Super-enhancer inhibiting proliferation and differentiation of chicken preadipocyte and application thereof
Patent Information
- Application Number
- CN202610979237.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-02
- Publication Date
- 2026-09-25
AI Technical Summary
[0005]二、存在缺陷
[0017]有益效果：本发明提供一种FABP4基因的核心增强子序列，并且在永生化鸡前脂肪细胞系中敲除核心增强子序列后可以抑制鸡前脂肪增殖和分化。本发明确定了一个新型的调控鸡脂肪生长发育的作用靶点，为实现鸡重要经济性状的遗传改良提供强大的资源。同时，本发明构建的调控鸡FABP4基因的核心增强子敲除细胞株。
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Figure CN122811176A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a super enhancer that inhibits the proliferation and differentiation of chicken preadipate and its applications. Background Technology
[0002] Fatty acid-binding proteins (FABPs) are an important class of intracellular fatty acid transporters that play a crucial role in adipocyte differentiation and lipid metabolism. Members of the FABP family exhibit significant tissue-specific expression, which is closely related to their physiological functions. Chickens are not only important economic animals but also commonly used model organisms in developmental biology and immunology research. However, while modern broiler breeding has significantly improved growth rate and meat production performance, it has also brought about the prominent problem of excessive abdominal fat deposition. Excessive abdominal fat not only reduces feed utilization and meat quality but also directly harms the economic benefits of poultry farms and weakens consumer acceptance of the product. Therefore, elucidating the molecular mechanisms of broiler fat generation and development, and providing a theoretical basis for reducing abdominal fat deposition and breeding low-fat breeds, is crucial for the sustainable development of the broiler breeding industry.
[0003] Currently, the main methods for reducing excessive fat deposition in broilers and inhibiting the proliferation and differentiation of abdominal preadipocytes include three categories: nutritional regulation, genetic selection and breeding, and molecular intervention, but each has obvious limitations.
[0004] I. Main Technologies Nutritional additive regulation: Adding conjugated linoleic acid (CLA), n-3 polyunsaturated fatty acids (fish oil / EPA / DHA), butyric acid, or plant extracts (such as oregano oil) to the diet can downregulate adipogenic genes such as PPARγ, C / EBPα, and FABP4, and in vitro studies have shown that it can inhibit the clonal expansion and lipid droplet accumulation of chicken preadipocytes.
[0005] II. Existing Defects Nutritional interventions are inconsistent in their effectiveness: additives have limited effects on reducing abdominal fat (usually 10%–25%), and are greatly affected by breed, age, and dosage; high doses of CLA or PUFA may interfere with the absorption of fat-soluble vitamins, affect growth performance, or produce off-odors, and most only inhibit one aspect of proliferation or differentiation, making it difficult to achieve dual and effective blockade. An effective method is urgently needed. Summary of the Invention
[0006] The purpose of this invention is to inhibit the proliferation and differentiation of chicken preadipocytes.
[0007] This invention provides a super enhancer that inhibits the proliferation and differentiation of chicken preadipate, the core enhancer of which is shown in SEQ ID NO.1.
[0008] The present invention provides a recombinant vector or recombinant microbial cell containing the above-mentioned core enhancer knocked out.
[0009] This invention provides the application of knocking out the above-mentioned core enhancer, recombinant vectors containing the above-mentioned core enhancer, or recombinant microbial cells in regulating the proliferation and differentiation of chicken preadipocytes.
[0010] This invention provides the application of knocking out the above-mentioned core enhancer, recombinant vectors containing the above-mentioned core enhancer, or recombinant microbial cells in the preparation of biological reagents that inhibit the proliferation and differentiation of chicken preadipocytes.
[0011] This invention provides an application of the above-mentioned core enhancer in enhancing the transcriptional activity of the chicken FABP4 gene.
[0012] This invention provides a method for preparing a chicken cell model with suppressed expression of the above-mentioned core enhancer. The method involves ligating gRNA into a PX458 vector to obtain recombinant vectors, and then co-transfecting the recombinant vectors into chicken preadipocytes to obtain a chicken cell model. The gRNA is shown in SEQ ID NO.6 and SEQ ID NO.7.
[0013] To further specify, the chicken preadipocytes are immortalized chicken preadipocyte line (ICP2).
[0014] This invention provides a method for inhibiting the proliferation and differentiation of chicken preadipocytes by knocking out the core enhancer shown in SEQ ID NO.1 in chicken preadipocytes.
