A method for constructing a mettl3 transgenic knockout mouse model

Abstract

The application relates to a construction method of a Mettl3 transgenic mouse model with specific knockout, enriches the understanding of the function and action mechanism of Mettl3 in mature pancreatic islet beta cells, and provides a new thought and target for the pathogenesis and treatment of diabetes.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a construction method of a Mettl3 knockout transgenic mouse model. BACKGROUND

[0002] In recent years, the prevalence of diabetes has been increasing year by year, and has become a global public health problem. Among them, type 2 diabetes (T2D) accounts for more than 90%, and population studies show that the prevalence of prediabetes in Chinese adults has exceeded 50%. In addition to insulin resistance, beta cell function failure is the main pathological basis for the occurrence and development of T2D. In the progression of T2D, islet beta cells mainly undergo the following two stages of pathological changes: (1) islet beta cells respond to metabolic stress, and secrete more insulin to maintain blood glucose stability; (2) when metabolic stress persists and cannot be alleviated, islet beta cells are transformed from compensation to decompensation, and islet beta cells will transdifferentiate or dedifferentiate, or even undergo apoptosis. Ultimately, islet beta cells fail, accelerating the progression of the disease. It is currently believed that genetic and environmental factors jointly cause T2D. Epigenetics mainly studies the mechanism of changes in gene expression without changing the DNA sequence. Targeting epigenetic regulation provides a new perspective for finding diabetes diagnosis and treatment targets. SUMMARY

[0003] The technical problem to be solved by the application is to provide a construction method of a Mettl3 knockout transgenic mouse model specific to islet beta cells.

[0004] The construction method of the Mettl3 knockout transgenic mouse model specific to islet beta cells provided by the application comprises the following steps:

[0005] Mettl3 flox / flox mice are mated to obtain heterozygous Mettl3 flox / + MIP-Cre mice; and then the heterozygous Mettl3 flox / + MIP-Cre mice are mated with Mettl3 flox / flox mice to obtain MKO-MIP mice.

[0006] RIP-Cre mice are mated with Mettl3 flox / flox mice to obtain heterozygous Mettl3 flox / + RIP-Cre mice; and then the heterozygous Mettl3 flox / + RIP-Cre mice are mated with Mettl3 flox / flox mice to obtain MKO-RIP mice.

[0007] The primer sequence used for PCR identification of the MKO-MIP mice is shown in SEQ ID NO. 1-4.

[0008] The MKO-RIP mouse is identified by PCR using primer sequences as shown in SEQ ID NO. 1-2, SEQ ID NO. 5-6.

[0009] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0010] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0011] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0012] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0013] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0014] The application discloses a specific Mettl3 knockout transgenic mouse model.

[0015] Beneficial effects

[0016] The application successfully constructs two kinds of beta cell specific Mettl3 knockout mouse models, although the MKO-RIP knockout efficiency is higher than that of the MKO-MIP, but the MKO-MIP does not appear weight difference and is more specific, and is more suitable for studying the influence of Mettl3 knockout on mature islet beta cells.

[0017] The application simultaneously constructs MKO-MIP and MKO-RIP two kinds of islet beta cell specific Mettl3 knockout mice by using two kinds of insulin promoter driven Cre mouse models, and studies the metabolic phenotype of the knockout mice, so as to explore the influence and specific molecular mechanism of Mettl3 on mature islet beta cells.

[0018] The application discloses a specific Mettl3 knockout transgenic mouse model. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1Construction of Mettl3 transgenic mice specifically knocked out in pancreatic beta cells; (A) Body weight monitoring of 8-week-old Mettl3 flox / flox mice and MKO-RIP mice (n=5); (B) Body weight monitoring of 8-week-old Mettl3 flox / flox mice and MKO-MIP mice (n=5); (C) Mettl3 flox / flox mice and MIP-Cre and RIP-Cre mice mating strategy diagram;

[0020] (D) mRNA levels of primary pancreatic islet cell identity genes in 10-week-old MKO-RIP mice and control mice (n=3); (E) mRNA levels of primary pancreatic islet cell identity genes in 14-week-old MKO-MIP mice and control mice (n=4); (F) Pancreatic immunofluorescence section staining of 10-week-old control mice, MKO-RIP mice and MKO-MIP mice, red represents Mettl3, green represents insulin, and blue represents DAPI; *p<0.05, **p<0.01. Data are presented as mean ± standard deviation;

[0021] Figure 2 Mice with beta cell-specific knockout of Mettl3 develop hyperglycemia and impaired glucose tolerance; (A) Fasting blood glucose levels of MKO-RIP mice and control mice (n=5); (B) Fasting blood glucose levels of MKO-MIP mice and control mice (n=5); (C) Intraperitoneal glucose tolerance test of 6-week-old MKO-RIP mice and control mice (n=5); (D) Intraperitoneal glucose tolerance test of 10-week-old MKO-MIP mice (n=5); (E) Intraperitoneal insulin tolerance test of 6-week-old MKO-RIP mice (n=5); (F) Intraperitoneal insulin tolerance test of 10-week-old MKO-MIP mice (n=5); * represents p<0.05, ** represents p<0.01, *** represents p<0.001;

