CN111118064B

CN111118064B - Animal model for spermatogenesis dysfunction and preparation method and application thereof

Info

Publication number: CN111118064B
Application number: CN201911346372.9A
Authority: CN
Inventors: 赵杰; 单嘉杰; 王键; 于才勇; 方莲; 曹倚慧
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2022-10-25
Anticipated expiration: 2039-12-24
Also published as: CN111118064A

Abstract

The invention provides a spermatogenesis deficiency animal model and a preparation method and application thereof. The invention discovers that the over-expression of PD-L1 has interference effect on the process of spermatogenesis and maturation for the first time, and proposes that PD-L1 can be used as a target for treating spermatogenesis deficiency diseases. Meanwhile, a construction method of a spermatogenesis deficiency animal model is provided, the animal model is obtained by over-expressing PD-L1; the construction method only changes the expression of the PD-L1 gene, does not directly influence the expression of other genes of the animal model, and does not influence the change of other phenotypes of the animal model; meanwhile, the method has good repeatability, is simple and feasible, can specifically and efficiently enhance the expression of the PD-L1 gene, and the constructed animal model is stable. The invention provides important animal basis, experimental materials and new ideas for related scientific research and clinical diagnosis and treatment of spermatogenesis dysfunction, and has important value and significance for clinical treatment of infertility.

Description

Animal model for dysspermia and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical engineering, and particularly relates to a spermatogenesis deficiency animal model and a preparation method and application thereof.

Background

At present, two main techniques are used for constructing mice with spermatogenesis deficiency: firstly, inducing the animal model of the spermatogenesis dysfunction mainly by feeding or injecting medicines, hormones and other methods; however, the induced animal model is easily unstable and the experimental repeatability is poor due to the influence of external interference factors, such as the batch of experimental animals and the stability of chemical reagents. Secondly, the animal model mostly forming gene defect by knocking out certain transcription factor, such as Sox30 ^-/- A mouse model, wherein a transcription factor gene knockout mouse model is formed by interfering a certain transcription factor(s); however, since the transcription factor generally involves the expression regulation of a plurality of genes, the expression of a plurality of genes regulated by the transcription factor is changed, and the phenotypes of the constructed animal models are diversified, the phenotype generated by the transcription factor gene knockout mouse model may not be merely the spermatogenesis deficiency, and more negative effects are accompanied.

Therefore, how to construct and obtain a spermatogenesis deficiency animal model with definite phenotype and stable model by a method which is simple and easy to implement and good in repeatability has important significance for discovery of pathogenic genes of animal spermatogenesis and maturation inhibition and research of pathogenic mechanisms and clinical treatment of male infertility.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing the application of PD-L1 as a target spot in screening or preparing the medicine for treating the spermatogenesis deficiency disease.

The invention also aims to provide a construction method of the animal model of the spermatogenesis deficiency.

It is yet another object of the present invention to provide an animal model of dysspermia.

The invention also aims to provide application of the animal model for the spermatogenesis deficiency.

The purpose of the invention is realized by the following technical scheme:

the application of PD-L1 (programmed death ligand-1) as a target point in screening or preparing a medicament for treating spermatogenesis deficiency diseases; the inventor firstly discovers that the PD-L1 overexpression has interference effect on the process of spermatogenesis and maturation.

A construction method of a spermatogenesis deficiency animal model is obtained by over-expressing PD-L1 in an animal body.

The concrete operation of the construction method of the spermatogenesis deficiency animal model is preferably as follows: constructing a recombinant expression vector containing a PD-L1 gene coding region sequence, injecting the expression vector into animal cells to over-express PD-L1 and performing embryo transplantation, thereby obtaining the animal model with the spermatogenesis deficiency.

The expression vector is preferably a mammalian cell expression vector; more preferably a pCAGGS vector.

The injection is preferably a prokaryotic injection.

The animal cell is preferably a fertilized egg.

The animal is preferably a murine animal.

The specific operation steps of constructing the recombinant expression vector containing the PD-L1 gene coding region sequence are preferably as follows:

(1) Amplifying a PD-L1 coding region DNA fragment, carrying out TA cloning by using a pMD18-T vector to obtain pMD18-PD-L1, carrying out enzyme digestion by using EcoRI and Xho I to obtain a PD-L1 DNA fragment, filling up by using T4 DNA Polymerase, and then recovering to obtain a PD-L1 coding region DNA fragment;

(2) After cutting the pCAGGS vector by EcoR I enzyme, filling in by T4 Polymerase to obtain a linearized pCAGGS vector;

(3) And (3) connecting the DNA fragment of the PD-L1 coding region obtained in the step (1) with the linearized pCAGGS vector obtained in the step (2) to construct the recombinant expression vector containing the PD-L1 gene coding region sequence.

The primer for amplifying the DNA fragment of the PD-L1 coding region in the step (1) is preferably:

an upstream primer: CGACTCGAGATGAGGATTTGC;

a downstream primer: CAGGAATTCTTACGTCTTCCTCGA.

The DNA fragment of the PD-L1 coding region in the step (1) can also be connected with a fluorescent protein gene.

The fluorescent protein gene is preferably EGFP gene.

An animal model of spermatogenesis deficiency is prepared by the construction method of the animal model of spermatogenesis deficiency.

The application of the animal model of the spermatogenesis dysfunction in the research of the spermatogenesis dysfunction disease or the screening of the drug of the spermatogenesis dysfunction disease.

