CN112881711A - Construction method and application of mouse model based on regulation and control signal path - Google Patents
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Abstract
The invention discloses a construction method and application of a mouse model based on a regulation signal channel, and belongs to the technical field of biology. A method for constructing a mouse model comprises the following steps: constructing mouse models of beta-catenin knockout, Notch1 knockout and double knockout of the beta-catenin knockout and the Notch1 knockout. Injecting tamoxifen and EdU when the age of a mouse is 0 or 1 day, dissecting a basement membrane of the mouse to perform an immunofluorescence experiment when the age of the mouse is 7 days, labeling hair cells with a myostatin 7a antibody, labeling support cells with a Sox2 antibody, and counting the cells positive to both EdU + Sox2 and EdU + Myo7 a; basement membranes of mice aged 7 days, i.e., wild-type control group, beta-catenin overexpression (. beta. -cat OE), Notch1-KO and beta-cat OE/Notch1-KO, were separated, and RNAs were extracted and sequenced.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing a mouse model based on a regulation signal channel and application thereof.
Background
Currently, the deafness problem is of concern to people. In 2015, about 5 hundred million people had hearing loss, accounting for about 6.8% of the world population. These figures are much higher than the estimated figures published before 2013, and the number of people showing hearing loss is increasing, indicating that the importance of global hearing health care is becoming more prominent. In addition, the data of WHO2018 show that more than 4.32 hundred million adults and 3400 ten thousand children suffer from disabled hearing loss, so that the data become a main burden affecting social health and economy. Hearing impairment can lead to social isolation, depression and reduced professional ability.
At present, no inner ear targeting medicament with exact curative effect can be clinically used for treating sensorineural deafness. Since cochlear hair cells of mammals are not reproducible, how to repair and regenerate hair cells after injury, and thus fundamentally treat sensorineural deafness is the focus of research in the hearing field in recent years. And the provision of a non-human animal model as a research and test platform to characterize hair cell repair and regeneration is also of great significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for constructing a mouse model based on a regulation and control signal path and application thereof.
The purpose of the invention can be realized by the following technical scheme:
a method for constructing a mouse model comprises the following steps:
constructing mouse models of beta-catenin knockout, Notch1 knockout and double knockout of the beta-catenin knockout and the Notch1 knockout. Injecting tamoxifen and EdU when the age of a mouse is 0 or 1 day, dissecting a basement membrane of the mouse to perform an immunofluorescence experiment when the age of the mouse is 7 days, labeling hair cells with a myostatin 7a antibody, labeling support cells with a Sox2 antibody, and counting the cells positive to both EdU + Sox2 and EdU + Myo7 a;
separating the wild control group, beta-catenin overexpression (beta-cat OE), Notch1-KO and beta-cat OE/Notch1-KO mice basement membranes aged 7 days respectively, and extracting RNA of the mice basement membranes for sequencing;
constructing an Atoh1 over-expressed mouse, injecting tamoxifen, taking out a basement membrane from the mouse for an immunofluorescence experiment, marking hair cells with a myostatin 7a antibody, marking support cells with a Sox2 antibody, and counting the cells which are double positive to EdU/Myo7 a;
regulating a Hippo signal pathway by inhibiting the expression of YAP, and analyzing the proliferation capacity of the Hippo signal pathway on progenitor cells expressing Lgr5 by a balling experiment;
optionally, the method further comprises the following steps:
separating and culturing inner ear stem cells expressing Lgr5 from a single cell suspension prepared by digesting a mouse cochlea basement membrane positive for Lgr5-EGFP-IRES-CreERT2, Lgr5-EGFP-CreERT2 and Foxg1-loxp/loxp, carrying out a cell balling experiment on the inner ear stem cells expressing Lgr5, and carrying out immunofluorescence staining to detect the proliferation condition of the inner ear stem cells expressing Lgr 5;
carrying out ABR detection on a mouse with the age of 30 days;
the mouse is 2 days old and is positive to Sox2-CreERT 2; the R26SmoM2 transgenic mouse cochlea is separated and cultured in PBS culture solution, neomycin injury is carried out, RNA is separated in the cochlea, and quantitative real-time PCR detection is carried out.
