Rapid leukemia incidence model based on over-expression transgenic line zebrafish

Abstract

The invention discloses a rapid onset leukemia model based on over-expression transgenic line zebrafish. The rapid onset leukemia model is constructed by injecting an expression vector for expressing human MYC gene or mouse myc gene into a zebrafish single-cell embryo. Compared with an existing mouse model and other leukemia animal models, the model provided by the invention has the advantages that the disease time is greatly shortened, leukemia phenotypes with abnormal cell expansion and cell distribution are easy to observe and analyze, and higher efficiency is achieved.

Description

Technical Field

[0001] This invention relates to a rapid onset leukemia model based on overexpression transgenic zebrafish. Background Technology

[0002] Acute myeloid leukemia (AML) is a type of myeloproliferative disease characterized by the excessive proliferation of leukemia cells, manifesting primarily as infection, anemia, and bleeding, posing a serious threat to human health. While significant progress has been made in the diagnosis and treatment of AML with the advent of the big data era and advancements in targeted drug development, the development of precise diagnosis and treatment, along with a comprehensive prognostic assessment system, still requires further exploration and validation through clinical trials and basic research.

[0003] Currently, basic research on AML mainly relies on in vitro culture of primary patient cells, mouse AML models induced by radiation and harmful chemicals, primary tumor cell implantation models in immunodeficient mice, animal models based on specific leukemia-causing gene mutants (such as mice and zebrafish), and mouse models based on multiple consecutive transplantations of genetically modified hematopoietic stem cells. The in vitro culture of primary cells and the primary tumor cell implantation models primarily rely on the ex vivo culture of tumor cells, lacking an optimal in vivo environment. Therefore, while they can effectively and rapidly explore the basic cellular properties of tumor cells, they cannot explore the actual behavior and related mechanisms of tumor cells in the in vivo microenvironment, nor can they verify the true state of tumor cells in vivo or identify suitable therapeutic targets. While most current mouse models can observe and study the true cellular behavior of tumor cells and their interactions with the microenvironment in vivo, and explore potential therapeutic targets and conduct drug screening, their limitations include long model establishment cycles, cumbersome and low-reproducibility procedures for multiple tumor cell transplantation experiments, and the ability to explore related molecular mechanisms and potential therapeutic targets only based on specific gene mutations. Currently widely used mouse AML models are mostly based on introducing genetic abnormalities such as PML-RARA fusion gene, RUNX1-ETO fusion gene, MLL-AF9 fusion gene mutations, NRAS12D, FLT3-ITD, and NPM1c using transgenic and viral transfection technologies. These models observe the progression of hematopoietic abnormalities to AML through long-term feeding. The main drawbacks are high feeding costs, long observation periods, low incidence rates, and unstable leukemia phenotypes. Mouse models using primary cell transplantation from patients, due to limitations in experimental techniques, can only observe tumor-related behaviors in specific areas, and the human cell microenvironment is not entirely suitable for mouse microenvironment, further limiting research.

[0004] Therefore, establishing an in vivo model with rapid onset, prominent phenotype that is easy to analyze experimentally, and suitable for screening relevant diagnostic and treatment methods is crucial. Compared to mouse models, zebrafish offer advantages such as lower breeding costs and shorter experimental cycles as an in vivo animal model for AML research. However, most currently popular AML zebrafish models are based on gene editing and transgenic technologies using specific leukemia-related pathogenic genes, resulting in relatively long onset cycles, lower leukemia incidence rates, and relatively unstable phenotypes. In conclusion, establishing an AML animal model that is not based on specific pathogenic genes, can rapidly and stably induce disease, and has a prominent phenotype that is easy to analyze in vivo is urgently needed. Summary of the Invention

[0005] This invention provides a rapid onset leukemia model based on overexpression transgenic zebrafish.

[0006] Specifically, a rapid onset leukemia model based on overexpressing transgenic zebrafish is constructed by injecting an expression vector expressing the human MYC gene or the mouse myc gene into zebrafish embryos.