A medicine for treating spermatogenesis dysfunction takes PD-L1 as a target point.

The invention adopts the fertilized egg pronucleus injection and the pseudopregnant mouse embryo transplantation to obtain the transgenic mouse, the culturing observation lasts for 190 days, the mouse is killed respectively in different culturing durations, the testis and epididymis tissues are picked up for observation and measurement, the main detection aspect comprises the observation of the fluorescent tissue shape and size, the tissue weight measurement, the tissue slice cell staining, the immunohistochemistry and the immunofluorescence staining under a mirror, and the successfully constructed sperm generation obstacle model is screened. Mainly shows that the testis shows strong green fluorescence, and the weight and the size are obviously lower than those of the wild type testis; the weight difference of epididymis is not obvious, but the immunofluorescence fluorescence staining of tissue sections of epididymis shows strong green fluorescence, and cell staining shows that no obvious cast-off cells (mostly round spermatids) exist in epididymis of transgenic PD-L1 mice.

Compared with the prior art, the invention has the following advantages and effects:

(1) The invention discovers for the first time that the over-expression of PD-L1 has an interference effect on the sperm maturation process, so that the spermatogenesis dysfunction of male mice is caused, a new treatment target is provided for the spermatogenesis dysfunction and the infertility diseases, and the treatment of the spermatogenesis dysfunction and the infertility diseases is hopefully realized through the regulation and control of PD-L1.

(2) The invention successfully establishes a spermatogenesis deficiency animal model by using PD-L1 overexpression, and performs fertilized egg prokaryotic injection on the established vector to establish a PD-L1 transgenic animal. The construction method of the invention can induce and produce the animal model with the dysspermia only by changing the expression of one gene, only increases the expression of the PD-L1 gene, does not directly influence the expression of other genes of the animal model, and does not influence the change of other phenotypes of the animal model; meanwhile, the constructed animal model is stable. The construction method of the invention is greatly different from the traditional gene knockout model in method and use, develops a new idea for researching animal spermatogenesis deficiency and infertility, can make up for instability of a chemical method induced animal model, and can reduce influence of multiple phenotypes and the like caused by interference of certain transcription factors.

(3) The expression vector used by the animal model construction method is convenient and easy to obtain, the vector construction method is simple and feasible, and the expression of the PD-L1 gene can be specifically and efficiently enhanced; the animal model shows that the barrier is generated in the process of generating sperms, the constructed animal model of the barrier generated by the sperms is stable, and a foundation is provided for discovery of pathogenic genes of barrier generation and maturation of the sperms of animals and research of pathogenic mechanisms.

(4) The invention creatively provides an animal model efficiently and stably constructed through PD-L1 overexpression, provides important animal basis and experimental materials for related scientific experiments and clinical diagnosis and treatment of spermatogenesis dysfunction, can be used for deeply researching molecular mechanisms of dysfunction in spermatogenesis and maturation processes, provides an animal model for clinical treatment of male infertility, and provides a theoretical basis for clinical treatment of infertility.

Drawings

FIG. 1 is a schematic view showing the preparation of a boat in example 2.

FIG. 2 is a schematic representation of the embryo handling in example 2.

FIG. 3 is a diagram showing the results of gel electrophoresis of the gene fragment in example 2.

FIG. 4 is a graph showing comparative analysis of testis and epididymis weights of a wild-type mouse (WT) and a PD-L1 transgenic group mouse (Tg).

FIG. 5 is a photograph of testis and tubules of wild type mice (WT) and PD-L1 transgenic group mice (Tg).

FIG. 6 is a graph of the immunohistochemical results of cryo-sections of testis and tubules (200X) of wild type mice (WT) and PD-L1 transgenic group mice (Tg).

FIG. 7 is a graph showing immunofluorescent staining of frozen sections of seminiferous tubules and epididymis of wild type and transgenic groups.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental animal materials included male C57BL/6 (B6) mice, female B6 mice and female ICR mice (purchased from shanghai slyke experimental animal center). The experimental animals were kept in the animal house of the college of medicine of university of science and technology in south China.

The experimental materials referred to in the examples are as follows

1. Main instrument

TABLE 1

2. Reagent, consumable material and strain

TABLE 2

EXAMPLE 1 construction of transgenic vectors

1. Amplification of PD-L1 coding region DNA fragment

1.1 primers

Forward primer：CGACTCGAGATGAGGATATTTGC

Reverse primer：CAGGAATTCTTACGTCTCCTCGA

1.2PCR amplification of PD-L1 fragments

Spleen tissues and RNA were extracted from mice (female B6 mice), and the full length of cDNA of the PD-L1 coding region was obtained by RT-PCR using the RT-PCR system as follows:

TABLE 3

The reaction conditions were as follows: 15min at 37 ℃, 5s at 85 ℃ and 4 DEG C

Carrying out PCR on the obtained cDNA by using the designed primer to obtain a target fragment of the target gene PD-L1, wherein the reaction system is as follows:

TABLE 4

The reaction conditions were as follows:

the obtained PCR product was subjected to nucleic acid electrophoresis, and the nucleic acid electrophoresis gel was prepared by dissolving 0.25g of agarose in 25mL of 1 XTAE solution, dissolving at high temperature in a microwave oven, adding 2.5. Mu.L of EB reagent, pouring into a mold, and standing at room temperature for 1 hour. The fragment was mixed with 6 × electrophoresis loading buffer at a ratio of 1.