Optionally, the method for sorting inner ear stem cells expressing Lgr5 is a flow sorting method.
Optionally, the method of inhibiting YAP is: YAP expression was inhibited by verteporfin, dobutamine as YAP inhibitors.
Optionally, the ABR detection experimental conditions are: three fine needle electrodes were inserted into the cranial crown, under the ear to be tested and at the back near the tail of the mouse. ABR sound points of 4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz were generated.
Optionally, the culture conditions of the cochlea are: the cells were cultured in DMEM/F12 supplemented with 1% N2, 2% B27 and 50. mu.g/ml ampicillin at 37 ℃ under 5% CO2, respectively.
In addition, the invention also provides application of the mouse model research in hair cell regeneration
The invention has the beneficial effects that:
in conclusion, after Wnt, Notch and Atoh1 signal channels are regulated, whether promoting effect on hair cell regeneration is achieved or not is researched, the regulation mechanism is understood, meanwhile, Lgr5-EGFP-CreERT2 tool mice are used for screening Lgr5+ progenitor cells, and Hippo signals are regulated to promote proliferation or differentiation capacity of inner ear progenitor cells, so that the methods for regulating the signal channels are applied to promoting the regeneration of viable cell hair cells. To determine the mechanism of involvement of Foxg1 in HC regeneration, tdTomato +/Sox 2+ stem cells were isolated by flow cytometry and mRNA was extracted for real-time quantitative qPCR to detect the expression levels of the relevant genes. Two transcription factors Atoh1 and Gfi1 mRNA for regulating HC generation are up-regulated, Notch signal pathways such as Notch 1-3, Hes1, Hes5, Jag2 and Hey1 are all obviously down-regulated, and other genes are not obviously changed. Knockout of Foxg1 therefore generates new HCS primarily by down-regulating cell cycle pathways and Notch signaling pathways. The mouse model of the application reflects the proliferation and regeneration conditions of hair cells, provides new evidence for Foxg1 to regulate HC regeneration of cochlear support cells and progenitor cells of newborn mice, and has important research significance.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic diagram showing the comparison of mouse hair cell proliferation after gene knockout according to the present application;
FIG. 2 is a hierarchy of global comparisons of gene expression and gene expression patterns for four different transgenic mice of the present application;
FIG. 3 is a schematic representation of the hair cell regeneration promoting profile in β -cat OE/Notch1 KO/Atoh1 OE mice as used herein;
FIG. 4 is a schematic illustration of the study of the current application of modulating the Hippo signaling pathway to investigate the proliferative differentiation of flow-sorted inner ear progenitor cells;
FIG. 5 is a mouse model specific knockout Foxg1 regenerated inner hair cell immunofluorescence map of the present application;
FIG. 6 shows the experimental mean field pattern and statistical figures of number and diameter of Lgr5 cells after Foxg1 specific knockout in the mouse model of the present application.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 6, in an embodiment of the present invention, a method for constructing a mouse model based on a regulatory signal pathway and an application thereof are provided, which may include the following steps.
Interaction between Wnt and Notch signaling pathways
Mice were constructed with a β -catenin knockout (β -cat KO), a Notch1 knockout (Notch1-KO), and a double knockout of both. Tamoxifen was injected at day 0 or day 1 of age, EdU was injected every two days from day 1 to day 6 of age, mice were dissected for immunofluorescence experiments with mouse basement membranes at day 7 of age, hair cells were labeled with myosin7a antibody, support cells were labeled with Sox2 antibody, and cells that were EdU/Sox2 double positive and EdU/Myo7a double positive were counted.
Secondly, transcriptomics analysis is carried out on the regulation of Wnt and Notch signal pathways, and the specific steps can be that basement membranes of mice aged 7 days are separated respectively from a wild control group, beta-catenin overexpression (beta-catenin OE), Notch1-KO and beta-catenin OE/Notch1-KO, 6-8 basement membranes are taken from each group, RNA is extracted by using Trizol, and then RNA sequencing is carried out. And performing bioinformatics analysis on the obtained RNA sequencing result to find out obvious difference genes and related biological functions.
