Construction method and application of a transgenic zebrafish model of hypoxia response

Abstract

The invention discloses a method for constructing a transgenic zebrafish model of hypoxia response. The method comprises the following steps: firstly assembling an sv40mp promoter after a hypoxia response element HRE sequence and cloning the promoter into a pT2AL200R150G plasmid carrying green fluorescent protein (GFP), then microinjecting the recombinant plasmid and transposase mRNA into zebrafish embryos, and inserting the recombinant sequence into the genome of F0 generation zebrafish by Tol2 transposon technology; raising the F0 generation fish to adult fish, hybridizing with wild-type zebrafish to produce F1 generation, hybridizing the F1 generation with the wild-type fish again to produce single-copy transgenic zebrafish F2 generation, self-pollinating male and female sibling zebrafish of the F2 generation, and screening out transgenic zebrafish Tg (HRE-sv40mp:GFP) of hypoxia response of F3 generation that can be stably inherited. The transgenic zebrafish Tg(HRE‑sv40mp:GFP) constructed in the present invention can be used as a simple and efficient hypoxia response model for biological research in hypoxia-related fields, as well as for screening molecular targeted drugs for HIF‑1α, providing a living animal screening model for the development of anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for constructing a transgenic zebrafish model of hypoxia response and application thereof. Background Art

[0002] Oxygen is vital for living organisms, and hypoxia affects cell proliferation, division, metabolism, redox and other life activities. Cells respond to hypoxia by activating and expressing many protein factors, among which hypoxia-inducible factor (HIF) plays an important role in maintaining oxygen homeostasis. As an important transcription factor, HIF regulates downstream target genes involved in life processes such as angiogenesis, vascular remodeling, erythropoiesis, glucose transport, glycolysis, cell proliferation, apoptosis and aging. When the expression and regulation of HIF are disordered, it may lead to many diseases, cancer being one of them. Cancer cells are characterized by rapid differentiation and proliferation, resulting in a hypoxic microenvironment inside the tumor tissue, and hypoxia can accelerate the proliferation and migration of cancer cells, leading to cancer deterioration. HIF plays an important role in this process.

[0003] HIF belongs to the bHLH-PAS transcription factor superfamily. It functions in the form of a heterodimer composed of α subunits and β subunits. The β subunit is constitutively expressed, while the α subunit is oxygen-dependent. Therefore, the transcriptional activity of HIF mainly depends on the stability of the α subunit. Three α subunits have been found: HIF-1α, -2α, and -3α. Among them, HIF-1α is the earliest member discovered in the HIF family and is also the most important regulatory factor for cell response to hypoxia. The expression and accumulation of HIF-1α in cancer cells under hypoxic conditions are significantly increased. For example, high expression of HIF-1α and activation of downstream target genes have been detected in renal cell carcinoma, colon cancer, glioma, breast cancer, and cervical cancer. Therefore, inhibiting the expression or activity of HIF-1α can inhibit or slow tumor growth. Studies have shown that the mTOR inhibitor CCI-779 can inhibit the production of HIF-1α-dependent VEGF in breast cancer cells under both normoxic and hypoxic conditions. CCI-779 can also rapidly inhibit the generation, angiogenesis and tumor growth of human rhabdomyosarcoma transplanted tumors by inhibiting the mTOR / HIF-1 / VEGF pathway. Mabjeesh et al. reported that 2-methyloxyestradiol can inhibit the expression of HIF-1α, tumor angiogenesis and tumor growth in human glioma transplanted tumor models (Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME 2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. [J]. Cancer Cell, 2003, 3: 363-375.). Welsh et al. found that PX-478 can inhibit the accumulation and transcriptional activation of HIF-1α and the expression of its target gene VEGF in various tumor cell lines (Welsh S, Williams R, Kirkpatrick L, et al. Antitumor activity and pharmacodynamic properties of PX-478, an inhibitor of hypoxia-inducible factor-1alpha[J]. Mol Cancer Ther, 2004, 3: 233-244.). HIF-1α has become a popular potential anti-tumor drug target.

