CN111424053B

CN111424053B - Construction method and application of axonal type peroneal muscular atrophy drosophila model

Info

Publication number: CN111424053B
Application number: CN202010311611.3A
Authority: CN
Inventors: 陈文锋
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2021-12-28
Anticipated expiration: 2040-04-20
Also published as: CN111424053A

Abstract

The invention relates to a construction method and application of a drosophila model for simulating human diseases, in particular to a construction method and application of a drosophila model for axonal type peroneal muscular atrophy (CMT 2). The amino acid mutation of the endogenous corresponding humanized ATP1A1 gene of the drosophila is constructed by a genome editing means, and the pathological phenotype is evaluated by utilizing the changes of brain nervous system loop and biological clock related behaviors, life and motor ability. The model and the pathological evaluation means established by the method have the advantages of simple operation and strong repeatability, and the obtained CMT2 drosophila model can be used for researching the pathological process and mechanism of the disease, can also be used for large-scale screening and verification of medicines for delaying or treating CMT2 diseases, and has good application prospect.

Description

Construction method and application of axonal type peroneal muscular atrophy drosophila model

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a drosophila model of axonal peroneal muscular atrophy (CMT 2).

Background

Peroneal muscular atrophy (CMT), also known as Hereditary Motor Sensory Neuropathy (HMSN), was discovered as early as 1886. CMT is a hereditary peripheral nervous system disorder characterized by defects in the peripheral nervous system, including defects in sensory and motor neurons, often manifested as progressive loss of the sense of touch in muscle tissue and various parts of the body. The main clinical features are progressive muscle weakness and atrophy with sensory disturbances distal to the extremities. The incidence of CMT is about 1/2500, one of the most common inherited peripheral neuropathies. Based on clinical and electrophysiological characteristics, CMT is classified as primary demyelinating neuropathy (CMT 1, CMT3 and CMT 4) and primary axonal neuropathy (CMT 2), mostly inherited in autosomal dominant, but also autosomal recessive or X-linked inheritance.

CMT disease is usually caused by mutations that are deficient in certain proteins in neurons. Neural signals are conducted by axons, surrounded by myelin sheaths. Most mutations in CMT affect myelin, such as the common duplication of a large region on the short arm of chromosome 17 that contains the gene PMP 22. Some mutations affect axons, such as MFN2, which is a gene encoding a mitochondrial protein. Cells contain separate genomes in their nuclei and mitochondria. In nerve cells, mitochondria propagate along the long axis process. In certain forms of CMT, the mutant MFN2 causes mitochondria to form large clusters or clumps that cannot move synaptically along axons. This may prevent synapses from functioning. To date, at least 50 gene mutations were found to cause CMT. However, there are great limitations to direct research using diseased human individuals, and it is particularly important to study the functions of these genes using model organisms.

Animal models of human diseases have long been used in the fields of medicine, pharmacy, etc., and increasingly exhibit advantages that cannot be replaced. Fruit fly (black belly) ()Drosophila melanogaster) Is one of the most important model insects in biological research, and plays a very important role in establishing a genetic chromosome theory. Meanwhile, the human genome contains 2-3 ten thousand genes, and homologous genes of more than 60% of the human genes can be found in the drosophila, so that the drosophila can become an important model organism for researching human diseases. To date, more than 50 CMT-causing mutations were found to have more than 35 of their cognate genes present in drosophila. Na (Na)⁺-K⁺ATPase is a protein ion pump responsible for the maintenance of intracellular sodium and potassium ion balance in response to the sodium and potassium ion transport activity at the plasma membrane. Human ATP1A1 encodes Na⁺-K⁺The α 1 subunit of ATPase, widely expressed in almost all tissues, and recent studies by our and others revealed that ATP1a1 mutation could trigger CMT 2. However, no suitable animal model simulating ATP1A1 mutation is available for the study of the disease mechanism and the screening of related drugs. The human ATP1a1 homologue in drosophila is Atp α, with a high degree of homology at the amino acid level. Therefore, the invention discloses a pair of optimized targeting sites based on CRISPR/Cas9 genome editing technology, and also discloses a group of primers for constructing donor DNA and a group of primers for identifying successful gene editing. Based on the primer sequence and the operation method disclosed by the invention, the drosophila with specific amino acid mutation of the Atp alpha transmembrane region can be efficiently constructed, and the human axonal peroneal muscular atrophy (CMT 2) can be simulated. Meanwhile, a method for evaluating pathological phenotype by using changes of brain clock nervous system circuit and biological clock related behaviors, life span and motor ability is established. The model and the pathological evaluation means established by the invention have the advantages of simple operation and repeatabilityThe Atp alpha mutant CMT2 drosophila model has strong advantages, can be used for researching the pathological process and mechanism of the disease, can also be used for screening drugs for delaying or treating the disease in a large scale, and has good application prospect.