[0015] Further specifying, the gRNA was ligated into the PX458 vector to obtain a recombinant vector, and then the recombinant vector was co-transfected into chicken preadipocytes, wherein the gRNA is shown in SEQ ID NO.6 and SEQ ID NO.7.
[0016] This invention provides a chicken preadipocyte that inhibits proliferation and differentiation, and is a cell line obtained by knocking out the core enhancer shown in SEQ ID NO.1 in chicken preadipocytes.
[0017] Beneficial Effects: This invention provides a core enhancer sequence of the FABP4 gene, and knocking out this core enhancer sequence in immortalized chicken preadipocyte cell lines can inhibit preadipocyte proliferation and differentiation. This invention identifies a novel target for regulating chicken adipocyte growth and development, providing a powerful resource for the genetic improvement of important economic traits in chickens. Simultaneously, this invention constructs a core enhancer knockout cell line that regulates the chicken FABP4 gene. Attached Figure Description
[0018] Figure 1Schematic diagram of the enrichment regions modified by FABP4-SE and its components with enhancers H3K4me1 and H3K27ac.
[0019] Figure 2 FABP4 Schematic diagram of the activity of SE component enhancers in chicken preadipocytes.
[0020] Figure 3 Identification of FABP-SE-E1 homozygous knockout cell clones. Figure 4 FABP4 Results of FABP4 gene mRNA expression level detection after SE-E1 knockout.
[0021] Figure 5 FABP4 The effect of SE-E1 knockout on the proliferation of chicken preadipocytes; A is the effect of FABP-SE-E1 knockout on ICP2 cell proliferation as detected by CCK-8 assay; B is the effect of FABP-SE-E1 knockout on the expression of proliferation marker genes as detected by Real-time PCR; C is the effect of FABP-SE-E1 knockout on ICP2 cell DNA synthesis as detected by EdU staining. Figure 6 FABP4 The effect of SE-E1 knockout on the differentiation of chicken preadipocytes; A shows the Oil Red O staining results of wild-type ICP2 cells and FABP-SE-E1 knockout cells at 24, 48, and 72 h of induced differentiation; B shows the Oil Red O quantification of wild-type ICP2 cells and FABP-SE-E1 knockout cells at 24, 48, and 72 h of induced differentiation; C shows the expression of adipogenesis marker genes in wild-type cells and FABP-SE-E1 knockout cells at 24, 48, and 72 h of induced differentiation detected by Real-time RT-PCR. Detailed Implementation
[0022] Example 1. Obtaining a super enhancer of the FABP4 gene 1. This invention, based on high-throughput sequencing data of histone modifications H3K4me1 and H3K27ac Cut&tag from early adipose tissue, first discovered a super enhancer targeting FABP4 (Genbank NM_204290.2) using the ROSE algorithm, and named it FABP4-SE. Combining histone modifications and PPARγ-enriched regions, FABP4-SE was divided into five component enhancers: E1, E2, E3, E4, and E5 (…). Figure 1The nucleotide sequences of E1 are shown in SEQ ID NO:1, E2 in SEQ ID NO:2, E3 in SEQ ID NO:3, E4 in SEQ ID NO:4, and E5 in SEQ ID NO:5. E1 is located at chromosome 120996301 of the chicken genome. E2 is located at position 120998000 on chromosome 2 of the mouse genome, specifically positions 121000800-121002500; E3 is located at position 121008800-121010700 on chromosome 2 of the mouse genome; E4 is located at position 121017800-121019600 on chromosome 2 of the mouse genome; and E5 is located at position 121020500-121022200 on chromosome 2 of the mouse genome.
[0023] 2. Chicken preadipocytes were cultured in DMEM / F12 medium containing 10% fetal bovine serum and incubated at 37°C with 5% carbon dioxide, with the medium changed every two days. The enhancers of each component were cloned into the STARR-seq luciferase validation vector, and the plasmid was co-transformed with pRL-TK into chicken preadipocytes [immortalized chicken preadipocyte line (ICP2)]. Dual-luciferase reporter gene assays were performed to evaluate the activity of each component enhancer. The results showed that enhancer E1 was the core enhancer fragment of FABP4-SE and exhibited the strongest activity. Figure 2 ).