[0004] Under normal oxygen conditions, HIF-1α protein is very easy to degrade, so the content in cells is very low. When cells are hypoxic, the expression of HIF-1α increases significantly, and its protein stability is significantly enhanced. In the nucleus, HIF-1α and HIF-β form heterodimers and bind to the hypoxia response elements (HRE) upstream of the target gene promoter to initiate the transcription of many target genes. HRE is the binding site of HIF-1α on DNA, generally less than 100 bp, and consists of a conservative core sequence "(A / G)CGT(G / C)C" and variable flanking sequences (Rhim T, Lee DY, Lee M.Hypoxia as a target for tissue specific gene therapy [J]. J Control Release, 2013, 172 (2): 484-494).

[0005] Currently, most of the models that can be used to screen compounds related to the HIF-1 signaling pathway utilize in vitro cultured cells. Summary of the invention

[0006] The purpose of the present invention is to provide a simple and efficient method for constructing a transgenic zebrafish model of hypoxia response, which is obtained by Tol2 transposon technology and indicates its hypoxia state by the expression intensity of green fluorescent protein (GFP). The model can be used for scientific research in hypoxia-related fields or for drug screening with HIF-1α as the target.

[0007] The invention clones an HRE sequence and a part of the sv40 promoter (sv40 mini promotor, sv40-mp) into a pT2AL200R150G plasmid with a GFP label, and introduces them into the zebrafish genome through the Tol2 transposon technology to construct a transgenic zebrafish that responds to hypoxia; when HIF-1α is activated, green fluorescence is induced in the zebrafish body, which is used to screen HIF-1α molecular targeted drugs.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] (1) In vitro amplification of the insert fragment: Synthesize primers, use plasmid p2.1 as a template, and amplify the hypoxia response element HRE and SV40 promoter in vitro by high-fidelity PCR technology to obtain the HRE-sv40mp sequence;

[0010] (2) Construction of recombinant plasmid: The pT2AL200R150G plasmid with green fluorescent protein (GFP) and the HRE-sv40mp sequence amplified in step (1) were digested, ligated, and transformed into Escherichia coli to construct the recombinant plasmid pT2AL200R150G (HRE-sv40mp-GFP);

[0011] (3) Embryo microinjection: zebrafish fertilized eggs were collected, microinjected with transposase mRNA and recombinant plasmid pT2AL200R150G (HRE-sv40mp-GFP), and incubated at a constant temperature;

[0012] (4) Screening transgenic strains: The embryos incubated in step (3) are raised to sexual maturity and marked as F0 generation zebrafish; the F0 generation is hybridized with the wild type, and embryos that can produce green fluorescence are selected, and they are further raised to sexual maturity and marked as F1 generation; the F1 generation is hybridized with the wild type again, and embryos that can produce green fluorescence are selected, and they are further raised to sexual maturity and marked as F2 generation; the male and female sibling zebrafish of the F2 generation are self-fertilized, and embryos that can produce green fluorescence are selected, and they are further raised to sexual maturity and marked as F3 generation, which is the hypoxia-responsive transgenic zebrafish model Tg (HRE-sv40mp: GFP).

[0013] The primer sequences in step (1) are as follows:

[0014] Forward primer sequence: 5′-TAACATACCCCGGGAGCATGCAGGGCCGGACGTGG-3′;

[0015] Reverse primer sequence: 5′-CGCGGATCCTTTACCAACAGTACCGG-3′.

[0016] In order to prove the effectiveness of the constructed transgenic zebrafish Tg (HRE-sv40mp: GFP) in response to hypoxia, the present invention adopts the following methods to verify:

[0017] Verification method 1: Tg(HRE-sv40mp:GFP) embryos were incubated in embryo culture medium with low oxygen concentration (hypoxic stress treatment), and it was found that the GFP expression in the myotome was significantly enhanced.

[0018] Verification method 2: Tg(HRE-sv40mp:GFP) embryos were incubated in embryo culture medium containing 100 μM CoCl2 (chemically simulated hypoxia), and it was found that GFP expression in the myotome was significantly enhanced.

[0019] Verification method three: HIF-1α mRNA was microinjected into Tg(HRE-sv40mp:GFP) embryos, and it was observed that GFP expression in the myotome was significantly enhanced.