Disclosure of Invention

The invention aims to solve the technical problems of establishing Atp alpha mutant CMT2 drosophila models and providing a construction method and application of an axon type peroneal muscular atrophy (CMT 2) drosophila models.

In order to solve the technical problems, the invention adopts the following technical scheme:

atp alpha mutation sites, three mutations corresponding to human ATP1A1 were simultaneously selected for simulation, including p.A597T, p.P600T and p.D601F, and the sites corresponding to drosophila Atp alpha gene were p.A576T, p.P579T and p.D580F.

According to the genome sequence information of the site to be mutated, a pair of CRISPR/Cas9 genome editing targets is optimally designed, wherein the target 1: 5'-GCACGTGGGGGATCAATCA-3' and target 2: 5'-GTCGGCACTTGGCAACGGCAT-3' are provided. Two target sequences are respectively constructed on a pU6-BbsI-gRNA (Addgene: 45946) vector and sequenced and identified, and finally a medium-amount DNA extraction kit is used for extracting a target plasmid.

Simultaneously, a group of primers for constructing donor DNA required by gene editing is designed, and 5' homology arm primers Atp alpha-5 arm-f: 5'-CACATCGACCATCTGCTCCGATA-3', respectively; atp α -5 arm-r: 5'-TGACGGCGGTACGTGGGGGATCGGTCATGGACATAAGGCCGACGAAAC-3' and 3' homology arm primer Atp α -3 arm-f: 5'-CCCCACGTACCGCCGTCACGTTCGCCGTTGCCAAGTGCCGATC-3', respectively; atp α -3 arm-r: 5'-TTACCTGCTTGGACACATCGGAAC-3' are provided. And (3) respectively amplifying the 5 'homologous arm and the 3' homologous arm by using PCR, recovering amplification product glue, then carrying out bridging PCR connection, recovering the 5 'homologous arm and the 3' homologous arm connection product glue, cloning to a blunt-end cloning vector, sequencing and identifying, and finally extracting donor DNA plasmid by using a medium-amount DNA extraction kit.

Two target and donor DNA plasmids linked to pU6-BbsI-gRNA were injected into any Drosophila embryo expressing Cas9 protein from endogenous germ cells in a mixture (e.g.: y [1] M { w [ + mC ] = nos-Cas9.P } ZH-2A w [). F0 Drosophila crosses only one of the phylogenetically balanced lethal genes, such as ywR122 (ywR 122= yw 122; sp/Cyo (y +); TM2/TM 6B), and the progeny were subjected to PCR using a set of primers, ATP α -KI-F: 5'-CACGTACCGCCGTCACGTTC-3' and ATP α -KI-r: 5'-CCTAATGGAAGGCCAATTTTGGTG-3' are provided.

And (3) constructing a drosophila model to carry out brain clock nervous system loop immunostaining and biological clock related behavior observation, life observation and motor ability observation to evaluate pathological phenotypes. The back projection of PDF on LNvs clock neurons is observed by scanning through a laser confocal microscope by using mouse anti-PDF antibody (1: 200, DSHB) for immunostaining, and the back axon projection of the PDF clock neuron loop of the model drosophila can be found to be abnormal. The observation of the relative behavior of the biological clock adopts a fruit fly behavior Monitor (DAM), and the data analysis adopts a software package based on Matlab, so that the circadian rhythm of the model fruit fly can be found to be disordered. Life-span observations and locomotor activity observations can reveal a shortened life-span and a decreased locomotor activity of model fruit flies.