[0007] The expression vectors are coronin1a:hMYC-H2B-GFP or coronin1a:mmyc-H2B-GFP. The non-erythroid marker coronin1a is used as the promoter to drive gene expression. Subsequently, the coding sequences of human MYC or mouse myc genes are ligated using the Gibson ligase system. The nuclear localization protein H2B and green fluorescent protein GFP are fused together using the P2A short peptide to achieve real-time observation and dynamic tracking of cells.

[0008] In practical applications, the vector plasmids constructed for transient expression are injected into zebrafish embryos at the single-cell stage. Subsequent continuous observation of zebrafish embryos and larvae at different developmental stages revealed that zebrafish embryos and larvae expressing the MYC gene exhibited characteristics consistent with leukemia.

[0009] Another objective of this invention is to provide a method for constructing a rapid-onset leukemia model based on overexpressing transgenic zebrafish.

[0010] Specifically, the method for constructing a rapid-onset leukemia model based on overexpression transgenic zebrafish includes constructing an expression vector expressing the human MYC gene or the mouse myc gene, and then injecting the expression vector into a zebrafish single-cell embryo.

[0011] The expression vectors are coronin1a:hMYC-H2B-GFP or coronin1a:mmyc-H2B-GFP.

[0012] The present invention has the following advantages:

[0013] The inventors constructed three MYC expression vectors that can express the human MYC gene or the mouse myc gene, respectively. Compared to gene editing technologies used in other animal models (such as CRISPR / Cas9), the construction and expression of these three expression vectors are technically mature and simple, with a high success rate and a faster time to obtain stable animal models. Green fluorescence signals can be observed one day after microinjection. Three days after fertilization, the inventors observed that approximately 50% of the juvenile fish in the MYC expression vector group expressed green fluorescence signals, and approximately 30% of the juvenile fish began to show abnormal cell aggregation and expansion phenotypes. In the control expression vector group, 65% of the juvenile fish expressed green fluorescence signals, but the distribution and number of green fluorescent cells in all juvenile fish were similar to those of normal hematopoietic cells. Seven days after fertilization, approximately 50% of the juvenile fish in the MYC expression vector group showed a focal aggregation of abnormally increased green fluorescent cells in the tail hematopoietic island region, while the control expression vector group maintained a normal hematopoietic development phenotype. Compared to existing mouse models and other animal models of leukemia, this model significantly shortens the onset time, makes the leukemia phenotype of abnormal cell proliferation and distribution easier to observe and analyze, and has high efficiency.

[0014] Further cytological analysis of MYC-expressing cells revealed that the co-localization of MYC-expressing green fluorescent cells with some lyz and mfap4 cells may indicate abnormal myeloid progenitor cells. Morphological analysis of the MYC-expressing group showed that these were typically larger, with a larger nucleus-to-cytoplasm ratio and more deeply stained cytoplasm, characteristic of abnormal immature myeloid cells. These results demonstrate that MYC-expressing cells conform to the morphological characteristics of AML.

[0015] In transplantation experiments, the inventors further discovered that, compared to the control group where no significant cell proliferation was observed daily after transplantation and most zebrafish larvae no longer showed green cells two days post-transplantation, MYC-expressing green fluorescent cells, after injection into zebrafish larvae, showed a daily increase in cell number, reaching 10 times the initial transplanted cell number by three days post-transplantation, and exceeding 100 cells by five days post-transplantation. Cell proliferation was observed in 80% of the MYC-expressing zebrafish larvae. This demonstrates that MYC-expressing cells have the potential for rapid proliferation, and their growth curve conforms to the behavioral characteristics of AML tumor cells. Attached Figure Description

[0016] Figure 1 This is a green fluorescence distribution map of zebrafish larvae at different developmental stages after microinjection of the control expression vector and the human MYC gene expression vector. It can be seen that abnormal cell proliferation and aggregation are observed after MYC expression, and the larvae expressing MYC have a shorter lifespan.

[0017] Figure 2After microinjection of the mouse myc gene expression vector, zebrafish larvae exhibited abnormal cell proliferation and aggregation similar to that observed after human MYC gene expression.