And (3) cutting and recycling the strips displayed after electrophoresis, and specifically comprising the following steps:

(1) Column equilibration step: adding 500 μ L of balance liquid BL into adsorption column CA2 (the adsorption column is placed into the collection tube), centrifuging at 12,000rpm for 1min, pouring out waste liquid in the collection tube, and replacing the adsorption column into the collection tube.

(2) A single band of the target DNA was cut out of the agarose gel (excess was removed as much as possible) and placed in a clean centrifuge tube and weighed.

(3) To the gel piece was added an equal volume of solution PN (100. Mu.L of PN solution if the gel weight is 0.1g, the volume can be considered 100. Mu.L) and placed in a 50 ℃ water bath with the centrifuge tube gently turned upside down to ensure that the gel piece was fully dissolved. If there is still undissolved lumps, it may be left for a few minutes or some more until the lumps are completely dissolved (if the lumps are too large, the lumps may be cut into pieces beforehand).

(4) Adding the solution obtained in the previous step into an adsorption column CA2 (the adsorption column is placed into a collecting pipe), standing at room temperature for 2min, centrifuging at 12,000rpm for 60s, pouring the waste liquid out of the collecting pipe, and placing the adsorption column CA2 into the collecting pipe.

(5) Adding 600 μ L of rinsing solution PW (added with anhydrous ethanol) into adsorption column CA2, standing at room temperature for 3min, centrifuging at 12,000rpm for 60s, removing waste liquid from the collection tube, and placing adsorption column CA2 into the collection tube. And repeating the steps once.

(6) The adsorption column CA2 was returned to the collection tube and centrifuged at 12,000rpm for 2min to remove the rinse as much as possible. The adsorption column CA2 was left at room temperature for several minutes and completely air-dried.

(7) Placing the adsorption column CA2 into a clean centrifuge tube, suspending and dripping 60 μ L of elution buffer EB into the middle position of the adsorption film, and standing at room temperature for 2min. The DNA solution was collected by centrifugation at 12,000rpm for 2min.

(8) The concentration of the PD-L1 fragment was measured and stored in a refrigerator at-20 ℃.

The obtained PD-L1 band has the size of about 870bp, and the target gene is recovered.

2. Construction of fragments for ligation vectors

2.1TA cloning of the ligation fragments of interest

The obtained PD-L1 sequence is connected to a vector pMD18-T through TA cloning to obtain pMD18-PD-L1. The specific experimental steps are as follows:

(1) The following DNA solutions were prepared in a microcentrifuge tube in a total volume of 5. Mu.L.

TABLE 5

(2) Add 5. Mu.L (equal volume) of Solution I.

(3) The reaction was carried out at 16 ℃ for 30min.

(4) The total amount (10. Mu.L) was added to 50. Mu.L of DH5a competent cells and kept on ice for 30min.

(5) After heat shock at 42 ℃ for 90s, it was placed in ice for 2min.

(6) 1mL of LB medium was added, and the mixture was cultured at 37 ℃ for 60min with shaking at 200 rpm.

(7) To LB agar plates containing Amp (100. Mu.g/mL) were added dropwise 40. Mu.L of X-Gal stock solution (20 mg/mL) and 5. Mu.L of IPTG (0.84 mol/L), respectively, and then LB medium containing the bacterial solution was plated.

(8) Culturing on the culture medium to form a single colony. Blue and white colonies were counted.

(9) Selecting white colony, adding 1mL LB liquid culture medium, oscillating and culturing at 37 deg.C and 200rpm for 60min, extracting 500 μ L bacterial liquid, and storing at-80 deg.C.

(10) The bacterial suspension 100. Mu.L with the correct sequencing result was selected and amplified in 10mL LB liquid medium containing ampicillin at 37 ℃ for 6h with shaking at 200 rpm.

(11) 1.5mL of the bacterial solution was centrifuged at 12,000rpm for 1min, and the supernatant was discarded.

(12) The bacterial pellet was suspended homogeneously by adding 250. Mu.L of buffer S1 to which RNaseA had been added.

(13) Adding 250 mu L of Buffer S2, gently and fully turning over the mixture for 4 to 6 times, and uniformly mixing the mixture to fully crack the thalli until a transparent solution is formed. This step should not be carried out for more than 5min.

(14) Add 350. Mu.L Buffer S3, mix gently and thoroughly by tumbling up and down 6-8 times, centrifuge at 12,000rpm for 10min.

(15) The centrifuged supernatant from step (14) was aspirated and transferred to a preparation tube (placed in a 2mL centrifuge tube), centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(16) The tube was placed back into the centrifuge tube, 500. Mu.L of Buffer W1 was added, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(17) Placing the prepared tube back into a centrifuge tube, adding 700 μ L Buffer W2, centrifuging at 12,000rpm for 1min, and discarding the filtrate; the column was washed once more with 700. Mu.L Buffer W2 in the same manner. The filtrate was discarded.

(18) The preparation tube was placed back into a 2mL centrifuge tube and centrifuged at 12,000rpm for 1min.

(19) The preparation tube was transferred to a new 1.5mL centrifuge tube (provided in the kit), 60-80. Mu.L of Eluent or deionized water was added to the center of the preparation tube membrane, and the mixture was allowed to stand at room temperature for 1min. Centrifuge at 12,000rpm for 1min.