[0020] Verification method 4: Tg (HRE-sv40mp: GFP) embryos were first treated with hypoxia stress according to verification method 1, and then 100 μM LW6 (HIF-1α inhibitor) was added to the embryo culture medium for incubation. It can be seen that the GFP expression in the myometrium after LW6 treatment was significantly weakened compared with the untreated group.

[0021] The model constructed by the method of the present invention is used for biological research in hypoxia-related fields and for screening molecular targeted drugs for HIF-1α.

[0022] Advantages of the present invention:

[0023] The transgenic zebrafish Tg (HRE-sv40mp: GFP) constructed by the present invention can be used as a simple and efficient hypoxia response model for biological research in hypoxia-related fields, as well as for screening molecular targeted drugs for HIF-1α, providing a live animal screening model for anti-tumor drug development. The live animal model of hypoxia response constructed by the present invention utilizes the characteristics of zebrafish embryos developing in vitro, transparent embryos, and large egg production, and conveniently and efficiently screens molecules acting on the HIF signaling pathway in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a construction diagram of the transgenic vector pT2AL200R150G (with Tol2 transposon) in the present invention.

[0025] Figure 2 This is a diagram showing the GFP expression pattern of Tg(HRE-sv40mp:GFP) embryos after normoxia and hypoxia treatment.

[0026] Figure 3 This is a diagram of the GFP expression pattern of Tg(HRE-sv40mp:GFP) embryos after CoCl2 treatment.

[0027] Figure 4 This is a diagram of the GFP expression pattern after Tg(HRE-sv40mp:GFP) embryos were microinjected with HIF-1α mRNA.

[0028] Figure 5 This is a diagram showing the GFP expression pattern in Tg(HRE-sv40mp:GFP) embryos after treatment with the HIF-1α inhibitor LW6. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings and through specific embodiments.

[0030] Unless otherwise specified, the technical means involved in the following embodiments are conventional means well known to those skilled in the art.

[0031] Embodiment 1:

[0032] This embodiment specifically includes the following steps:

[0033] 1. PCR amplification of target fragment

[0034] 1.1 Design primers, use the existing plasmid p2.1 as a template, and amplify the hypoxia response element HRE and SV40 promoter (sv40mp) in vitro by PCR. The primer sequences are as follows:

[0035] Forward primer sequence:

[0036] 5'-TAACATACCCCGGGAGCATGCAGGGCCGGACGTGG-3';

[0037] Reverse primer sequence: 5′-CGCGGATCCTTTACCAACAGTACCGG-3′.

[0038] 1.2 The reaction system (100 μL) and reaction procedure are as follows:

[0039] Table 1 PCR reaction system and reaction procedure

[0040]

[0041] 2. Connect the target fragment to the plasmid

[0042] 2.1 The PCR amplification product was about 300 bp, which was recovered using a DNA gel recovery kit, then double-digested with BamHI and SphI restriction endonucleases, and then purified using a DNA purification kit.

[0043] 2.2pT2AL200R150G plasmid was also double-digested with BamHI and SphI, and the target band was about 4500bp, which was recovered from gel.

[0044] 2.3 Ligate the two DNA fragments using T4 DNA ligase at 4°C overnight.

[0045] 3. Plasmid Transformation into E. coli

[0046] 3.1 Add the above ligation product to E. coli competent cells JM109, ice bath for 30 minutes, then heat shock at 42°C for 90 seconds, ice bath again for 5 minutes, add 600 μL liquid LB medium (Amp-), and culture at 37°C in a shaker at 180 rpm for 50 minutes.

[0047] 3.2 Spread the bacterial solution on LB plate (Amp+) and culture at 37℃ for 12 hours. Select the single clone colony and expand the culture to 5mL liquid LB medium (Amp+).

[0048] 3.3 Extract plasmid and sequence it to identify the correct recombinant plasmid as pTol2-HRE-sv40mp-eGFP ( Figure 1 As shown). The plasmid uses the Tol2 vector as the backbone, and Tol2 is the transposon recognition sequence. The HRE sequence in the human enolase promoter is selected. The HRE sequence and the partial promoter of SV40 are used as the regulatory elements for initiating the expression of eGFP. eGFP is green fluorescent protein. SV40-PA is the polyadenylic acid translation termination signal.