[0018] Figure 3 This involves co-localization analysis of MYC expression in different blood lineages;

[0019] Figure 4 Flow cytometry and morphological analysis of MYC-expressing cells showed that they conformed to the morphology of leukemia cells.

[0020] Figure 5 The transplantation experiment was conducted after MYC expression, and continuous observation was performed. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] To better study the pathogenesis of acute myeloid leukemia (AML), understand the interaction between leukemia cells and other cells in the AML microenvironment, and explore new targeted therapies targeting specific molecules and pathways, it is essential to establish animal models with rapid and stable disease progression and easily analyzable phenotypes. The MYC gene, a crucial gene regulating cell proliferation, is overexpressed during the development of AML. Previous large-scale studies have also shown that the MYC gene is frequently highly expressed in AML patients. Therefore, the inventors designed a method to overexpress the MYC gene in hematopoietic cells using transgenic technology and labeled it with GFP fluorescent protein for real-time tracking.

[0023] The inventors specifically expressed the MYC gene in blood cells to observe whether an AML model could be constructed. They used the non-erythroid marker coronin1a as a promoter to drive gene expression, subsequently ligating the coding sequence of the human MYC gene using a Gibson ligase system. A P2A peptide was then used to link the nuclear localization protein H2B to the green fluorescent protein GFP fusion protein, enabling real-time observation and dynamic tracking of cells. The resulting coronin1a:hMYC-H2B-GFP expression vector will be used for subsequent microinjection, and phenotypic changes at different time points will be observed. Correspondingly, the inventors also constructed a control vector without the hMYC coding sequence: coronin1a:H2B-GFP. Simultaneously, they also constructed related vectors expressing the mouse myc gene: coronin1a:mmyc-H2B-GFP and a non-nuclear localization expression vector: coronin1a:mmyc-GFP. The specific implementation steps are as follows:

[0024] 1. Construction of the expression carrier:

[0025] PBLK-coronin1a-SV40 was used as the backbone vector. Restriction enzyme digestion with ClaI and NotI was performed, and the linearized plasmid fragment was purified by PCR (Qiagen, 28104) for subsequent vector ligation. The coding sequences of the human MYC and mouse myc genes were determined using PCR (Phusion). TM Plus DNA polymerase, Thermo Scientific TM Amplification was performed using F630L. The primers used were:

[0026] Human MYC gene (NM_002467) amplification:

[0027] FP: 5'-TTGCAGCTGCTTTAGACGCTGG-3';

[0028] RP: 5'-CGCACAAGAGTTCCGTAGCTG-3'.

[0029] Mouse myc gene (NM_010849) amplification:

[0030] FP: 5'-ATGCCCCTCAACGTGAACTTC-3';

[0031] RP: 5'-TGCACCAGAGTTTCGAAGC-3'.

[0032] The H2B-GFP fragment sequence is consistent with previous reports, and the PCR-purified fragment (Qiagen, 28104) was used for subsequent vector ligation. The primers used were:

[0033] FP: 5'-CAGCTACGGAACTCTTGTGCGCCCGGGGGATCCGGAGCTACTAATTTC-3'; RP: 5'-GTAATACGGCTAGCAAGCGCGGCCGCATCTACGTAATACGGCTAGC-3'.

[0034] The Phusion Plus PCR reaction system is as follows:

[0035] 5X Phusion Plus buffer*1X 10mM dNTPs 200μM each

[0036] Forward primer 0.5μM Phusion Plus DNA Polymerase 0.5μL

[0037] Reverse primer 0.5μM Nuclease-free water Add to 50μL final

[0038] The Phusion Plus PCR reaction procedure is as follows:

