Construction method and application of human TFEB gene transgenic fruit fly model
By constructing a human TFEB gene transgenic fruit fly model, the problem that TFEB research in the existing technology is mainly carried out at the cell line level, and the physiological function and tumorigenesis mechanism of TFEB are studied in the fruit fly model, providing a tool for disease drug screening.
Patent Information
- Application Number
- CN202510086500.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-05-02
AI Technical Summary
The research on TFEB in the prior art is mainly carried out at the cell line level, and the driving mechanism of TFEB in tumor or immune diseases has not been fully analyzed.
A human TFEB gene transgenic fruit fly model was constructed, and the expression of TFEB was driven in different tissues by inserting the human TFEB gene coding region into the genome of the fruit fly.
This model provides an effective biological model for analyzing the mechanism of tumorigenesis and disease drug screening, which can induce phenotypes such as increased autophagy levels similar to those in mammals.
Smart Images
Figure CN119913203A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of biotechnology and genetic engineering, and in particular to a method for constructing and applying a human TFEB gene transgenic fruit fly model. Background Art
[0002] The human microphthalmia family of transcription factor (MiT / TFE) is a highly conserved transcription factor, consisting of four factors: MITF, TFEB, TFEC, and TFE3. MiT / TFE can recognize and specifically bind to the promoter region of lysosome-related genes, thereby promoting their transcription, increasing the synthesis of lysosomes, and playing an important role in autophagic degradation. Under normal physiological conditions, lysosomes degrade intracellular denatured proteins or damaged organelles into amino acids, lipids, and nucleotides through autophagic degradation to maintain the normal function of cells; nutritional deficiency can induce the occurrence of autophagic degradation and provide nutrition for cell survival. Metabolically abnormal tumor cells can use autophagy to promote the redistribution of nutrients, providing a basis for the rapid proliferation of tumor cells. Studies have shown that MiT / TFE family proteins such as TFEB and TFE3 are highly expressed in tumor tissues or immune-related diseases, suggesting that TFEB may be a target for the development of therapeutic drugs for related diseases. At present, many studies on TFEB have been completed at the cell line level, and the mechanism driving the occurrence of tumors or immune diseases has not yet been fully resolved. Summary of the invention
[0003] In view of the problem that the above-mentioned research on TFEB is mostly completed at the cell line level and the mechanism driving the occurrence of tumors or immune diseases has not been fully analyzed, the present invention provides a method for constructing and using a human TFEB gene transgenic fruit fly model. The establishment of the TFEB transgenic fruit fly model will provide a good foundation for analyzing the physiological function of TFEB.
[0004] In order to achieve the above object, the present invention provides a method for constructing a human TFEB gene transgenic fruit fly model, which comprises the following steps: S1. Insert the coding region sequence of human TFEB gene into UASp-HA-attB vector to construct UASp-TFEB-HA-attB plasmid; S2, introducing the UASp-TFEB-HA-attB plasmid constructed in step S1 into Drosophila embryos carrying attP; S3. The hatched G0 generation fruit flies are hybridized with white-eyed fruit flies to screen out red-eyed fruit flies, which are UASp-TFEB-HA transgenic fruit flies.
[0005] Specifically, in step S1, the construction method of the UASp-TFEB-HA-attB plasmid is: S1.1. PCR amplification was performed using cDNA of human 293T cells as a template, and the products were collected. The TFEB forward primer was GTCCTGTTCATTGGAATTCatgacagcaagctcaggct, and the TFEB reverse primer was CCTCCACCGCGGGGTACCcacagcacatcgccctcct; S1.2, double-digest the UASp-HA-attB vector with the restriction enzymes EcoRI and KpnI, and then collect the vector; S1.3. Connect the product of step S1.1 with the vector of S1.2 to obtain the UASp-TFEB plasmid.