[0024] Example 2. Preparation of a cell line that inhibits the differentiation and proliferation of chicken preadipocytes 1. Constructing the carrier: For FABP4 SE E1 was used to design and construct gRNA vectors. GRNAs targeting and knocking out upstream regulatory sequences were designed. 1 and downstream gRNA 2. The DNA sequences corresponding to upstream gRNA1 and downstream gRNA2 were ligated into the PX458 vector, named PX458-gRNA1 and PX458-gRNA2, respectively. 1: TGTTCGCGGGGCATATAAGA (SEQ ID NO.6); gRNA 2:TTGATATGTAACTATTGTCC (SEQ ID NO.7); 2. Cell line construction: PX458-gRNA1 and PX458-gRNA2 vectors were co-transfected into chicken preadipocytes. Monoclonal cells were screened using flow cytometry, and genomic DNA was extracted. PCR amplification of the CRISPR / Cas9-edited sites was performed (upstream primer: GTTTTATCCCCGTTGCTTGTG, SEQ ID NO.8; downstream primer: GCATGTGAAGTTGCTACAGGTATG, SEQ ID NO.9) and sequencing was performed to obtain knockout monoclonal cell lines. Figure 3 RNA was extracted from knockout cells, and the expression level of FABP4 mRNA was detected. The results showed that the expression level of FABP4 was significantly decreased compared with WT cells (immortalized chicken preadipocytes). Figure 4 ).
[0025] 3. CCK8 results showed that, compared with the WT group, knocking out FABP4... SE-E1 significantly inhibited the proliferation of chicken preadipocytes. Figure 5 A). RT-qPCR analysis further showed that the expression levels of proliferation marker genes CyclinD1, PCNA, and Ki67 were significantly downregulated in knockout cells. Figure 5 B). Furthermore, DNA synthesis activity was detected by EdU staining, revealing a significant decrease in EdU incorporation in FABP4-SE-E1 knockout cells. Figure 5 C). The above results consistently indicate that FABP-SE E1 knockout significantly inhibited the proliferative capacity of preadipocytes. (The text abruptly ends here, likely due to an incomplete translation or a formatting error.) SE-E1 knockout group and WT cells underwent oleic acid-induced differentiation. Oil Red O extraction and colorimetric results are as follows: Figure 5 The results showed that lipid droplet accumulation in FABP-SE-E1 knockout cells was significantly lower than that in WT cells. RT-PCR results indicated that the expression levels of PPARγ, AdipoQ, C / EBPα, and LPL were significantly downregulated in knockout cells. Figure 6 ).
[0026] E1 sequence: (SEQ ID NO.1) E2 sequence: (SEQ ID NO.2) E3 sequence: (SEQ ID NO.3) E4 Sequence: (SEQ ID NO.4) E5 sequence: (SEQ ID NO.5)
Claims
1. A super enhancer that inhibits the proliferation and differentiation of chicken preadipocytes, characterized in that, The core enhancer of the super enhancer is shown in SEQ ID NO.
1.
2. A recombinant vector or recombinant microbial cell containing the core enhancer of claim 1 knocked out.
3. The application of knocking out the core enhancer of claim 1, a recombinant vector containing the knockout of the core enhancer of claim 1, or recombinant microbial cells in regulating the proliferation and differentiation of chicken preadipocytes.
4. The use of the core enhancer of claim 1, the recombinant vector containing the core enhancer of claim 1, or the recombinant microbial cell in the preparation of a biological reagent that inhibits the proliferation and differentiation of chicken preadipocytes.
5. The application of the core enhancer described in claim 1 in enhancing the transcriptional activity of the chicken FABP4 gene.
6. A method for preparing a chicken cell model with suppressed core enhancer expression as described in claim 1, characterized in that, Recombinant vectors were obtained by ligating gRNA into the PX458 vector, and then co-transfected the recombinant vectors into chicken preadipocytes to obtain a chicken cell model. The gRNAs are shown in SEQ ID NO.6 and SEQ ID NO.
7.
7. The preparation method according to claim 6, characterized in that, The chicken preadipocytes are an immortalized chicken preadipocyte line.
8. A method for inhibiting the proliferation and differentiation of chicken preadipocytes, characterized in that, Knock out the core enhancer shown in SEQ ID NO.1 in chicken preadipocytes.
9. The method according to claim 8, characterized in that, The gRNA was ligated into the PX458 vector to obtain a recombinant vector, which was then transfected into chicken preadipocytes. The gRNA is shown in SEQ ID NO.6 and SEQ ID NO.
7.
10. A chicken preadipocyte that inhibits proliferation and differentiation, characterized in that, The cell line obtained by knocking out the core enhancer shown in SEQ ID NO.1 in chicken preadipocytes.