CN113273545B

CN113273545B - Construction method of GPA, Espin and Ikzf2 mouse model

Info

Publication number: CN113273545B
Application number: CN202110194377.5A
Authority: CN
Inventors: 柴人杰; 陈蔚; 张莎莎; 程诚; 高下
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2021-02-20
Filing date: 2021-02-20
Publication date: 2022-08-16
Anticipated expiration: 2041-02-20
Also published as: CN113273545A

Abstract

The invention discloses a construction method of GPA, Espin and Ikzf2 mouse models, and belongs to the technical field of biology. A construction method of GPA, Espin and Ikzf2 mouse models is characterized by comprising the following steps: generating an Lgr5-CreER positive mouse, a GPA positive mouse and a tdTomato positive mouse, specifically expressing the GPA in cochlear support cells expressing Lgr5, injecting tamoxifen into the mice when the mouse ages for 3 days and 4 days, and dissecting the cochlea when the mouse ages for 10 days; marking Myo7a positive cells and tdTomato positive cells, and counting the number of the Myo7a positive cells and tdTomato positive cells; hair cell generation in the cochlea of the mouse was identified.

Description

Construction method of GPA, Espin and Ikzf2 mouse model

Technical Field

The invention relates to the technical field of biology, in particular to a construction method and application of GPA, Espin and Ikzf2 mouse models.

Background

According to data published by world health organization in 2019, the population of hearing disabilities is 4.66 hundred million, which accounts for about 6.1% of the total population. Hearing is important for maintaining daily activities of human beings, and hearing impairment can seriously reduce the life quality of human beings, and brings certain negative effects to patients, families and even the society.

The inner ear stem cells can regenerate hair cells, thereby achieving the repair of cochlear structure and function, and fundamentally restoring hearing. The search for means or substances for inducing capillary race regeneration has been an important research topic, and it is also important to provide a non-human animal model as a research and test platform for the topic.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a construction method of GPA, Espin and Ikzf2 mouse models.

The purpose of the invention can be realized by the following technical scheme:

a construction method of GPA, Espin and Ikzf2 mouse models comprises the following steps:

generating an Lgr5-CreER positive mouse, a GPA positive mouse and a tdTomato positive mouse, specifically expressing the GPA in cochlear support cells expressing Lgr5, injecting tamoxifen into the mice when the mouse ages for 3 days and 4 days, and dissecting the cochlea when the mouse ages for 10 days;

marking Myo7a positive cells and tdTomato positive cells, and counting the number of the Myo7a positive cells and tdTomato positive cells;

the generation of hair cells in the cochlea of the mouse was identified.

Optionally, further comprising the steps of performing patch clamp recordings of cochlea of mice of wild type and GPA aged 33 days; measuring calcium current by applying a voltage ramp to the cochlea and recording the resulting current;

whole cell membrane capacitance measurements were performed to quantify exocytosis of inner ear hair cells.

Optionally, further comprising, fixing the temporal bone of said mouse in paraformaldehyde and decalcifying in 0.5mM EDTA at room temperature; samples were cut into small pieces and immersed in blocking medium containing 10% donkey serum in PBS and 0.5% Triton-X100 at room temperature; the cochlear tissue was then incubated with Espin antibody at 4 ℃; after complete washing with 0.01M PBS, the samples were incubated with the corresponding Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H + L) secondary antibody for 1 hour at room temperature; labeling F-actin with a fluorescent dye;

the distribution of Espin under the membrane and in the cochlea was observed.

Alternatively, Lgr5 cree-positive, Atoh 1-OE-positive, and tdTomato-positive mice were generated as a control group of Lgr5 creer-positive, GPA-positive, and tdTomato-positive mice.

Alternatively, Espin and F-actin are co-immunoprecipitated.

Alternatively, vgout 3 and Prestin were used to identify newly generated hair cells in Lgr5-GPA-Tom mice.

The mouse model construction method is applied to research on inner ear cell regeneration.

In addition, the invention also provides application of GPA, Espin and Ikzf2 genes in hair cell regeneration.

Drawings

The invention will be further described with reference to the accompanying drawings.