[0039] 1.Initial denaturation 98℃;30sec 4.Extension 72℃30sec / kb

[0040] 2.Denaturation 98℃; 10sec Step2-4; Cycle 40

[0041] 3.Annealing 60℃10sec 35 5.Final extension 72℃5min

[0042] After obtaining human MYC, mouse myc, and H2B-GFP fragments, they were purified using the same PCR purification method (Qiagen, 28104) for later use. The obtained backbone vector, human MYC and mouse myc gene sequences, and H2B-GFP sequence were ligated using Gibson Assembly. Based on the purified fragment concentrations, the DNA fragments were added to a premixed Gibson Assembly Master Mix ligation reaction system at a vector:target fragment ratio of 1:3. The premixed Master Mix was 7.5 μL, and the total amount of ligation DNA fragments added did not exceed 2.5 μL. The resulting 10 μL ligation reaction system was incubated in a PCR instrument at 50°C for 60 min, followed by transfection of competent cells. 5 μL of the ligation reaction system was added to 50 μL of competent cells, thoroughly mixed, and incubated on ice for 30 min, followed by a 42°C heat shock for 90 s, and then returned to ice for later use. Ampicillin-resistant bacterial plates were used for plating, and after incubation overnight at 37°C, colonies were picked for identification.

[0043] 2. Expression vector microinjection:

[0044] After successfully constructing the vector and confirming the correct base sequence of the linked fragment via Sanger sequencing, the inventors injected a mixture of the expression vector coronin1a:hMYC-H2B-GFP and the control vector coronin1a:H2B-GFP with transposase mRNA into single-cell zebrafish embryos via microinjection. The final concentration of the expression vector used for microinjection was 40 ng / μL, and the final concentration of the transposase mRNA was 50 ng / μL, injected into single-cell zebrafish embryos at a volume of 1 nmL for continuous observation experiments. In addition to microinjecting the expression vector coronin1a:hMYC-H2B-GFP, the expression vectors for the mouse myc gene, coronin1a:mmyc-H2B-GFP and the non-nuclear localization expression vector, coronin1a:mmyc-GFP, were also mixed with transposase mRNA at a final concentration of 50 ng / μL and microinjected into single-cell zebrafish embryos at a final concentration of 40 ng / μL for continuous observation experiments.

[0045] Figure 1 The study showed that after microinjection, the inventors conducted continuous fluorescence microscopy observations, recording the number of zebrafish larvae that showed GFP green fluorescence signals daily, as well as the number and distribution characteristics of cells expressing or not expressing the MYC gene and showing green fluorescence signals. Fluorescence microscopy was performed on days 1, 3, and 7 post-fertilization to record phenotypic changes in the zebrafish embryos. Statistical analysis showed that green fluorescence signals were observed in zebrafish embryos injected with either the control expression vector or the myc expression vector on day 1 post-fertilization. At this time, the number and distribution of green fluorescent cells in both groups were similar to those of normal hematopoietic cells, with no obvious abnormalities. However, on day 3 post-fertilization, the inventors observed that approximately 50% of the larvae in the myc expression vector group expressed green fluorescence signals, and approximately 30% of the larvae began to exhibit abnormal cell aggregation and amplification. In the control expression vector group, 65% of the larvae expressed green fluorescence signals, but the distribution and number of green fluorescent cells in all larvae were similar to those of normal hematopoietic cells. Seven days post-fertilization, approximately 50% of the juvenile fish in the myc expression vector group exhibited focal aggregations of abnormally increased green fluorescent cells in the tail hematopoietic island region, while the control expression vector group maintained a normal hematopoietic development phenotype. Furthermore, the inventors conducted survival analysis on zebrafish injected with the MYC expression vector and the control expression vector. The results showed that the survival time of MYC-expressing zebrafish juveniles was significantly shorter than that of the control group, with rapid mortality occurring after 10 days post-fertilization. Simultaneously, the inventors also observed the expression vectors for the mouse myc gene, coronin1a:mmyc-H2B-GFP and non-nuclear localization vectors: coronin1a:mmyc-GFP. Figure 2As shown, the number and phenotype of zebrafish juveniles expressing green fluorescence are similar to those of juveniles expressing the human MYC gene. Both exhibit progressively increasing abnormal cell aggregation in the tail hematopoietic islands starting 3 days after fertilization. Therefore, the inventors believe that zebrafish juveniles expressing the MYC vector exhibit a leukemia-like phenotype with abnormal cell proliferation and distribution.