[0006] As a classic model organism for biological research, Drosophila melanogaster has the advantages of simple cultivation, numerous phenotypes, and clear genetic background, and plays an important role in the analysis of gene functions. Many important signaling pathways and regulatory factors that regulate the growth and development of mammals have corresponding homologous proteins in Drosophila, and Drosophila plays an important role in the analysis of signaling pathways such as JNK, Hippo, and Wnt. The present invention uses transgenic technology to introduce the coding sequence of the human TFEB gene into Drosophila to prepare UASp-TFEB-HA transgenic Drosophila; driving the expression of TFEB in different tissues can induce cells to have phenotypes such as increased autophagy levels similar to overexpression of TFEB in mammalian cells, indicating that TFEB has physiological functions in Drosophila and mammals, and can provide a good model for analyzing the tumorigenesis mechanism caused by TFEB activation, and also provide tools for drug screening of diseases caused by abnormal TFEB activity.
[0007] Preferably, in step S2, the UASp-TFEB-HA-attB plasmid is injected into Drosophila embryos carrying attP by microinjection.
[0008] Further preferably, the Drosophila embryo carrying attP is an early embryo within 2 hours of egg-laying.
[0009] Preferably, in step S2, the fruit fly embryo with attP is a white-eyed fruit fly embryo with attP40 site; in step S3, the white-eyed fruit fly is a white-eyed fruit fly with chromosome 2 carrying SM6 balance chromosome.
[0010] The second aspect of the present invention provides the application of the human TFEB gene transgenic fruit fly model constructed by the above-mentioned construction method in analyzing the tumorigenesis mechanism caused by TFEB activation.
[0011] The third aspect of the present invention provides the use of the human TFEB gene transgenic fruit fly model constructed by the above-mentioned construction method in screening drugs for diseases caused by abnormal TFEB activity.
[0012] Through the above technical solution, the present invention achieves the following beneficial effects: The present invention utilizes transgenic technology to introduce the coding sequence of the human TFEB gene into Drosophila to prepare UASp-TFEB-HA transgenic Drosophila; driving the expression of TFEB in different tissues can induce cells to exhibit phenotypes such as increased autophagy levels similar to overexpression of TFEB in mammalian cells, indicating that TFEB has the same physiological functions in Drosophila as in mammals, and can provide a good model for analyzing the tumorigenesis mechanism caused by TFEB activation, and also provide a tool for screening drugs for diseases caused by abnormal TFEB activity. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is the construction route of the TFEB transgenic fruit fly model in Example 1 of the present invention; Figure 2 This is the result of the TFEB transgenic fruit fly model in Example 2 of the present invention in verifying the level of TFEB promoting autophagy in fat body cells; Figure 3 This is the result of the TFEB transgenic fruit fly model in Example 3 of the present invention in verifying that TFEB promotes intestinal hyperplasia and intestinal cell proliferation. DETAILED DESCRIPTION
[0014] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.