(20) Storing at-80 deg.C.

2.2 connection of the target fragment to the EGFP Gene fragment

In order to facilitate observation of phenomena in the later experimental process, an EGFP gene connection target gene segment can be obtained from pIRES2-EGFP plasmid, and the segment is constructed by adopting a double enzyme digestion method. The specific experimental steps are as follows:

(1) Selecting the restriction enzymes Xho I and EcoR I to cut pMD18-PD-L1 and pIRES2-EGFP. The plasmids were digested separately using the following system: ecoRI 1 mu L, xho I1 mu L,10 XH Buffer2 mu L, DNA less than or equal to 1 mu g, and sterilized water up to 20 mu L. The reaction conditions were as follows: react at 37 ℃ for 3.5h.

(2) Connecting the enzyme-digested PD-L1 and pIRES2-EGFP to obtain a vector pPD-L1-IRES2-EGFP of 20 mu L in total, wherein the reaction system is as follows: 250ng of PD-L1 fragment after enzyme digestion, 50ng of pIRES2-EGFP plasmid vector after enzyme digestion, and equal volume of ligation Mix. The reaction conditions were as follows: the reaction was carried out at 16 ℃ for 30min.

(3) The total amount (20. Mu.L) was added to 50. Mu.L of DH5a competent cells and kept on ice for 30min.

(4) After heat shock at 42 ℃ for 90s, it was placed in ice for 2min.

(5) 1mL of LB medium was added thereto, and the mixture was cultured at 37 ℃ for 60min with shaking at 200 rpm.

(6) The single colony is formed by culturing on an LB agar plate medium containing X-Gal, IPTG and Amp. Blue and white colonies were counted.

(7) Selecting white colonies, adding 1mL LB liquid medium, shaking culturing at 37 deg.C and 200rpm for 60min, extracting 500 μ L bacterial liquid, sequencing to detect sequence, and storing 500 μ L bacterial liquid at-80 deg.C.

(8) The bacterial suspension 100. Mu.L having the correct sequencing result was selected and amplified by 10mL of LB liquid medium (100. Mu.g/mL) to which ampicillin was added, and shaking-culturing at 37 ℃ and 200rpm for 6 hours.

(9) 1.5mL of the bacterial solution was centrifuged at 12,000rpm for 1min, and the supernatant was discarded.

The following steps were performed according to the procedures of the Axygen plasmid miniprep kit:

(10) Adding 250 mu L of Buffer S1 added with RNaseA to evenly suspend the bacterial sediment; adding 250 mu L of Buffer S2, gently and fully turning the mixture up and down for 4 to 6 times, and uniformly mixing the mixture to fully crack the thalli until a transparent solution is formed. This step should not be carried out for more than 5min.

(11) Adding 350 mu L of Buffer S3, gently and fully overturning and mixing the mixture for 6 to 8 times up and down, and centrifuging the mixture for 10min at 12,000rpm.

(12) The centrifuged supernatant from step (11) was aspirated and transferred to a preparation tube (placed in a 2mL centrifuge tube), centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(13) The tube was placed back into the centrifuge tube, 500. Mu.L of Buffer W1 was added, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(14) Placing the prepared tube back into a centrifuge tube, adding 700 μ L Buffer W2, centrifuging at 12,000rpm for 1min, and discarding the filtrate; the same procedure was followed with a further 700. Mu.L Buffer W2 wash. The filtrate was discarded.

(15) The preparation tube was placed back into a 2mL centrifuge tube and centrifuged at 12,000rpm for 1min.

(16) The preparation tube was transferred to a new 1.5mL centrifuge tube (provided in the kit), 60-80. Mu.L of Eluent or deionized water was added to the center of the preparation tube membrane, and the mixture was allowed to stand at room temperature for 1min. Centrifuge at 12,000rpm for 1min.

(17) Storing at-80 deg.C.

2.3 construction of PD-L1-IRES-EGFP fragment

(1) And selecting an endonuclease Xho I and a Not I to cut the pPD-L1-IRES2-EGFP. The plasmid was digested with the following system: not I1. Mu.L, xho I1. Mu.L, 10 XH Buffer 2. Mu.L, DNA Not more than 1. Mu.g, sterile water up to 20. Mu.L. The reaction conditions were as follows: react at 37 ℃ for 3.5h.

(2) Fragment concentrations were determined using a microplate reader.

(3) The end was blunted with T4 Polymeras, and the reaction solution was in a microcentrifuge tube at a total volume of 9. Mu.L, as follows:

overhang DNA fragment > 0.1pmol,10 XT 4 DNA Polymerase Buffer 1. Mu.L, 0.1% BSA 1. Mu.L, 1.7mmol/L dNTP mix 1. Mu.L, sterilized water up to 9. Mu.L.

The reaction conditions were as follows: the temperature is maintained for 5min at 70 ℃, and then the mixture is moved into a thermostatic bath at 37 ℃. mu.L of T4 DNA Polymerase was added, gently mixed with a sampler, and incubated at 37 ℃ for 5 minutes. The reaction solution was placed on ice and immediately ligated to obtain a PD-L1-IRES-EGFP fragment.