[0046] After observing that juvenile zebrafish expressing the MYC vector exhibited a leukemia-like phenotype, the inventors further analyzed the cellular characteristics of MYC expression by injecting the vector into transgenic zebrafish expressing different hematopoietic markers. Figure 3 As shown, the inventors observed that MYC expression driven by the non-erythroid promoter coronin1a or the control vector expression did not colocalize with the erythroid transgenic line gata1:dsRed. In the control expression vector group, the coronin1a:H2B-GFP signal mostly colocalized with lyz:dsRed (granulocytes), partially colocalized with lck:LRLG (lymphocytes), and only a few cells colocalized with mfap4:LRLG (macrophages). Compared with the control expression vector group, the abnormally increased cells in the MYC expression group mostly colocalized with lyz:dsRed, and in MYC-expressing juvenile fish, the lyz:dsRed signal also showed a different abnormal increase and aggregation compared to the control group. In MYC-expressing juvenile fish, in the mfap4:LRLG positive signal, cells with multi-pseudopodia did not colocalize with the green fluorescence of MYC expression, but the signal of some weakly expressed mfap4:LRLG was more increased than that of the control group and colocalized with MYC expression. In MYC-expressing juvenile fish, MYC-expressing green fluorescent cells showed partial co-localization with lck:LRLG, similar to the control group. These results suggest that MYC-expressing green fluorescent cells are myeloid cells. Under normal hematopoietic development, lyz-labeled granulocytes and mfap4-labeled macrophages do not co-localize. However, in MYC-expressing zebrafish juveniles, MYC-expressing green fluorescent cells were found to co-localize with some lyz and mfap4-labeled cells. The inventors speculate that these co-localized cells may be abnormal myeloid precursor cells.

[0047] To better explore whether MYC-expressing cells conform to AML cell characteristics, the inventors collected 7-day-old juvenile fish expressing the MYC vector and juvenile fish expressing the control vector, respectively, and performed flow cytometry analysis to sort green fluorescent positive cells. Subsequent morphological analysis was then conducted, and the results are shown below. Figure 4 and Figure 5 The specific implementation steps are as follows:

[0048] 1. Flow cytometry analysis of MYC-expressing cells

[0049] Zebrafish injected with the MYC expression vector and control vector during the single-cell embryonic stage were raised to 7 days post-fertilization. Zebrafish larvae exhibiting green fluorescence were selected using a fluorescence microscope for subsequent flow cytometry sample preparation. The MYC-expressing zebrafish larvae and control larvae were collected separately into 1.5 ml centrifuge tubes. After washing with an equal volume of phosphate-buffered saline (1xDPBS), 1 ml of phosphate-buffered saline containing 5% fetal bovine serum (FBS / 1xDPBS) was added. The yolk sac was removed by pipetting and aspirating the liquid. Subsequently, the tubes were centrifuged at 500 g at 4°C for 5 min. After discarding the supernatant, the remaining tissue was digested at 30°C using 3 ml of 0.25% trypsin for approximately 30 min in both the MYC-expressing group and the control group. During digestion, frequent pipetting and aspiration were performed to ensure thorough digestion. After the digestion time was up, the cell suspension was observed using a stereomicroscope to confirm complete tissue digestion. When the cell suspension was a single-cell suspension, 300 μL of FBS and 6 μL of CaCl2 were added sequentially and thoroughly mixed by pipetting to terminate the digestion. The cell suspension was then centrifuged at 500 g for 5 min at 4°C. After discarding the supernatant, 1 ml of 5% FBS / 1xDPBS was added for cell resuspending. The cells were then centrifuged again at 500 g for 5 min at 4°C. After discarding the supernatant, 1 ml of 5% FBS / 1xDPBS was added for cell resuspending. The cells were then filtered through a 40 μM filter before analysis and sorting. The single-cell suspension obtained in the previous step was subsequently processed using BDFACSDiscover. TM S8Cell Sorter was used for flow cytometry analysis and sorting. After removing adherent and dead cells by gate cytometry, the green fluorescent positive cells obtained by gate cytometry were sorted by flow cytometry and transferred to 500 μL of 5% FBS / 1xDPBS for subsequent experiments.