[0022] Figure 3Knocking out Mettl3 specifically in β-cells decreased glucose-stimulated insulin secretion and insulin content; (A) Intraperitoneal glucose insulin release test of 8-week-old MKO-RIP mice and control mice (n=5); (B) Intraperitoneal glucose insulin release test of 12-week-old MKO-MIP mice and control mice (n=5); (C) Primary GSIS test of 48-week-old MKO-MIP mice and control mice (n=3); (D) Insulin content in pancreatic islets of 48-week-old MKO-MIP mice and control mice (n=4); (E) mRNA level of Ins1 and Ins2 genes in primary islets of 10-week-old MKO-RIP mice and control mice (n=3); (F) mRNA level of Ins1 and Ins2 genes in primary islets of 14-week-old MKO-MIP mice and control mice (n=4); * represents p<0.05, ** represents p<0.01, *** represents p<0.001. Data are presented as mean ± standard deviation;

[0023] Figure 4 Knocking out Mettl3 specifically in β-cells decreased the proliferation of pancreatic β-cells and promoted the apoptosis of pancreatic β-cells; (A) Pancreatic immunofluorescence section staining of 8-week-old MKO-RIP mice and control mice, green represents insulin, red represents Ki67, and blue represents DAPI; (B) Statistics of the percentage of Ki67-positive cells in insulin-positive cells in Figure A, insulin-positive cells > 2000 / mouse (n=4); (C) TUNEL staining of pancreatic tissue sections, statistics of the percentage of TUNEL-positive cells in insulin-positive cells, insulin-positive cells > 8000 / mouse (n=3); * represents p<0.05 compared with the control. Data are presented as mean ± standard deviation;

[0024] Figure 5 Knocking out Mettl3 specifically in β-cells affected β-cell differentiation; (A) Pancreatic immunofluorescence section staining of 8-12-week-old MKO-RIP mice and control mice, green represents insulin, red represents Gcg, Sst and Ppy respectively, and blue represents DAPI. (B) Statistics of the proportion of multiple hormone cells in Figure A, insulin-positive cells > 3000 / mouse (n=3). * represents p<0.05, *** represents p<0.001. Data are presented as mean ± standard deviation;

[0025] Figure 6Knocking out Mettl3 in β-cells affects β-cell identity gene expression; (A) mRNA levels of β-cell identity genes in primary islet cells from 10-week-old MKO-RIP mice and control mice (n=3); (B) mRNA levels of β-cell identity genes in primary islet cells from 14-week-old MKO-MIP mice and control mice (n=4); (C) Pancreatic immunofluorescence sections staining of 10-week-old MKO-RIP mice and control mice, green represents insulin, red represents Mettl3 and Pdx1 respectively, blue indicates DAPI; (D) mRNA levels of transdifferentiation-related genes in primary islet cells from 10-week-old MKO-RIP mice and control mice (n=3); * represents p<0.05 compared with control, ** represents p<0.01 compared with control. Data are presented as mean ± standard deviation;

[0026] Figure 7 Knocking out Mettl3 in β-cells leads to endoplasmic reticulum stress; (A) mRNA levels of insulin synthesis and endoplasmic reticulum stress-related genes in primary islets from 14-week-old MIP-Cre KO mice and control mice (n=4); (B) Xbp1 and Ire1a immunohistochemical staining of pancreatic sections from 10-week-old MKO-RIP mice and control mice; * represents p<0.05, ** represents p<0.01, *** represents p<0.001. Data are presented as mean ± standard deviation;

[0027] Figure 8 . The level of m6A modification of Ire1a is reduced after knocking out Mettl3: MeRIP qPCR detects the level of m6A modification of Ire1a in islets from 38-week-old Mip-Cre βKO mice and control mice (islets >3500). 329 and 788 are two m6A modification sites of Ire1a;

[0028] Figure 9 ERS model is induced in vitro by thapsigargin: 8-week-old wild-type mouse primary islets are treated with thapsigargin, and mRNA levels of endoplasmic reticulum stress-related genes (n=4); * represents p<0.05, ** represents p<0.01;

[0029] Figure 10 Inhibition of Ire1a expression can partially alleviate endoplasmic reticulum stress; (A) 8-week-old wild-type mice are treated with thapsigargin, and mRNA levels of endoplasmic reticulum stress genes after adding 4μ8c to inhibit Ire1a after treatment (n=3); (B) 8-week-old wild-type mice are treated with thapsigargin, and GSIS results after adding 4μ8c to inhibit Ire1a after treatment (n=3); * represents p<0.05, ** represents p<0.01. Data are presented as mean ± standard deviation;

[0030] Figure 11 To alleviate endoplasmic reticulum stress by overexpressing Mettl3; (A) 8-week-old wild-type mice were treated with Thapsigargin, and the mRNA levels of endoplasmic reticulum stress genes after infection with adenovirus overexpressing Mettl3 (n = 3); (B) 8-week-old wild-type mice were treated with Thapsigargin, and GSIS test after infection with adenovirus overexpressing Mettl3; * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. Data are expressed in the form of mean ± standard deviation;

[0031] Figure 12 Genotyping of tail DNA of Mettl3flox / flox, MIP-Cre and RIP-Cre carrying mice;

[0032] Figure 13 To construct a map of MIP-Cre mice. DETAILED DESCRIPTION

[0033] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught by the application, and these equivalent forms also fall within the scope of the appended claims of the application.