FIG. 1 is a graph showing the generation of new hair cells in the inner and outer hair cell regions by induction of GPA expression in the construction method of the present application, which can effectively convert Lgr5 positive support cells into new hair cells in the inner and outer hair cell regions. In the figure, the group A is that three-day or four-day mice are injected with tamoxifen, and the mice are dissected when the age of the mice reaches ten days to obtain cochlea epithelium. Images of cochlear epithelium from Lgr5iCreER positive tdTomato positive, Lgr5iCreER positive tdTomato positive Atoh1 positive and Lgr5iCreER positive tdTomato positive GPA positive mice are shown in group B-I. Group G is statistics of cochlear Myo7a positive tdTomato positive cells of these three groups.

FIG. 2 is a graph showing that expression of GPA in the construction methods of the present application promotes the generation of new hair cells in Lgr5 positive progenitor cells. Expression of GPA promotes maturation of new hair cells in Lgr5 positive progenitors, in which both the inner hair cell-specific end marker vgout 3 and the outer hair cell-specific end marker Prestin are expressed. Panel a shows cochlear epithelium dissected at mouse age up to 33 days after tamoxifen injection at P3 and P4. Panel B-C is images of Prestin positive neohair cells transferred in Lgr5 creer positive GPA positive tdTomato positive mice. D-F: images of Otof-positive and vgout 3 new hair cells at cochlear transit in Lgr5 icarer-positive GPA-positive tdTomato-positive mice. Group G are triple immunofluorescence images of Myo7a, Tuj1 and tdTomato in Lgr5 icarer positive GPA positive tdTomato positive mouse cochlea.

FIG. 3 shows that functional neoinner hair cell production occurs in GPA-expression-inducing Lgr 5-positive progenitor cells constructed according to the methods of the present application. Functional neoinner hair cells were generated from Lgr 5-positive progenitor cells that induced GPA expression. In the figure, group A is a group in which tamoxifen is injected into mice three or four days old, and cochlear epithelium is obtained by dissection when the mice age reaches 33 days. Group B are phase contrast and differential interference contrast images and fluorescence images of new inner hair cells in cochlea of the top circle of an Lgr5-CreER positive GPA positive tdTomato positive mouse. Newly generated inner hair cells were identified by their tdTomato fluorescence, one of which was patch-clamp recorded with a pipette with visible phase difference and differential interference contrast. Group C is representative calcium current (ICa) of control group (WT) and new inner hair cells (GPA) induced by voltage ramps applied under voltage clamps. D: representative exocytosis ("Δ" "C" _ "m") of control (WT) and new inner hair cells (GPA) was induced by a progressive depolarization of 200 ms. The E-F groups are the combined data of ICa and "Δ" "C" _ "m" in control (WT) and neoinner hair cells (GPA). The G-H group is a triple immunofluorescence image of Ctbp2, Myo7a and tdTomato in Lgr5iCreER positive GPA positive tdTomato positive mouse cochlea. Both the confocal images (G) and the 3D reconstruction (H) show new and native inner hair cells. Group I is the quantification of Ctbp2 positive points for each inner hair cell.

Fig. 4 is the distribution of Espin of the present application under the membrane and in the cochlea of mice. Group A is a confocal image of espin and F-actin in outer and inner hair cells of mice 14 days old. Group B are confocal and stimulated emission depletion fluorescence microscopy images of Espin and F-actin in outer and inner hair cells of the boxed area shown in group A. Group C is a partially enlarged image of the square region shown in group B.

Fig. 5 shows the distribution of Espin in different areas of cochlea in the construction method of the present application. Group a is a representative confocal image of espin and F-actin in apical, and basolateral transition of cochlea from a mouse aged 14 days. Group B are confocal and stimulated emission depletion fluorescence microscopy images of Espin and F-actin in inner hair cells at the top, middle and bottom corners of the cochlea. The intensity curve of the corresponding dashed line is shown on the right.