[0050] 2. Morphological analysis of MYC-expressing cells:

[0051] Cell suspensions obtained by flow cytometry sorting were centrifuged at 500g for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in 100 μL of 5% FBS / 1xDPBS. Cell slides were then spun at 300g for 3 min using a cell slide spinner, followed by Wright-Giemsa staining. Cells were first stained with Wright's solution at room temperature for 5 min, rinsed with a small flow of running water, air-dried, and then counterstained with Giemsa solution at room temperature for 15 min. The resulting stained slides were then subjected to morphological analysis and photographed under 100x oil immersion.

[0052] After removing adherent and dead cells through a gate, further analysis of the FSC and SSC of the green fluorescent cells obtained through the gate revealed that MYC-expressing green fluorescent cells had a larger FSC value than the control group, indicating that MYC-expressing cells were larger than normal blood cells in the control group. MYC-expressing green fluorescent cells also exhibited a more uniform single-peak SSC value compared to the bimodal SSC value of the control group, indicating that MYC-expressing cells were simpler in complexity than normal blood cells in the control group. Giemsa staining analysis showed that the blood cells in the control vector expression group were mature cells with segmented nuclei and folded nuclei, exhibiting morphological characteristics of mature granulocytic and mononuclear cells. In contrast, the MYC expression group commonly showed abnormally large, immature myeloid cells with a large nucleus-to-cytoplasm ratio and deeply stained cytoplasm. These results indicate that MYC-expressing cells conform to the morphological characteristics of AML.

[0053] To further explore whether MYC-expressing cells are transplantable like AML cells, the inventors designed a transplantation experiment for verification. The specific implementation steps are as follows:

[0054] Seven days post-fertilization, the inventors used flow cytometry, following the experimental procedures described above, to separate green fluorescent cells from zebrafish larvae in the control and MYC expression groups. After removing adherent and dead cells through the gate, the green fluorescent cells were separated into control and MYC expression cells, with a total of 30,000 cells in each group. The separated cells were centrifuged at 4 degrees Celsius for 5 minutes, the supernatant was removed, and the cell pellet was resuspended in 10 μL of 5% FBS / 1xDPBS. This was then used for blood circulation microinjection into wild-type zebrafish larvae two days post-fertilization. Under a 5x objective lens of a standard white light stereomicroscope, the droplet size was adjusted to 1 mL. The number of droplets was adjusted based on the total number of cells injected into the larvae, ensuring a final cell count of 2-5 cells per larvae.

[0055] The inventors continuously observed the number and distribution of green fluorescent cells in zebrafish larvae daily after microinjection. Statistical analysis showed that, compared to the control group where no significant cell proliferation was observed daily after transplantation, and where most zebrafish larvae no longer showed green cells after 2 days, the number of MYC-expressing green fluorescent cells increased daily after injection into zebrafish larvae, reaching 10 times the initial transplanted cell number by 3 days post-transplantation, and exceeding 100 cells by 5 days post-transplantation. Cell proliferation was observed in 80% of the MYC-expressing zebrafish larvae. The inventors mathematically modeled the number of green fluorescent cells observed in the control group or MYC-expressing zebrafish larvae at different post-transplantation days, finding that the resulting curves generally conformed to a logarithmic growth curve (log growth curve). Figure 4-5 ).

Claims

1. A rapid-onset leukemia model based on overexpression transgenic zebrafish, characterized in that, It was constructed by injecting an expression vector expressing the human MYC gene or the mouse myc gene into a zebrafish single-cell embryo.

2. The rapid onset leukemia model based on overexpressing transgenic zebrafish according to claim 1, characterized in that, The expression vector is coronin1a:hMYC-H2B-GFP or coronin1a:mmyc-H2B-GFP.

3. A method for constructing a rapid-onset leukemia model based on overexpression transgenic zebrafish, characterized by: This includes constructing an expression vector that expresses the human MYC gene or the mouse myc gene, and then transiently microinjecting the expression vector into a zebrafish single-cell embryo.

4. The method for constructing a rapid-onset leukemia model based on overexpressed transgenic zebrafish according to claim 3, characterized in that, The expression vector is coronin1a:hMYC-H2B-GFP or coronin1a:mmyc-H2B-GFP.

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