3. Expression vector linearization and end filling

3.1pCAGGS expression plasmid restriction enzyme

(1) The endonuclease EcoRI was chosen to cleave the pCAGGS plasmid (which is disclosed in Zhang ZU MEI, huanghaibi, palmiperi et al. Construction of eukaryotic expression vectors expressing 3 foreign genes [ J ]. Chinese preventive veterinary bulletin, 2011,33 (4): 281-284.). The plasmids were digested separately with the following enzymes: ecoRI 1 μ L,10 XH Buffer2 μ L, DNA less than or equal to 1 μ g, sterile water up to 20 μ L. The reaction conditions were as follows: react at 37 ℃ for 3.5h.

(2) Fragment concentrations were determined using a microplate reader.

3.2 the end of the digestion fragment is filled up

(1) The ends were filled in with T4 Polymerase, and the reaction solution was added to a microcentrifuge tube in a total volume of 9. Mu.L, as follows: overhang DNA fragment > 0.1pmol,10 XT 4 DNA Polymerase Buffer 1. Mu.L, 0.1% BSA 1. Mu.L, 1.7mmol/L dNTP mix 1. Mu.L, sterilized water to 9. Mu.L.

(2) The reaction conditions were as follows: the temperature is maintained for 5min at 70 ℃, and then the mixture is moved into a thermostatic bath at 37 ℃. mu.L of T4 DNA Polymerase was added, gently mixed with a sampler, and incubated at 37 ℃ for 5 minutes. The reaction solution was placed in ice. Ready for immediate connection.

4. Preparation of recombinant clones

4.1 preparation of competent cells

The permeability of the competent cells is increased through treatment, so that the exogenous gene or vector can enter the competent cells conveniently.

(1) Extracting 25 mu L of DH5 alpha strain, evenly inoculating the strain on LB solid culture medium without antibiotics, and culturing for 16-20 h at 37 ℃.

(2) A single colony is picked from the cultured plate by the sterilized gun head, transferred into an LB liquid culture medium containing 15mL, and shaken for 10-12 h at the temperature of 37 ℃ and the rpm of 200.

(3) Standing on ice for 10min.

(4) Centrifuge at 6000rpm for 10min at 4 ℃, discard the supernatant and invert the tube for 1min to drain the last traces of culture.

(5) By using a pre-heaterCold 0.1mol/L CaCl ₂ Solution (80 mmol/L MgCl) ₂ ，20mmol/L CaCl ₂ ) The cell pellet was resuspended at a ratio of 2.5.

(6) Centrifuge at 6000rpm for 10min at 4 ℃, discard the supernatant and invert the tube for 1min to drain the last traces of culture.

(7) With 2mL of precooled 0.1mol/L CaCl ₂ Resuspend each cell pellet and let stand on ice for 10min.

(8) Adding 2mL 30% glycerol, mixing, packaging into 100 μ L tubes, and storing at-80 deg.C.

4.2PCR product ligation linearized expression vector

And (3) connecting the PD-L1-IRES-EGFP fragment recovered in the step 2.3 to the linearized pCAGGS plasmid to obtain a transgenic vector pCAG-PD-L1.

The enzyme linked system (10. Mu.L system) was as follows:

TABLE 6

Finally adding enzyme, repeatedly blowing and sucking by a gun, mixing uniformly, and violently shaking.

Overnight at 16 ℃ (water is added to the lower layer of the thermal insulation lunch box, ice is added to the upper layer, the enzyme connecting body is put on the water, and the lunch box is covered tightly).

4.3 transformation

(1) From the-80 ℃ refrigerator, 200 u L competent cell suspension, at room temperature to thaw, thawing immediately after ice.

(2) Adding the plasmid DNA solution obtained in the step 4.2 (the content is not more than 50ng, the volume is not more than 10 mu L), gently shaking, and standing on ice for 30 minutes.

(3) Heat shock is carried out for 90 seconds at 42 ℃, and the mixture is quickly placed on ice for cooling for 3 to 5 minutes after heat shock.

(4) Adding 1mL LB liquid culture medium (without Amp) into the tube, mixing, shaking culturing at 37 deg.C for 1 hr to restore normal growth state of bacteria, and expressing plasmid-encoded resistance gene (Amp) ^r )。

(5) Centrifuging the bacterial liquid at 25 ℃ and 3000rpm for 5min, discarding 900 mu L of supernatant, shaking up, taking 100 mu L of supernatant, coating the 100 mu L of supernatant on a screening plate containing Amp, standing for half an hour with the front side upward, inverting the culture dish after the bacterial liquid is completely absorbed by the culture medium, and culturing at 37 ℃ for 16-24 hours.

Control 1-the DNA solution was replaced with the same volume of sterile double distilled water, and the other operations were the same as above. This group should normally have no colonies present on the LB plates containing the Amp.

Control 2, 10ng of the enzyme-cleaved purified product was used in place of the DNA solution, and the other operations were the same as above. This group should normally show no more than 50 colonies on the LB plates containing the Amp.

5. Positive clone identification

Sequencing: depending on the number of colonies of the negative control (i.e., control 1 and control 2 in step 4.3), 5 to 10 colonies from the positive colonies were added to 1mL of LB liquid medium, shaken for 5 hours, and 500. Mu.L of the mixture was taken and tested.

Carrying out PCR of bacterial liquid, preparing a PCR system (10 mu L): taking the bacterial colony as a template for amplification, selecting the monoclonal antibody to be in 20 mu L sterile double distilled water, and uniformly blowing the solution to prepare bacterial liquid.