[0049] 4. In vitro transcription of transposase mRNA

[0050] 4.1 The plasmid pCS2FA-transposase carrying the transposase gene was digested with restriction endonuclease NotI, extracted with phenol:chloroform:isoamyl alcohol (12:12:1), centrifuged at 12000 rpm for 10 min, and the supernatant was retained.

[0051] 4.2 Add 0.1 times volume of sodium acetate (3M, pH 5.2) and 2.5 times volume of pre-cooled anhydrous ethanol to the supernatant, incubate at -20°C overnight, centrifuge at 13000rpm at 4°C for 15min, discard the supernatant, wash the precipitate with 1ml of pre-cooled 75% ethanol, centrifuge and discard the supernatant.

[0052] 4.3 Dissolve the precipitate in 12 μl DEPC water, take 1 μl for agarose gel electrophoresis to detect DNA quality, take another 1 μl to measure the concentration, and use the remaining 10 μl as an in vitro transcription template.

[0053] 4.4 Use mMESSAGE mMACHINE kit to synthesize mRNA.

[0054] 4.5 Add 1.5ul Turbo DNase to the reaction system, mix well, and place at 37℃ for 30min. Add 30ul LiCl (7.5M) and 30ul DEPC water, mix well, and place in a -20℃ refrigerator overnight. Centrifuge at 13000rpm for 15min at 4℃, discard the supernatant, wash the precipitate with 70% ethanol, and centrifuge for 5min. Discard the supernatant, aspirate the excess liquid, blow dry in a clean bench, and dissolve the precipitate with DEPC water.

[0055] 5. Microinjection

[0056] The microinjection solution was placed on ice and mixed: including transposase mRNA, recombinant plasmid pTol2-HRE-sv40mp-Egfp at a concentration of 50ng / ul, as well as phenol red and enzyme-free water. Zebrafish were provided by the School of Medicine of Ocean University of China, Tubingen strain. One-cell embryos of zebrafish were collected, and fertilized eggs were microinjected under a microinjection instrument, with 1nl of microinjection mixture injected. The injection site is generally in the yolk near the animal pole of the embryo. After the injection, the embryo was gently blown into a culture dish containing embryo culture medium and cultured in a 28.5℃ incubator. At the same time, embryos that were not injected were collected as a control group. After culture to a certain developmental stage, the phenotype was observed under a microscope and photographed and recorded.

[0057] 6. Screening of Transgenic Zebrafish Responses to Hypoxia

[0058] After the microinjected zebrafish embryos reach sexual maturity at 3 months, they are crossed with Tubingen wild-type zebrafish, and the fluorescence expression of zebrafish is observed at 48 hpf (hours postfertilization, hpf). The embryos expressing green fluorescent protein after hypoxia are selected and raised to establish the F1 generation of zebrafish, and the corresponding parents are the founder fish or F0 generation (Founderfish). The same founder fish may have multiple insertion sites, so it may produce offspring with different fluorescence spatiotemporal expression profiles. The same fluorescence expression is classified into one category to establish the F1 family. After the F1 generation is raised to adult fish, it is crossed with wild-type zebrafish to separate possible multiple insertion sites to obtain the F2 generation of transgenic zebrafish. After the F2 generation is raised to adult fish, it is crossed with wild-type zebrafish to obtain the F3 generation of transgenic zebrafish, which is the hypoxia-responsive transgenic zebrafish model Tg (HRE-sv40mp: GFP).

[0059] Embodiment 2:

[0060] In order to prove the effectiveness of the transgenic zebrafish model Tg(HRE-sv40mp:GFP) constructed in Example 1 in response to hypoxia, the present invention adopts the following methods to verify:

[0061] Verification method 1: Tg(HRE-sv40mp:GFP) embryos were placed in embryo culture medium with low oxygen concentration, and the dissolved oxygen was 0.7±0.5mg / L (hypoxic stress treatment). The results showed that under normal dissolved oxygen concentration (7.0±0.5mg / L), 48hpf zebrafish embryos showed weak green fluorescence, while after 24 hours of hypoxia treatment for 24hpf embryos, the expression of GFP in the myotome of zebrafish embryos was significantly enhanced (e.g. Figure 2 ), indicating that the transgenic zebrafish can respond to physical hypoxia.