The constructed drosophila model can be used for large-scale screening and verification of drugs for delaying or treating the CMT2 disease. Generally, the drug to be screened is mixed in fruit fly food for feeding, and the drug effect is evaluated by the pathological phenotype evaluation means described above, and the evaluation can be divided into different drug series and different concentration series of the same drug for comparison.

One characteristic of the axonal type peroneal muscular atrophy fruit fly model constructed by the invention is that the fruit fly has a serious pathological phenotype. The fruit fly model has multiple phenotypes which are easy to observe, the medicine feeding mode is simple and convenient, the medicine screening work is relatively quick, and a large amount of medicine screening can be rapidly carried out.

Drawings

Objects, advantages and features of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. The examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

FIG. 1 ATP alpha Drosophila Gene editing design protocol.

FIG. 2A flow chart of the ATP alpha Drosophila gene editing target cloning.

FIG. 3 molecular identification diagram of ATP alpha Drosophila model.

FIG. 4 ATP α Drosophila's bell circuit dorsal axonal projection abnormalities.

FIG. 5 ATP alpha fruit fly circadian rhythm disorder.

FIG. 6 life shortening of ATP α Drosophila model.

FIG. 7 the ATP α Drosophila model is reduced in locomotor ability.

FIG. 8 results of application of ATP α Drosophila model.

FIG. 9 is a flow chart of the technology for screening drugs by ATP alpha Drosophila model.

Detailed Description

Example 1

Gene editing and result identification of drosophila ATP alpha simulation human ATP1A1 amino acid mutation

Atp alpha mutation sites, as shown in FIG. 1, we selected three mutations corresponding to human ATP1A1 at the same time to simulate, including p.A597T, p.P600T, and p.D601F, and the sites corresponding to Drosophila Atp alpha gene are p.A576T, p.P579T, and p.D580F.

According to the genome sequence information of the site to be mutated, as shown in fig. 1, a pair of CRISPR/Cas9 genome editing targets, target 1: 5'-GCACGTGGGGGATCAATCA-3' and target 2: 5'-GTCGGCACTTGGCAACGGCAT-3' are provided. As shown in FIG. 2, two target sequences were constructed separately on pU6-BbsI-gRNA (Addgene: 45946) vectors. Two target primers (forward: 5'-CTTC (target sequence) -3' and reverse: 3'- (target sequence) CAAA-5') were synthesized for each target based on the template, and the 5'-CTTC-3' and 3'-CAAA-5' overhang sequences of the forward and reverse primers were complementary to the overhangs generated by digestion of pU6-BbsI-gRNA vector with BbsI endonuclease. Annealing the forward and reverse primers of the target spot: mu.L of forward primer (100. mu.M), 1. mu.L of reverse primer (100. mu.M), 1. mu.L of 10x T4 ligation buffer, and 7. mu.L of deionized water, and performing a 95 ℃ reaction for 5min, and then reducing to 25 ℃ at a rate of-0.1 ℃ per sec.

Digesting the pU6-BbsI-gRNA vector with BbsI endonuclease: mu.g of pU6-BbsI-gRNA vector is cut by BbsI endonuclease (NEB company), the cutting scheme refers to the endonuclease instruction, and the cut product is recovered and purified by glue.

Connecting the annealing product of the target primer with the enzyme-cut pU6-BbsI-gRNA vector: 1 mu L of BbsI enzyme-digested pU6-BbsI-gRNA (50ng), 1 mu L of target primer annealing product, 1 mu L of 10x T4 connecting buffer solution, 1 mu L T4 DNA ligase (NEB) and 6 mu L of deionized water, converting escherichia coli competent cells after connecting for 1 hour at 25 ℃, performing insertion identification by using a T7 or T3 primer, and finally extracting plasmids by using a medium DNA extraction kit.