FIG. 6 is a schematic diagram showing how actin filaments are bound in cilia by Espin in the construction method of the present application. Group A are representative confocal images of Espin-mNeon Green, F-actin and DAPI in Espin-mNeon Green transfected cells. White asterisks indicate untransfected cells surrounding the transfected cells. Group B are representative confocal images of Espin-HA and F-actin in Espin-HA transfected cells. The bottom right bright field image shows a single in the capture areaA cell. White asterisks indicate untransfected cells surrounding the transfected cells. Group C was co-immunoprecipitated with Espin and actin. Human HEK293T cells were transfected with FLAG, Actb-FLAG or Espin-HA constructs. Immunoprecipitation was performed with FLAG antibody, and then western blotting was performed using HA antibody to detect the co-expressed protein. Representative images from three independent experiments are shown. Group D are representative confocal images of Espin-HA and F-actin in Espin-HA transfected cells, with the white square region on the right magnified. The dashed lines are numbered and show the corresponding intensity curves along the dashed lines. Group E is the transmission electron microscope image of the bulk microscope and its magnification. Red arrows indicate actin-membrane junctions. Group F is a representative stimulated emission depletion fluorescence microscopy image with magnification of stereoscopic impression co-labeled with Espin and F-actin.Yellow arrowRepresenting the point of the rotor between the edge of the actin filament and the membrane. Group G is representative amplified stimulated emission depletion fluorescence microscopy images of hair cells at different developmental stages after birth, labeled with Espin and F-actin together. And the group H is a functional model of Espin in stereoscopic vision.

Fig. 7 is a distribution of Espin in cochlea in the control group of the present application. Group A are confocal images of Espin and F-actin in Corti's apparatus from adult Brg 1-negative and Atoh1-Brg 1-negative mice. Fluorescence microscopy images of stimulated emission depletion of Espin and F-actin in OHC and IHC of adult Brg 1-negative and Atoh1-Brg 1-negative mice in group B. The area represented by the white closed curve shows the skin panel.

Fig. 8 is a graph of Espin distribution in cochlea in outer hair cells of mice of the control group of the present application. Group A in FIG. 8 are representative scanning electron microscope images of Corti organs from adult Brg1 negative and Atoh1-Brg1 negative mice. Group B are representative confocal images of Corti's apparatus from adult Atoh1-Brg1 negative mice. The number IV indicates different morphologic outer hair cell damage. White triangles indicate that adult Atoh1-Brg1 negative mice occasionally lacked inner hair cells. Group C are stimulated emission depletion fluorescence microscopy images of adult Atoh1-Brg1 negative Espin and F-actin under different outer hair cell morphologies. Scale bar: 1 μm.

FIG. 9 is a graph of the synergy of GPA, Espin, and Ikzf2 in mice of the present application to promote new outer hair cell generation. Mice aged 0 days were injected with AAV-ie-GPA, Espin, and Ikzf2, and the cochlea was harvested at 30 days of mouse age. In fig. 9, group a is a representative confocal projection image of GPA or GPA positive Espin positive Ikzf2 injected cochlea. Group B is the number of Myo7a positive neohair cells per 100 μm of sensory area.

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.

In some embodiments, the invention relates to the construction of a mouse model, as shown in FIGS. 1-9.

First, the Effect of GPA expression on Lgr5 Positive support cell transformation

(1) Lgr5-CreER positive, GPA positive and tdTomato positive mice were generated, GPA was specifically expressed in Lgr5 positive cochlear SC, while Lgr5 CreER positive, tdTomato positive and GPA negative mice were used as controls. Mice were injected with tamoxifen at 3 and 4 days of age and the cochlea was dissected at 10 days of age.

(2) Lgr5-CreER positive Atoh1-OE positive tdTomato positive mice were generated and named as Lgr5-Atoh1 group. Mice were injected with tamoxifen at 3 and 4 days of age and the cochlea was dissected at 10 days of age.

(3) Myo7 a-positive tdTomato-positive cells were counted and counted.

(4) Lgr5-GPA-Tom mice cochlear epithelial cells aged 33 days were dissected and a subset of newly generated hair cells in Lgr5-GPA-Tom mice was identified using vGlut3 and Prestin.

Second, function of GPA-induced Lgr 5-positive hair cells supporting cell switching was examined

(1) Fresh cochlea of wild-type and GPA mice aged 33 days was dissected and immersed in oxygenated extracellular solution. The tissue was visualized by a 60-fold water immersion objective in a vertical microscope and patch-clamp recorded by an EPC10/2 amplifier driven by a Patchmaster.