Thirdly, Wnt, Notch and Atoh1 co-regulate and promote hair cell regeneration
Mice over-expressing Atoh1 (Atoh1-OE) and Atoh 1-OE/beta-cat OE, Atoh 1-OE/beta-cat OE/Notch1-KO were constructed, tamoxifen was injected at 0 or 1 day of mouse age, then the mice of these different genotypes were dissected at 5 days of mouse age and the basement membranes were removed for immunofluorescence experiments, hair cells were labeled with myosin7a antibody, support cells were labeled with Sox2 antibody, and cells double positive for EdU/Myo7a were counted.
Fourthly, the Hippo signal pathway is regulated and controlled to explore the influence of the proliferation and differentiation of the flow-typed selected inner ear progenitor cells
1) Small molecule drug regulates Hippo signal path by activating or inhibiting YAP expression
Using micromolecular drugs verteporfin and dobutamine as YAP inhibitors and XMU-MP-1 as YAP activators, extracting proteins after in vitro processing of basement membranes of mice aged for 3 days by using different doses of the three micromolecular drugs, and determining a proper experimental concentration through a Western blotting experiment;
2) balling experiments to analyze the proliferative capacity of Hippo signaling pathway on Lgr5+ progenitor cells
Lgr5+ progenitor cells are separated from Lgr5-EGFP-CreERT2 mouse cochlea through flow sorting, then the sorted progenitor cells are put into a 96-well plate, the average cell number of each well is 200, the cells are cultured for 5 days, four experimental groups of 0.5 mu M Verteporfin, 1 mu M dobutamine, 0.5 mu M XMU-MP-1 and one DMSO are designed to be used as control groups respectively, and the drugs are added for 5 consecutive days from the first day of the seed cells for culture. Counting the number and diameter of the cell balls by bright field shooting on the 5 th day;
3) effect of the Hippo signalling pathway on the differentiation Capacity of Lgr5+ progenitor cells
Lgr5+ progenitor cells were isolated from the cochlea of Lgr5-EGFP-CreERT2 mice by flow sorting, 5000 per well of the sorted Lgr5+ progenitor cells were added to a laminin-coated four-well dish for 10 days of culture, 10. mu.M EdU was added on days 4 to 7, and the same small molecule drug as in the spheronization experiment was added from day 3 to day 9. Immunofluorescence experiments were performed after the end of the 10 th day culture, hair cells were labeled with myosin7a antibody, and the number of hair cells, the number of cell balls, and the number of EdU +/Myo7a + hair cells were counted.
Fifthly, Foxg1 signal path promotes transdifferentiation of inner ear stem cells into hair cells
1) Conditional knock-out of Foxg 1.
Three positive progeny were obtained by crossing Sox2CreER/+ Foxg1loxp/loxp mice with Lgr 5-EGFPReER/+ Foxg1loxp/loxp mice, followed by injection of Tamoxifen to specifically knock out Foxg1 in the support cells and inner ear stem cells. Mice were sacrificed at different time points and cochlear detection was performed. The growth of hair cells, the proliferation of inner ear stem cells and the like are detected by immunofluorescence techniques, and the hair cells, the supporting cells and the proliferated cells are labeled with Myosin7a, Sox2 and EDU, respectively. New born mice (aged 0-7 days) were immunofluorescent tested for Myosin7a, Sox2, Foxg1, Ctbp2, PSD95, Tuj 1.
2) Mice were tested for hearing threshold by ABR assay.
The ABR test was performed in a sound isolation booth, and three fine needle electrodes were inserted near the tip, base and tail of the skull of the mouse. ABR tone points of 4kHz, 8kHz, 12kHz, 16kHz, 24kHz and 32kHz are generated. The hearing threshold is determined by subtracting the sound intensity from 90dB to 10dB until the lowest sound intensity of the first wave is identified. ABR data were analyzed using GraphPad Prism 6 software.
3) Cell proliferation was examined using a balling assay.