[0015] Example 1: Construction of TFEB transgenic fruit flies like Figure 1 As shown, the construction route is as follows: Take 5*10 5293T cells were centrifuged, the supernatant was removed, 1 mL of Trizol lysis buffer was added and mixed, 200 μL of chloroform was added, the mixture was allowed to stand for 10 min, 17000 g was centrifuged for 10 min, 300 μL of the upper supernatant was taken to a new centrifuge tube and 300 μL of isopropanol was added, the mixture was centrifuged at 17000 g for 10 min, the supernatant was removed, 1 mL of 75% ethanol was added to suspend the precipitate, 17000 g was centrifuged for 1 min, the supernatant was removed, and after drying, 50 μL of ultrapure water was added to dissolve the precipitate to obtain total RNA from human cells. 200 ng of total RNA, 2 μL of gDNA wiper Mix (Novozyme), ultrapure water was added to a volume of 10 μL, the mixture was mixed and incubated at 42 °C for 2 min; 4 × HiScript IV RT SμperMix (Novozyme) 5μL, Oligo (dT) 20 1 μL, 4 μL ultrapure water, mix well and incubate at 50℃ for 5 min to obtain cDNA from human cells. Referring to the human TFEB gene sequence, primers were designed to amplify the coding sequence of TFEB: TFEB forward primer: GTCCTGTTCATTGGAATTCatgacagcaagctcaggct TFEB reverse primer: CCTCCACCGCGGGGTACCcacagcacatcgccctcct Take 1 μL TFEB forward primer (10 μM), 1 μL reverse primer TFEB (10 μM), 10 μL 2×PhantaMax Master Mix, 1 μL cDNA, 7 μL ultrapure water, and mix well. After the reaction is carried out on the PCR instrument, the product is subjected to agarose electrophoresis, and the gel is recovered by gel recovery kit, and the product is washed and recovered with 30 μL deionized water. 1 μg UASp-HA-attB vector was double-digested with endonucleases EcoRI and KpnI (Baori Medical Biology), and the product was subjected to agarose electrophoresis, and the gel was recovered by gel recovery kit (Kangwei Biology), and the product was washed and recovered with 30 μL deionized water. Take 4 μL of UASp-attB vector recovered by enzyme digestion, 4 μL of amplified TFEB coding region product, 1 μL T4DNA ligase, and 1 μL reaction buffer, mix well, and incubate at 16°C overnight. The ligated plasmid was transformed into DH5α competent Escherichia coli, cultured in a plate medium containing ampicillin for 16 hours, and a single clone was picked for sequencing to obtain the UASp-TFEB-HA-attB plasmid.
[0016] Purify the UASp-TFEB-HA-attB plasmid using an endotoxin-free kit, and use a microinjector to inject the plasmid into the early embryos of white-eyed fruit flies at the attP40 site (within 2 hours of egg laying). After waiting for about 10 days for the fruit fly embryos to develop into adults, hybridize with white-eyed fruit flies with SM6 balance chromosomes on chromosome 2, and the red-eyed fruit flies obtained by screening are UASp-TFEB-HA transgenic fruit flies.
[0017] Example 2: Application of TFEB transgenic fruit flies—TFEB promotes autophagic degradation in fat body cells UASp-TFEB-HA transgenic flies were crossed with hsflp;Act>CD2>Gal4,UAS-GFP flies to obtain fruit fly larvae with the genotype of hsflp;Act>CD2>Gal4,UAS-GFP / UASp-TFEB-HA. The fat body tissues of the third instar larvae were dissected in PBS and fixed in 4% paraformaldehyde for 20 minutes. The fat bodies were rinsed three times with PBST (0.5% Triton X-100 in PBS) for 15 minutes each time. The fat bodies were incubated in a PBST dilution of mouse Rab7 antibody (1:1000) at 4°C for 12 hours. The fat bodies were rinsed three times with PBST for 15 minutes each time. The fat bodies were incubated in a PBST dilution of red fluorescent goat anti-mouse secondary antibody (1:1000) at 4°C for 4 hours. The fat bodies were rinsed three times with PBST for 15 minutes each time. The fat bodies were pressed into slices with a blocking solution containing DAPI. Observe under a fluorescence microscope. Since the recombinase FLP can drive Act>CD2>Gal4 in some fat body cells to remove CD2 and form active Act-Gal4, GFP and TFEB under the control of UAS are driven to express by Gal4 in these cells, thus forming cells that overexpress TFEB (express GFP, marked with green fluorescence) and control cells (do not express GFP, no green fluorescence mark). Rab7 is a protein specifically expressed on lysosomes and can be used to mark lysosomes. Figure 2 It can be seen that in the cloned cells overexpressing TFEB (GFP positive), the brightness and number of Rab7 markers were significantly increased, indicating that overexpression of TFEB promoted the level of autophagy in fat body cells.