TABLE 7

And (4) selecting positive bacteria liquid after agarose gel electrophoresis detection.

The PCR conditions were as follows:

TABLE 8

Example 2

The vector pCAG-PD-L1 obtained in example 1 was digested with SalI and Avr II, and the large fragment was recovered using the endotoxin-free Gel Extraction Kit QIA quick Gel (Qiagen) for use. Establishment of transgenic mice PD-L1 by prokaryotic injection ^ves 。

Prokaryotic injection of sperm eggs establishment of transgenic animals was carried out according to the method established by Nagy et al (Nagy A, gertsenstein M, vinterstein K.Maniating the Mouse Embryo: A Laboratory Manual [ M ].3 rd. New York.

1. Reagent consumable

(1) Surgical instruments: ophthalmic forceps (toothed or toothless), micro forceps (pair), ophthalmic scissors, suture line, suture needle, tissue clamp, injection needle (GD-1, narishige) self-made, and fixed needle (made in China)

(2) The instrument comprises the following steps: olympus inverted microscope, eppendorf operating arm, P-87 needle drawing instrument, MF-900 needle cutting instrument

(3) Reagent

M16 (cat M7167), M2 (cat M7292), mineral oil (cat M5310), hyaluronidase (cat H3506) (500 IU/mL deluted in M2), and Avermectin (cat T48402) were purchased from Sigma.

Injection buffer (from Millipore, cat # MR-095-F), microloader (from Eppendorf NO. 5242956.003)

(4) Donor and recipient mice

Male C57BL/6 (B6) mice, female B6 mice, female ICR mice (purchased from shanghai slaike laboratory animal center), and TgPD-L1 male mice.

2. Ligation male mouse

(1) C57BL/6 (B6) male mice were anesthetized (1.25% Avermectin, i.p.).

(2) The abdomen is supine, the hair of the mouse at the operation position is cut off, and the lower abdomen is disinfected by 70% alcohol.

(3) The skin and muscle layers were cut open and the incision was about 1cm long. The suture is penetrated at the opening of the body wall of the single side, so that the muscle layer can be found conveniently in the later stage.

(4) Ligation of vas deferens: the fat pad was grasped with blunt forceps and the testis, epididymis and vas deferens were pulled out gently.

(5) Identifying vas deferens: the vas deferens connects the epididymis and penis. Vas deferens is rich in longitudinally distributed blood vessels, and is freed from surrounding tissue with scissors or forceps. The vas deferens is clamped by forceps to form a ring, another blunt forceps is heated by an alcohol lamp until the ring turns red, and the red hot forceps are used for clamping the vas deferens ring to separate from the vas deferens and form two closed ends. The two burnt ends are separated and the whole organ is returned to the abdominal cavity with blunt forceps.

(6) Repeating the above steps to cut off the vas deferens at the other side.

(7) The muscle layer and skin incision were sutured.

(8) Monitoring after an operation: the mice are monitored until complete resuscitation and euthanasia should be given if significant pain, restlessness or postoperative reaction is observed. Mice should be observed daily for infection or other problems within one week after resuscitation and, once found, should be euthanized.

3. Prokaryotic injection

1. Hormone dispensing method

PMSG (pregnant horse serum gonadotropin) PMSG freeze-dried powder is dissolved in 0.9% NaCl sterile physiological saline with the concentration of 50IU/mL, and is subpackaged into 1.5mL EP tubes according to the quantity required by the experiment (the dosage of 5 or 10 mice can be subpackaged into each tube), and the dosage of each mouse is 0.1mL (5 IU). Can be stored at-20 deg.C for at least one month.

HCG (human chorionic gonadotropin) lyophilized powder was dissolved in 0.9% NaCl sterile physiological saline at a concentration of 50IU/mL and dispensed into 1.5mL EP tubes (5 or 10 mice per tube) at a dose of 0.1mL (5 IU) per mouse, according to the amount required for the experiment. Can be stored at-20 deg.C for at least one month.

(2) Superovulation procedure

TABLE 9

Monday

Zhou Di

Wednesday

Week four

Zhou Wu

Saturday medicine

(Sunday)

PMSG

HCG

Suppository detection/injection

PMSG

HCG

Suppository detection/injection

PMSG

HCG

Suppository detection/injection

PMSG

HCG

Suppository detection/injection

2. Isolation of oocytes

(1) Three days afternoon before the experiment: b6 female mice of 3-4 weeks old are injected in the abdominal cavity, and PMSG 5IU is added for each mouse.

(2) Afternoon of the day before the experiment: PMSG is injected for 46-48 hours, and HCG is injected in the abdominal cavity for 5 IU/mouse.

(3) The superovulated female mouse is put into a single-seed mouse cage with 1 mouse per cage.

(4) An estrus ICR female mouse is picked out and put into a ligation male mouse cage according to the proportion of 1 cage and 2 mice.

(5) On the day of injection: the ligation was checked and the record of the presence/absence of ligation was registered in the ligation male mouse cage plate. Multiple M16 microdroplets were prepared in petri dishes, covered with mineral oil, and placed in CO ₂ And balancing in the incubator.

(6) Preparing a 35mm culture dish, and placing M2 liquid drops in the culture dish for storing and taking out the oviduct; a60 mm petri dish was prepared for digestion and washing of fertilized eggs.