Lgr 5-EGFPReER/+ Foxg1loxp/loxp mice and Lgr 5-EGFPReER/+ control groups were injected with Tamoxifen at 1 day of age and sacrificed at 3 days of age. The cochlea was dissected, trypsinized into single cells, and FAC sorting was performed on Lgr5+ cells. The selected cells were cultured in DMEM/F12 in its entirety at a density of 2 cells/. mu.1 for 5 days. Then, the cells were digested with trypsin and the next generation was cultured in the same manner. At the end of the incubation, all spheres in each well of each passage were imaged with a zeiss microscope (HAL 100) and the number and diameter of spheres was quantified.
4) Cell sphere differentiation experiments were used to detect differentiation.
Much like the balling experiment. EdU was added to the culture medium on days 2, 3, and 4 of the culture to examine the proliferation of Lgr 5-positive inner ear stem cells.
5) The molecular mechanism of the Foxg1 gene participating in hair cell regeneration is explored by adopting the technologies of Q-PCR and the like.
Sixth, Effect of Shh Gene on proliferation and differentiation of Lgr5 inner ear Stem cells
1) And (3) digesting the single-cell suspension prepared from the basilar membrane of the mouse cochlea with positive Lgr5-EGFP-IRES-CreERT2, Lgr5-EGFP-CreERT2 and Foxg1-loxp/loxp, and sorting the inner ear stem cells expressing Lgr5 by using a flow cytometer for culturing.
2) Cell balling experiment
Shh recombinant proteins with different gradients are added, and then the number and the size of cell spheres are counted, so that the influence of the existence of the Shh gene on the cell sphere forming capacity is observed. The spheronizing capacity of Lgr5 positive cells from Foxg1 cKD was also observed to quantify their number and size.
3) Performing immunofluorescence staining
Myosin7a, Sox2, Foxg1, Ctbp2, PSD95 and Tuj-1, and EdU is added after blocking to detect the proliferation of Lgr5 expressing inner ear stem cells. Meanwhile, through differentiation experiments, the number of hair cells, the number of cell balls, and EdU, Myo7a and Sox2 are observed to show positive changes of the number of hair cells.
4) The ABR test was performed on a mouse aged 30 days in a sound-proof room, and three fine needle electrodes were inserted into the cranial vertex, under the tested ear, and at the back near the tail of the mouse. ABR sound points of 4kHz, 8kHz, 12kHz, 16kHz, 24kHz, and 32kHz were generated. The threshold is determined by reducing the sound intensity from 90db in steps of 10db to the lowest sound intensity at which the first wave can be identified.
5) Cochlear explants were cultured and neomycin injury was performed.
The mouse is 2 days old and is positive to Sox2-CreERT 2; the R26SmoM2 transgenic mouse cochlea was isolated and cultured in PBS culture medium, and cultured in DMEM/F12 at 37 ℃ and 5% CO2 with the addition of 1% N2, 2% B27 and 50. mu.g/ml ampicillin, respectively. Cre activity was induced by 4-OH tamoxifen and cells were proliferated by EDU labeling. In the neomycin treatment group, the transplanted cochlear epithelial cells were treated with 0.5mM neomycin sulfate, and after 24 hours of culture, 0.5mM neomycin sulfate was added. Littermate cochlear explants without the SmoM2 allele were used as controls.
6) Total RNA was isolated from cochlea.
cDNA was synthesized by reverse transcription using 1. mu.g of total RNA as a template and the GoScriptTM reverse transcription system, and quantitative real-time PCR was performed.