[0018] Example 3: Application of TFEB transgenic fruit flies—TFEB promotes intestinal hyperplasia and intestinal cell proliferation Take UASp-TFEB-HA transgenic fruit flies and cross-link them with NP1-Gal4, Tub-Gal80 ts ,UAS-GFP(NP1 ts ) fruit flies were crossed and the genotype obtained was NP1-Gal4, Tub-Gal80 ts,UAS-GFP fruit fly eggs were placed in an 18°C incubator for culture. At this time, Gal80 inhibited the activity of Gal4, TFEB could not be expressed in intestinal epithelial cells, and the fruit flies could normally hatch into adults. The hatched fruit fly adults were placed in a 29°C incubator to release the inhibitory effect of Gal80 on NP1-Gal4, and the Gal4 expressed in intestinal epithelial cells drove the expression of TFEB under UAS control. After 10 days of culture, the fruit flies were fed with 10 mM EdU for 24 hours. The fruit fly intestines were dissected in PBS, and the fruit fly intestines were fluorescently stained using the EdU detection kit (Biyuntian Biotechnology). The basic operation is as follows: move the intestine to a 1.5 mL centrifuge tube and add 1 mL of 4% paraformaldehyde fixative, fix at room temperature for 15 minutes; remove the fixative, wash each well with 1 mL of PBST washing solution 3 times, 15 minutes each time; remove the washing solution, add 0.5 mL of Click reaction solution, incubate at room temperature in the dark for 30 minutes; aspirate the Click reaction solution, wash 3 times with washing solution, 5 minutes each time, seal the intestine with a blocking solution containing DAPI, and observe under a fluorescence microscope. EdU is an analog of thymidine deoxynucleoside. EdU can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis, so it is used to mark cells in the S phase. Figure 3 It can be seen that overexpression of TFEB in intestinal epithelial cells increases the number of cells in the S phase in the intestine and thickens the intestinal diameter, indicating that overexpression of TFEB promotes intestinal hyperplasia.
[0019] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.
[0020] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.
[0021] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.
Claims
1. A method for constructing a human TFEB gene transgenic fruit fly model, characterized in that: The steps include: S1. Insert the coding region sequence of human TFEB gene into UASp-HA-attB vector to construct UASp-TFEB-HA-attB plasmid; S2, introducing the UASp-TFEB-HA-attB plasmid constructed in step S1 into Drosophila embryos carrying attP; S3. The hatched G0 generation fruit flies are hybridized with white-eyed fruit flies to screen out red-eyed fruit flies, which are UASp-TFEB-HA transgenic fruit flies.
2. The construction method according to claim 1, characterized in that: In step S1, the construction method of the UASp-TFEB-HA-attB plasmid is: S1.
1. PCR amplification was performed using cDNA of human 293T cells as a template, and the products were collected. The TFEB forward primer was GTCCTGTTCATTGGAATTCatgacagcaagctcaggct, and the TFEB reverse primer was CCTCCACCGCGGGGTACCcacagcacatcgccctcct; S1.2, double-digest the UASp-HA-attB vector with the restriction enzymes EcoRI and KpnI, and then collect the vector; S1.
3. Connect the product of step S1.1 with the vector of S1.2 to obtain the UASp-TFEB plasmid.
3. The construction method according to claim 1, characterized in that: In step S2, the UASp-TFEB-HA-attB plasmid is injected into Drosophila embryos carrying attP by microinjection.
4. The construction method according to claim 3, characterized in that: The Drosophila embryos carrying attP are early embryos within 2 hours of egg laying.
5. The construction method according to claim 1, characterized in that: In step S2, the fruit fly embryo with attP is a white-eyed fruit fly embryo with attP40 site; in step S3, the white-eyed fruit fly is a white-eyed fruit fly with chromosome 2 carrying SM6 balance chromosome.
6. Use of the human TFEB gene transgenic fruit fly model constructed by the construction method of any one of claims 1 to 5 in analyzing the tumorigenesis mechanism caused by TFEB activation.
7. Use of the human TFEB gene transgenic fruit fly model constructed by the construction method according to any one of claims 1 to 5 in screening drugs for diseases caused by abnormal TFEB activity.