(7) Removing cervical vertebrae to kill fertilized eggs for mice, wetting the back of the fertilized eggs by using 70% alcohol, lifting skin of the back close to the tail end of the fertilized eggs by using forceps, cutting a transverse incision by using scissors, clamping the skin at the incision by using the forceps, peeling the skin to the head side, exposing muscles at the waist of the back side, opening a small opening at the position by using small scissors, clamping fat piles on the ovary in vivo by using the forceps, dragging the fat piles out of the body, clamping the uterus by using the forceps, cutting off the ovary by using the small scissors (paying attention to not cutting the oviduct), cutting off the uterine horn, and putting the cut-off oviduct into M2 liquid drops prepared in advance.

(8) The ampulla of the fallopian tube is torn by the ophthalmological forceps, the fertilized egg is extruded out of the tube, and if the fertilized egg does not flow out, the fallopian tube can be slightly extruded by the forceps.

(9) The hyaluronidase is digested for a few minutes until the granulosa cells are substantially shed, and if necessary, the drops can be blown several times, and the eggs are washed when all the granulosa cells are shed. But do not leave cumulus cell-free embryos in hyaluronic acid for too long, as this is detrimental to the embryos.

(10) The fertilized eggs were transferred to a new M2 solution with a mouth control tube.

3. Prokaryotic injection

The P-87 instrument is used to draw out prokaryotic injection needle and fixed needle with their tips bent at 20-30 deg. angle. Fixing the fixed needle on the operating arm; the boat was fabricated as shown in FIG. 1 by making an M2 strip approximately 2mm wide and coated with mineral oil. The enzyme-linearized vector pCAG-PD-L1 was introduced into the injection needle using a microsyringe, so that the formation of bubbles at the tip was avoided as much as possible. Portions of the embryos were washed several times with M2 microdroplets and transferred to injection dishes (FIG. 2), the handling dishes were placed on an inverted microscope, and the stage was moved until the embryos were visible under the field of view. Carefully lower the needle below the liquid level, see the needle in the field and position it close to the embryo injection needle in the same way. The injection needle collides with the fixing needle to generate a tiny fracture. The ovum is sucked by a fixed needle, and the position of the ovum is adjusted by slightly pulling the injection needle, so that the pronucleus can be clearly seen and the needle point and the pronucleus can be clearly positioned on the same focal plane. The injection tube is inserted into the pronucleus, and the DNA is injected by pressing the Inject key, the pronucleus is rapidly withdrawn after obvious expansion, and the embryo is dissolved by too much DNA. The ovum is pushed downwards by the injection needle and a new ovum is sucked for injection, and the process is repeated until all the ovum is injected. After injection, embryos were transferred to M16 dishes and placed in incubators for culture. This was repeated until all eggs were injected.

4. Embryo transfer

Embryos after injection are cultured appropriately to recover the embryos, and dead embryos are discarded. Transplanting a pseudopregnant female mouse with 0.5-day thrombus; if the number of the pseudopregnant mice is not enough, the mice can be combined in the morning of the day, and pseudopregnant female mice (0.25 dpc) with thrombus in the afternoon can be used as a receptor, and the embryos can be cultured to the next day and then transplanted to 2-cells. Abamectin is injected into abdominal cavity for anesthesia. Transferring the embryo into M2 liquid drop to clean for several times, firstly sucking a small bubble in the transfer tube, then sucking a small amount of culture solution, then next to a bubble, sucking 20-30 embryos and a small amount of culture solution, and finally sucking a small bubble and a small amount of culture solution. The abdominal cavity is opened, forceps are used for clamping fat pile on the ovary in vivo, the ovary, the oviduct and the uterine upper segment are pulled out of the body, the oviduct and the ampulla thereof are found under a body microscope, the oviduct is fixed by the forceps of ophthalmology, and a sharp needle is used for puncturing a small opening before the ampulla is enlarged. Then the transplantation tube is inserted into the opening for about 5mm, the embryo and the liquid are slightly blown out until all three bubbles enter the oviduct, and then the transplantation tube is pulled out. The uterus and the fallopian tube are returned. The muscle and skin were sutured separately with silk. The mice that had undergone the transplantation procedure were returned to the cage, and the cage was placed on a hot table and incubated (37 ℃) until the mice recovered.

In the experiment, 5 superovulated mice are fertilized to obtain 52 eggs, wherein 40 fertilized eggs are injected successfully, 35 fertilized eggs are returned to pseudopregnant mice to obtain 30 transgenic mice; the modeling method has high repeatability. Trizol was used to extract RNA from testis tissues of 6 transgenic male mice (lanes 1-6) and common male C57BL/6 (B6) mice (lanes 7-8), and RT-PCR was used to confirm that PD-L1 transgenic mice were successfully constructed (FIG. 3).

Example 3

The biological traits of the transgenic mice and B6 mice constructed in example 2 were measured: the body length, body weight and size of spleen and thymus were measured using electronic day and millimeter ruler in transgenic and wild type mice.

The transgenic mice and wild mice are bred in an SPF animal laboratory, at least 3 bred transgenic mice and wild mice are killed respectively at 40d, 60d, 70d, 90d, 100d, 120d, 140d, 160d and 190d in sequence, testis and epididymis tissues are taken immediately, and after photographing and weighing, the average weight of the testis and epididymis tissues of the two types of mice is calculated. Then, the testis and the epididymis are immediately taken and sliced respectively, and cell staining and immunohistochemical experiments are carried out.