[0062] Verification method 2: Cobalt chloride CoCl2 is used as an iron ion chelator to inactivate proline hydroxylase, thereby simulating hypoxia. Tg(HRE-sv40mp:GFP) embryos were incubated in control culture medium and embryo culture medium containing 100μM CoCl2 for 24 hours. The results showed that zebrafish embryos cultured in normal culture medium showed weak green fluorescence, while after 24 hours of CoCl2 treatment, zebrafish embryos showed strong green fluorescence in the head, myotome and other parts (such as Figure 3 ), further indicating that the transgenic zebrafish are able to respond to chemical hypoxia.

[0063] Verification method three, in order to further prove whether Tg (HRE-sv40mp: GFP) transgenic zebrafish respond to hypoxia-inducible factor HIF-1. HIF-1α mRNA was synthesized in vitro and microinjected into zebrafish one-cell embryos. The results showed that 800pg and 1600pg HIF-1α mRNA overexpression could promote zebrafish GFP expression (such as Figure 4 This indicates that the transgenic zebrafish can respond to the hypoxia-inducible factor HIF-1 and is a good in vivo animal model for screening drugs that act on HIF-1.

[0064] Verification method 4: Tg(HRE-sv40mp:GFP) embryos were first subjected to hypoxia stress treatment according to verification method 1, and then HIF-1 inhibitor LW6 (100 μM) was added to the embryo culture medium for 24 hours. It can be seen that the expression of GFP in the myometrium was significantly weakened after LW6 treatment compared with the untreated group (e.g. Figure 5 The results showed that the transgenic zebrafish can be used as a model for screening compounds targeting HIF-1.

[0065] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

Claims

1. A method for constructing a transgenic zebrafish model of hypoxia response, characterized in that: The following steps are involved: (1) In vitro amplification of the insert fragment: Synthesize primers, use plasmid p2.1 as a template, and amplify the hypoxia response element HRE and SV40 promoter in vitro by high-fidelity PCR technology to obtain the HRE-sv40mp sequence; the HRE is the HRE sequence in the human enolase promoter; the primer sequence is as follows: Forward primer sequence: 5'-TAACATACCCCGGGAGCATGCAG GGCCGGACGTGG-3′; reverse primer sequence: 5′-CGCGGATCCTTTACCAACAGTACCGG-3′; (2) Construction of recombinant plasmid: The pT2AL200R150G plasmid with green fluorescent protein and the HRE-sv40mp sequence amplified in step (1) were digested, ligated, and transformed into Escherichia coli to construct the recombinant plasmid pT2AL200R150G (HRE-sv40mp-GFP); (3) Embryo microinjection: zebrafish fertilized eggs were collected, microinjected with transposase mRNA and recombinant plasmid pT2AL200R150G (HRE-sv40mp-GFP), and incubated at a constant temperature; (4) Screening transgenic strains: The embryos incubated in step (3) are raised to sexual maturity and marked as F0 generation zebrafish; the F0 generation is hybridized with the wild type, and embryos that can produce green fluorescence are selected and further raised to sexual maturity, and marked as F1 generation; the F1 generation is hybridized with the wild type again, and embryos that can produce green fluorescence are selected and further raised to sexual maturity, and marked as F2 generation; the F2 generation zebrafish are self-fertilized, and embryos that can produce green fluorescence are selected and further raised to sexual maturity, and marked as F3 generation, which is a transgenic zebrafish model of hypoxia response.

2. The method according to claim 1, characterized in that The enzyme digestion in step (2) is double digestion using BamHI and SphI restriction endonucleases.

3. The method according to claim 1, characterized in that The ligation in step (2) is performed using T4 DNA ligase.

4. Use of the transgenic zebrafish model constructed by the method according to claim 1 for screening molecular targeted drugs for HIF-1α.

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