(2) The calcium current was measured by applying a voltage ramp from-90 mV of holding potential to positive 70mV over 0.3s and recording the resulting current.

(3) Whole cell membrane capacitance measurements were performed. The resulting current response is used to determine the full cell capacitance. The net increase of "C" _ "m" before and after stimulation ("Δ" "" C "_" m ") was used to quantify exocytosis of inner hair cells.

Thirdly, observing the distribution of Espin under membrane and in cochlea

(1) Mice were sacrificed with an excess of sodium pentobarbital (100mg/kg, i.p.) and the temporotemporal bone was dissected rapidly. Temporal bones were fixed in 4% paraformaldehyde (in PBS, pH 7.2) for 2 hours and decalcified in 0.5mM EDTA (pH 8.0) for 4-8 hours at room temperature. The samples were then cut into small pieces and immersed in blocking medium containing 10% donkey serum in PBS (pH 7.2) and 0.5% Triton-X100 for 2 hours at room temperature. Cochlear tissue was then incubated with Espin antibody overnight at 4 ℃. After complete washing with 0.01M PBS, the samples were incubated with the corresponding Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H + L) secondary antibody for 1 hour at room temperature. Phalloidin conjugated with a fluorescent dye (ATTO 488, ATTO-TEC) was used to label F-actin. Cochlear samples were fixed with Prolong Gold fixation medium.

(2) The distribution of Espin under the membrane and in the cochlea was observed with a confocal microscope.

(3) And (3) performing co-immunoprecipitation on Espin and F-actin. The cDNA sequence of interest was cloned into the vector from mouse cochlear cDNA. The C-terminal region of the target protein was linked to a 3x FLAG tag, a 3x HA tag or the fluorescent protein meneongreen for further co-immunoprecipitation or immunostaining in human HEK293T cells. Cells were homogenized in lysis buffer containing a mixture of protease inhibitors. The lysis buffer contained 50mM Tris, 120mM NaCl and 0.5% NP 40. The protein supernatant was incubated with Anti-FLAG M2 magnetic beads overnight at 4 ℃. After immunoprecipitation, protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose filters. The membrane was blocked with 5% nonfat dry milk for 2 hours at room temperature and incubated with primary antibody overnight at 4 ℃. The membrane was then incubated with HRP-conjugated secondary antibody for 2 hours at room temperature. The signal was visualized using enhanced chemiluminescence and captured on ImageQuant LAS 4000. Immunoprecipitation experiments were performed using rabbit anti-FLAG and rabbit anti-HA antibodies and repeated at least 3 times to verify the reproducibility of the data. The mixture was left at room temperature for 2 hours. Signals were visualized using enhanced chemiluminescence (ECL, Pierce, Rockford, IL) and captured on ImageQuant LAS 4000. Immunoprecipitation experiments were performed using rabbit anti-FLAG and rabbit anti-HA antibodies and repeated at least 3 times to verify the reproducibility of the data.

Fourthly, observing the distribution condition of Espin in cochlea of the deaf mouse

(1) The cochlea of the transgenic Atoh1-Brg1 negative mice was dissected, and Espin and F-actin in the cochlea were labeled with Espin antibodies and phalloidin, respectively, as in the immunofluorescence procedure described above.

(2) The distribution of Espin was observed under a confocal microscope and a scanning electron microscope.

Fifthly, the synergistic effect of GPA, Espin and Ikzf2 in vivo is observed

(1) Mice aged 0 days were injected with AAV-ie-GPA, Espin and Ikzf2, and the cochlea was collected at mouse age 30 days.

(2) The number and distribution of hair cells were observed under a confocal microscope.