The specific operation of immunohistochemistry is as follows:

immunohistochemistry detects PD-L1 expression in surrounding tissues: after incubating wild-type mice and transgenic mice epididymis frozen sections for 30min with 0.3% triton x-100PBS, rat anti-mouse PD-L1-IgG (purchased from Serotech) was added at a ratio of 1.

As shown in fig. 4, it was found by observation of wild type mice and PD-L1 transgenic for up to 190 days that the transgenic testis and epididymis weights were significantly lower than the wild type.

As shown in fig. 4 and fig. 5, the wild-type mice and PD-L1 transgenic mice were observed for 190 days, and the wild-type mice had a higher testis weight than the transgenic PD-L1 mice from 40 days, which was significantly different (P < 0.05). From day 70, the weight of epididymis of wild type mice was gradually higher than that of transgenic PD-L1 type mice, and there was a significant difference in weight between the two (P < 0.05). From a general observation that the testis size of the wild type mice is larger than that of the transgenic PD-L1 type mice, epididymis differences are not obvious.

As shown in FIG. 6, the epididymis tissue section was cyto-stained, and it was observed that there was a large number of cell loss in wild type mouse epididymis, and most of them were round spermatids. And no obvious desquamation cell exists in the epididymis of the transgenic PD-L1 type mouse.

As shown in figure 7, immunofluorescence staining of epididymis cryosections of transgenic PD-L1 mice and wild mice shows that the surfaces of seminiferous tubules and epididymis columnar epithelial cells of the transgenic PD-L1 mice highly express PD-L1, while the expression levels of PD-L1 on the surfaces of the seminiferous tubules and epididymis columnar epithelial cells of the wild mice are not obvious, and the two have obvious difference.

And (4) conclusion: the results show that PD-L1 can influence the shedding of sperm cells in seminiferous tubules, and in transgenic PD-L1 mice, the high expression of PD-L1 of supporting cells and sperm cells is an important factor for the sterile expression of the mice.

Through the steps, a PD-L1 overexpression transgenic mouse is constructed and verified, and the construction of a spermatogenesis deficiency mouse model is obtained.

For a passage PD-L1 transgenic mouse model, selecting a first established PD-L1 transgenic male mouse to mate with a wild female mouse, wherein the male mouse offspring which has the fertility has the reproductive capacity but shows the negative PD-L1 or positive PD-L1 and weak positive testicular EGFP; selecting a first established PD-L1 transgenic male mouse to mate with a wild type female mouse, wherein the male mouse has no fertility, the size and weight of the testis of the mouse are obviously smaller than those of the wild type mouse, and the testis of the mouse simultaneously displays strong green fluorescence; and (3) selecting the first established PD-L1 transgenic female mouse to mate with a wild type male mouse, wherein the offspring of the male mouse with fertility is represented by the reproductive capacity, and the observation conditions of testis and epididymis tissues are the same as the first established male mouse with the reproductive capacity. The result shows that the high expression PD-L1 in testis and epididymis tissues has high correlation with the spermatogenesis deficiency of male mice, the PD-L1 transgenic male mice have the phenotype of the spermatogenesis deficiency, and the phenotype change caused by the gene change has stable heredity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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<120> animal model of spermatogenesis dysfunction, preparation method and application thereof

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Claims

1. The application of the transgenic mouse in preparing the animal model with the spermatogenesis deficiency is characterized in that the transgenic mouse is a PD-L1 overexpression transgenic mouse;

the PD-L1 overexpression transgenic mouse is constructed by the following method: injecting a recombinant expression vector containing a PD-L1 gene coding region sequence into animal cells to enable PD-L1 to be over-expressed and carrying out embryo transplantation to obtain a PD-L1 over-expressed transgenic mouse; wherein, the construction steps of the recombinant expression vector containing the PD-L1 gene coding region sequence are as follows:

(1) Amplifying a PD-L1 coding region DNA fragment, carrying out TA cloning by using a pMD18-T vector to obtain pMD18-PD-L1, carrying out enzyme digestion by using EcoRI and Xho I to obtain a PD-L1 DNA fragment, filling in by using T4 DNA Polymerase, and then recovering to obtain a PD-L1 coding region DNA fragment;

(3) Connecting the DNA fragment of the PD-L1 coding region obtained in the step (1) with the linearized pCAGGS vector obtained in the step (2) to construct a recombinant expression vector containing the PD-L1 gene coding region sequence;

the expression vector contains a fluorescent protein gene EGFP.

2. Use according to claim 1, characterized in that:

the expression vector is a mammalian cell expression vector.

3. Use according to claim 1, characterized in that:

the injection is prokaryotic injection;

the expression vector is a pCAGGS vector.

4. Use according to claim 1, characterized in that:

the animal cell is a fertilized egg;

the animal is a murine animal;

the primers for amplifying the DNA fragment of the PD-L1 coding region in the step (1) are as follows:

an upstream primer: CGACTCGAGATGAGGATTTGC;

a downstream primer: CAGGAATTCTTACGTCTTCCTCGA.

5. The application of a transgenic mouse in preparing an animal model for screening a medicament effective on spermatogenesis deficiency is characterized in that the transgenic mouse is a PD-L1 overexpression transgenic mouse;

6. The use of claim 5, wherein the spermatogenic disorder is caused by overexpression of PD-L1.