As shown in FIG. 1, a set of primers for constructing donor DNA required for gene editing is designed at the same time, specifically 5' homology arm primer Atp alpha-5 arm-f: 5'-CACATCGACCATCTGCTCCGATA-3', respectively; atp α -5 arm-r: 5'-TGACGGCGGTACGTGGGGGATCGGTCATGGACATAAGGCCGACGAAAC-3' and 3' homology arm primer Atp α -3 arm-f: 5'-CCCCACGTaCcGCCGTcaCgttcGCCGTTGCCAAGTGCCGATC-3', respectively; atp α -3 arm-r: 5'-TTACCTGCTTGGACACATCGGAAC-3' are provided. 5' homology arm amplification: to a 50. mu.L reaction system, 25. mu.L of 2xPrimerSTAR Max DNA Polymerase (TaKaRa), 1.5. mu.L each of primers (10. mu.M), 1. mu.L (100 ng) of Drosophila genome, and the balance of deionized water were added; pre-denaturation at 98 ℃ for 10s before cycling: denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, 40 cycles, and final extension at 72 ℃ for 10 min; the 3 'homology arm amplification is the same as the 5' homology arm amplification.

And (3) carrying out bridging PCR connection after recovering the amplification product glue: to a 50. mu.L reaction system, 25. mu.L of 2xPrimerSTAR Max DNA Polymerase (TaKaRa), Atp α -5arm-f and Atp α -3arm-r primers were added, 1.5. mu.L each, 5 'homology arm amplification (100 ng) and 3' homology arm (100 ng), and the balance deionized water; pre-denaturation at 98 ℃ for 10s before cycling: denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, 40 cycles, final extension at 72 ℃ for 10 min, PCR product gel recovery, connection to blunt-end cloning vector and sequencing identification, and final extraction of donor DNA plasmid with medium DNA extraction kit.

Two target plasmids (50 ng/. mu.L each) and donor DNA plasmids (50 ng/. mu.L each) ligated to pU6-BbsI-gRNA were co-injected with Drosophila embryos (y [1] M { w [ + mC ] = nos-Cas9.P } ZH-2A w [) expressing Cas9 protein from endogenous germ cells. F0 Drosophila melanogaster was singly hybridized with the phylogenetically balanced lethal gene Drosophila ywR122 (ywR 122= yw 122; sp/Cyo (y +); TM2/TM 6B), and the progeny was subjected to PCR using a set of primers to identify whether gene editing was successful or not, the primers were ATP α -KI-F: 5'-CACGTACCGCCGTCACGTTC-3' and ATP α -KI-r: 5'-CCTAATGGAAGGCCAATTTTGGTG-3', adding 10 μ L of 2 xDeamaTaq Green PCR Master Mix (ThermoFisher), 1.0 μ L of each primer, 1 μ L of Drosophila genome (100 ng), and the balance of deionized water into a 20 μ L reaction system; pre-denaturation at 95 ℃ for 3min and circulation: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, 40 cycles, and final extension at 72 ℃ for 10 min. As shown in FIG. 3, the PCR product was electrophoresed and the band size was about 521 bp, and finally determined by sequencing, the sequencing result is shown in FIG. 1.

Example 2

Fruit fly ATP alpha simulation human ATP1A1 amino acid mutation CMT2 model pathology assessment

And constructing a drosophila model to carry out brain clock nervous system loop immunostaining and biological clock related behavior observation to evaluate pathological phenotypes. Immunostaining protocol: collecting the fruit fly imagoes for 3-5 days, and fixing the fruit fly imagoes in 4% paraformaldehyde fixing solution for 2 hours at room temperature. Dissect with a dissecting tip forceps, remove the treated Drosophila brains from the worm, and wash in 150 μ L PBST (PBS +0.5% Teiton X-100) at room temperature for 15min 3 times. Then transferred to a medium containing 150. mu.L PNT (PBST +10% blocking goat serum) and incubated for 2h at room temperature for blocking. The primary antibody (mouse anti-PDF 1: 200, DSHB) was then diluted with PNT and the blocked brain was transferred to the primary antibody overnight at 4 ℃. Wash 3 times with PBST at room temperature for 15min each. Finally, the PNT diluted fluorescent labeled secondary antibody was incubated overnight at 4 ℃. PBST room temperature washing 3 times, each time after 15min for mounting. Finally, scanning and observing the back projection of PDF on the LNvs clock neuron by using a confocal laser microscope, and as shown in fig. 4, the abnormal projection of the back axon of the PDF clock neuron circuit of the model drosophila can be found.