By constructing the mouse model, it is possible to obtain:

(1) generating Lgr5-CreER positive; GPA is positive; tdTomato positive mice were controlled with Lgr5-CreER positive, tdTomato positive (GPA negative) mice, while GPA was specifically expressed in Lgr5 positive cochlear support cells. Mice were injected with tamoxifen three or four days old and the cochlea was dissected ten days old. Immunofluorescent staining of cochlear with hair cell marker Myo7a revealed that in the Lgr5-GPA-Tom group, a large number of tdTomato-positive Myo7 a-positive cells were found throughout the inner and outer hair cell regions of the cochlear turn, i.e. induction of GPA expression could effectively transform Lgr 5-positive support cells into new hair cells in the inner and outer hair cell regions. While Lgr5-CreER positive, Atoh1-OE positive, tdTomato positive mice (Lgr5-Atoh1 group) were generated and treated identically, the number of new HCs was found to be much smaller than in the Lgr5-Atoh1 group. These results indicate that co-regulation of GPA greatly increases the efficiency of Lgr5 positive support cells to generate hair cells.

(2) Lgr5-GPA-Tom mice cochlear epithelial cells aged 33 days in mice were dissected and the subtypes of newly generated hair cells in mice were identified using vGlut3 and Prestin. Lgr5 positive support cell-derived new hair cells inside inner hair cells were found to express vgout 3, while Lgr5 positive support cell-derived new hair cells in the outer hair cell region express Prestin.

(3) Patch-clamp recordings of newly generated hair cells were found to be able to respond to acoustically induced depolarization and to cause intracellular calcium elevation, an increase in the whole-cell capacitance of GPA cells indicates that they can release synaptic vesicles, and there is heterogeneity in synaptic vesicle maturation in these newly generated inner hair cells. Scanning electron microscopy results also showed normal hair bundles of newly transformed inner hair cells. These results indicate that newly transformed inner hair cells, which are co-regulated by GPA, are functional.

(4) Dissecting a cochlea of a mouse, respectively marking Espin and F-actin by using an Espin antibody and phalloidin conjugated with fluorescent dye (ATTO 488, ATTO-TEC), observing the distribution of the Espin under a confocal microscope, and finding that the Espin is expressed in the three-dimensional cilia of outer hair cells and inner hair cells, and the Espin and the F-actin in the hair cells have strong co-marking. Espin appears to distribute across the entire three-dimensional cilia sub-membrane under 100 x objective lens and 9 x physical magnification.

(5) Through co-immunoprecipitation of Espin and F-actin, it was found that Espin molecules can interact with actin and maintain the cytoskeleton and three-dimensional cilia of actin.

(6) The cochlea of the transgenic Atoh1-Brg1 negative mice was dissected, and Espin and F-actin in the cochlea were labeled with Espin antibody and phalloidin, respectively. The distribution of Espin in the stereocilia of Atoh1-Brg 1-/-mouse OHCs was shown under a microscope. Espin signals are not only detected in the stereocilia, but also accumulate in the epidermal plates in OHCs, which severely damage the cilia or denature. The results of the study indicate that Espin is involved in ciliated actin organization and support the hypothesis that Espin is associated with actin maintenance and normal cilia morphology.

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

1. A construction method of GPA, Espin and Ikzf2 mouse models is characterized by comprising the following steps:

hair cell generation in the cochlea of the mouse was identified.

2. The method for constructing the mouse models of GPA, Espin and Ikzf2 according to claim 1, further comprising the steps of performing patch clamp recording on the cochlea of a mouse of wild type and GPA, the mouse ages of which are 33 days; applying a voltage ramp to the cochlea and recording the resulting current;

3. The method for constructing a mouse model of GPA, Espin, Ikzf2 according to claim 1, further comprising the steps of: marking F-actin by taking the temporal bone of the mouse through a fluorescent dye;

the distribution of Espin under the membrane and in the cochlea was observed.

4. The method of constructing a mouse model of GPA, Espin, Ikzf2 according to claim 1, wherein Lgr5iCreE positive, Atoh1-OE positive and tdTomato positive mice are generated as a control group of Lgr5iCreER positive, GPA positive and tdTomato positive mice.

5. The method for constructing a GPA, Espin, Ikzf2 mouse model according to claim 3, wherein Espin and F-actin are co-immunoprecipitated.

6. The method for constructing the mouse model of GPA, Espin, Ikzf2 according to claim 1, wherein vigout 3 and Prestin are used to identify newly generated hair cells in Lgr5-GPA-Tom mice.

7. The use of the mouse model construction method of any one of claims 1 to 6 for studying inner ear cell regeneration.

8. An application of GPA, Espin and Ikzf2 genes in hair cell regeneration.