In conclusion, after Wnt, Notch and Atoh1 signal channels are regulated, whether promoting effect on hair cell regeneration is achieved or not is researched, the regulation mechanism is understood, meanwhile, Lgr5-EGFP-CreERT2 tool mice are used for screening Lgr5+ progenitor cells, and Hippo signals are regulated to promote proliferation or differentiation capacity of inner ear progenitor cells, so that the methods for regulating the signal channels are applied to promoting the regeneration of viable cell hair cells. To determine the mechanism of involvement of Foxg1 in HC regeneration, tdTomato +/Sox 2+ stem cells were isolated by flow cytometry and mRNA was extracted for real-time quantitative qPCR to detect the expression levels of the relevant genes. Two transcription factors Atoh1 and Gfi1 mRNA for regulating HC generation are up-regulated, Notch signal pathways such as Notch 1-3, Hes1, Hes5, Jag2 and Hey1 are all obviously down-regulated, and other genes are not obviously changed. Knockout of Foxg1 therefore generates new HCS primarily by down-regulating cell cycle pathways and Notch signaling pathways. The mouse model of the application reflects the proliferation and regeneration conditions of hair cells, provides new evidence for Foxg1 to regulate HC regeneration of cochlear support cells and progenitor cells of newborn mice, and has important research significance. Therefore, the method for constructing a mouse model in this embodiment can be applied to the study of hair cell regeneration.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Claims (7)
1. A method for constructing a mouse model is characterized by comprising the following steps:
constructing mouse models of beta-catenin knockout, Notch1 knockout and double knockout of the beta-catenin knockout and the Notch1 knockout. Injecting tamoxifen and EdU when the age of a mouse is 0 or 1 day, dissecting a basement membrane of the mouse to perform an immunofluorescence experiment when the age of the mouse is 7 days, labeling hair cells with a myostatin 7a antibody, labeling support cells with a Sox2 antibody, and counting the cells positive to both EdU + Sox2 and EdU + Myo7 a;
separating the wild control group, beta-catenin overexpression (beta-cat OE), Notch1-KO and beta-cat OE/Notch1-KO mice basement membranes aged 7 days respectively, and extracting RNA of the mice basement membranes for sequencing;
constructing an Atoh1 over-expressed mouse, injecting tamoxifen, taking out a basement membrane from the mouse for an immunofluorescence experiment, marking hair cells with a myostatin 7a antibody, marking support cells with a Sox2 antibody, and counting the cells which are double positive to EdU/Myo7 a;
the Hippo signaling pathway was regulated by inhibiting YAP expression, and the spheronization assay analyzed the proliferative capacity of Hippo signaling pathway on Lgr 5-expressing progenitor cells.
2. The method for constructing a mouse model according to claim 1, further comprising the steps of:
separating and culturing inner ear stem cells expressing Lgr5 from a single cell suspension prepared by digesting a mouse cochlea basement membrane positive for Lgr5-EGFP-IRES-CreERT2, Lgr5-EGFP-CreERT2 and Foxg1-loxp/loxp, carrying out a cell balling experiment on the inner ear stem cells expressing Lgr5, and carrying out immunofluorescence staining to detect the proliferation condition of the inner ear stem cells expressing Lgr 5;
carrying out ABR detection on a mouse with the age of 30 days;
the mouse is 2 days old and is positive to Sox2-CreERT 2; the R26SmoM2 transgenic mouse cochlea is separated and cultured in PBS culture solution, neomycin injury is carried out, RNA is separated in the cochlea, and quantitative real-time PCR detection is carried out.
3. The method for constructing a mouse model according to claim 1, wherein the method for sorting inner ear stem cells expressing Lgr5 is a cell flow sorting method.
4. The method of constructing a mouse model according to claim 1, wherein the method of inhibiting YAP is: YAP expression was inhibited by verteporfin, dobutamine as YAP inhibitors.
5. The method for constructing a mouse model according to claim 2, wherein the experimental conditions for the ABR detection are as follows: inserting three fine needle electrodes into the cranial vertex of a mouse, the position below a tested ear and the position of the back close to the tail; generating ABR sound points of 4kHz, 8kHz, 12kHz, 16kHz, 24kHz and 32 kHz.
6. The method for constructing a mouse model according to claim 2, wherein the culture conditions of the cochlea are as follows: the cells were cultured in DMEM/F12 supplemented with 1% N2, 2% B27 and 50. mu.g/ml ampicillin at 37 ℃ under 5% CO2, respectively.
7. Use of the mouse model of any one of claims 1 to 6 for studying hair cell regeneration.
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