Biological clock-related behavior observation scheme: a fruit fly behavior Monitor (DAM, Drosophila Activity Monitor) purchased from Trikinetics, USA is adopted, a single fruit fly is put into a small glass tube (with the inner diameter of 5 mm and the length of 6.5 cm) with feed at one end, then the small glass tube is inserted into the behavior Monitor and then connected with a computer to record signals, and every time the fruit fly passes through the middle of the tube, an infrared light beam signal is blocked and recorded. Male fruit flies were exposed to light for 3-4 days at 25 ℃ for 12 h: 12 hours dark behavior followed by 6-7 days of monitoring when switched to full darkness. Behavior data were collected every 1min, and Matlab-based software package was used for data analysis, as shown in FIG. 5, it was found that the circadian rhythm of model fruit flies was disturbed.

The constructed fruit fly model can also be used for observing the service life and the movement capability. Life observation scheme: fresh food was changed every two days until all flies were counted dead, and a significant reduction in the life span of model flies was found, as shown in fig. 6. Observation scheme of motor ability: 10 fruit flies are placed in the hollow pipe, after the pipe is shaken for 2-3 times by people, the number of the fruit flies climbing at a speed of more than 6mm/s is observed and recorded, and as shown in figure 7, the movement capacity of the model fruit flies is found to be obviously reduced.

Example 3 application of Drosophila ATP alpha to model CMT2 for amino acid mutation of human ATP1A1

By using the constructed drosophila model, the neurogenic drug oxcarbazepine is evaluated. The specific scheme is as follows: first, a drug-containing food was prepared, and the neurogenic drug oxcarbazepine (O3764) from Sigma was dissolved in a solution containing 1 ‰ dimethyl sulfoxide to prepare a 120 mM stock solution, and then incorporated into a food containing 0.75% soybean powder (g/ml), 4.5% corn flour (g/ml), 1.5% yeast (g/ml), 0.5% propionic acid (v/v), 0.1% methylparaben (g/ml), 0.02% corn syrup (v/v), 1.25% sucrose (g/ml), 1.25% glucose (g/ml), 0.5% agar (g/ml) at a final concentration of 120. mu.M. The control diet was formulated with normal diet spiked with dimethyl sulfoxide at concentrations corresponding to the drug diet. Next, control and pathological Drosophila models 2-3 days after eclosion were collected and treated with drug-containing food or control food, and observed using the previously described motor ability observation protocol, as shown in FIG. 8, we found that the applied neurochemical improved the motor ability of the pathological Drosophila models to some extent.

Therefore, the drug to be screened is mixed in fruit fly food for feeding, the drug effect is evaluated by using a relevant pathological phenotype evaluation means, the drug screening can be carried out according to the approximate steps and the key characteristics of the technology of figure 9, and the scheme of finally obtaining the effective drug is feasible.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

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Claims

1. A method for constructing an axonal type peroneal muscular atrophy drosophila model is characterized by comprising the following steps: constructing amino acid mutation of drosophila endogenous Atp alpha gene corresponding to human ATP1A1 gene by a genome editing method, wherein the amino acid mutation comprises a pair of CRISPR/Cas9 targeting sites; a set of primers for constructing donor DNA; and a set of identifying primers;

the CRISPR/Cas9 targeting sites are as follows: target 1: 5'-GCACGTGGGGGATCAATCA-3' and target 2: 5'-GTCGGCACTTGGCAACGGCAT-3', respectively;

one set of primers for constructing donor DNA was: 5' homology arm primer Atp α -5 arm-f: 5'-CACATCGACCATCTGCTCCGATA-3', respectively; atp α -5 arm-r: 5'-TGACGGCGGTACGTGGGGGATCGGTCATGGACATAAGGCCGACGAAAC-3', respectively; and 3' homology arm primer Atp α -3 arm-f: 5'-CCCC ACG TACCG CCG TC ACG TTCG CCG TTG CC AAG TG CCG ATC-3', respectively; at p α -3a rm-r: 5'-TTACCTGCTTGGACACATCGGAAC-3', respectively;

the identification primer is AT P alpha-K I-f: 5'-C ACG T AC CG C CG TC ACG T TC-3' and AT P α -K I-r: 5'-CCTAATGGAAGGCCAATTTTGGTG-3' are provided.

2. The use of the axonal peroneal muscular atrophy drosophila model constructed by the method of claim 1 in the preparation of a medicament for delaying or treating CMT2 disease.