 mouse precursor adipocyte differentiation was observed by oil red O. The observations are shown at G, H, I, J in FIG. 3.
The present example further examined the effect of over-expressed ZBED6 on proliferation and differentiation of 3T3 mouse precursor adipocytes, and the results demonstrate that over-expressed ZBED6 can significantly promote proliferation and differentiation of 3T3 mouse precursor adipocytes. The effect of over-expression ZBED6 on the expression of 3T3 mouse precursor fat cell de novo synthesis gene was further examined, and the result shows that over-expression ZBED6 can significantly inhibit the expression of fat de novo synthesis gene. It was demonstrated that increasing the expression level 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%, inoculating the cell culture flask to a 12-hole cell culture plate, and carrying out adenovirus infection when the cell confluence reaches 40%, wherein 6 holes use ZBED6 adenovirus over-expression vectors, and the other 6 holes use adenovirus over-expression negative vectors, and the CDS region used for constructing the vectors is NM_001166552.2.
The construction method of the ZBED6 adenovirus overexpression vector is as follows:
1. Constructing a CDS region of the ZBED6 gene on a tool carrier PSB 50;
2. transferring the CDS region of the ZBED6 gene to a PSE6041 vector through homologous recombination for subsequent adenovirus packaging;
3. Adenovirus packaging was performed by AdMax adenovirus packaging system using helper vector pBHG loxΔe1,3 Cre;
4. Transfecting the vector into HEK293 cells for virus amplification, collecting the cells and collecting and purifying adenovirus by ultracentrifugation;
5. Purified adenovirus titer was measured at a viral titer of 1.14X10 11 IFU/mL.
The carriers used above were all purchased from Shanghai Biotechnology Co.
The cell infection method is as follows:
1) When the cell density reached 40%, penicillin/streptomycin free medium (12 well plate plus 500 μl) was replaced with 5% fetal bovine serum;
2) Adenovirus diluted by addition of OPTI-MEM reduced serum medium (available from Gibco) (moi=100);
3) Penicillin/streptomycin free medium with 5% fetal bovine serum was added to 1mL after 4 hours;
4) After 8 hours, the culture medium is replaced by a complete culture medium, and the culture is carried out until the subsequent induced differentiation.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Reference is made to:
[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 Oct 28;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 U S 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)
- 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. 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. 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. 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. 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. 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. The use according to any one of claims 1 to 6, wherein the animal is a mammal.
- 8. The use according to claim 7, wherein the mammal is selected from the group consisting of pigs, mice.
- 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. 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.
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| CN118685412A (en) * | 2024-08-28 | 2024-09-24 | 中国农业科学院北京畜牧兽医研究所 | Application of ZBED6 gene in regulation and control of skeletal muscle atrophy of animals |
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| CN118685412A (en) * | 2024-08-28 | 2024-09-24 | 中国农业科学院北京畜牧兽医研究所 | Application of ZBED6 gene in regulation and control of skeletal muscle atrophy of animals |
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