[0034] Construction of MIP-Cre mice:

[0035] The Ins1-Cre-Dsred transgene is assembled from an 8.5 kb fragment of the mouse Ins1 promoter (Ins1), a nuclear localization signal fused to a Cre gene fragment, an internal ribosome entry site (IRES) sequence and a Dsred T3 cDNA fragment, and a polyadenylation signal.

[0036] Mettl3 flox / flox Mice were provided by Shanghai Institute of Immunology.

[0037] Example 1

[0038] 1. Construction of mouse model:

[0039] 8-week-old mature male MIP-Cre (genotype: Mettl3 + / + MIP-Cre + ) or RIP-Cre (genotype: Mettl3 + / + RIP-Cre + ) mice were mated with 8-week-old mature female Mettl3 flox / flox Mice to obtain heterozygous Mettl3flox / + MIP-Cre (genotype: Mettl3 flox / + , MIPCre + ) and heterozygous Mettl3 flox / + RIP-Cre (genotype: Mettl3 flox / + , RIPCre + ) mice. Then, 8-week-old mature male heterozygous Mettl3 flox / + MIP-Cre and heterozygous Mettl3 flox / + RIP-Cre mice were mated with 8-week-old mature female Mettl3 flox / flox mice, resulting in MKO-MIP (genotype: Mettl3 flox / flox , MIP-Cre) and MKO-RIP (genotype: Mettl3 flox / flox , RIPCre) mice, which were the experimental groups. Homozygous Mettl3 flox / flox mice were the control group.

[0040] 2. Genotype identification of transgenic mice:

[0041] (1) Extraction of mouse genomic DNA;

[0042] (2) PCR of mouse genomic DNA

[0043] The primer names and sequences are as follows:

[0044]

[0045] The PCR reagents and reaction system (total volume 12 μl) are as follows:

[0046] Name Volume (μl) DNA template 0.5 Primer F 0.15 Primer R 0.15 3d H2O 5.45 Taq enzyme 6.25

[0047] The PCR program is as follows:

[0048]

[0049] After the PCR, agarose gel electrophoresis was performed, as shown in Figure 12 .

[0050] 3. General observation of mice and related experiments of sugar metabolism:

[0051] 3.1 General observation of mice: From the birth of mice, the birth weight, feeding condition and random blood glucose of mice were observed, and the body weight was weighed once a week at a fixed time;

[0052] 3.2 Related experiments of sugar metabolism:

[0053] (1) Intraperitoneal glucose tolerance test (IPGTT) (2) Intraperitoneal insulin tolerance test (ITT) (3) Insulin release test (IRT) (4) Glucose-stimulated insulin secretion test (GSIS):

[0054] 4. Mouse islet insulin content determination;

[0055] 5. Mouse islet morphological analysis: pancreas tissue sampling; pancreas section immunofluorescence staining; pancreas section TUNEL staining;

[0056] 6. Islet total RNA extraction, reverse transcription and real-time quantitative PCR.

[0057] All quantitative variables are expressed as mean ± standard deviation, and analyzed using Graph Pad (Version 8) software. Unless otherwise specified, Student's t test was used to compare the differences between two groups of variables, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and it is considered that p < 0.05, the difference between the two groups is statistically significant.

[0058] Results:

[0059] Two mouse models of Mettl3 knockout in mature pancreatic beta cells were obtained by Cre-LoxP recombination system driven by two insulin promoters Ins1 and Ins2. RIP-Cre uses the rat insulin 2 promoter, although the knockout efficiency of the corresponding gene in the islet is high, but there is leakage in the brain, which will affect the judgment of the metabolic phenotype of the knockout mice. And through observation, MKO-RIP mice appear low weight Figure 1 A), in order to avoid other factors interfering with the judgment of the metabolic phenotype, Mettl3 knockout mice were constructed using MIP-Cre (Ins1-Cre), and MKO-MIP mice did not appear weight difference Figure 1 B). Two mouse models of Mettl3 knockout in mature pancreatic beta cells were used to explore the effect of Mettl3 on mature pancreatic beta cells. The breeding strategy of mice was applied as shown in Figure 1 C. Mettl3 flox / flox The control group was set as Mettl3 Figure 1 D-F), and the knockout efficiency of MKO-RIP mice was higher than that of MKO-MIP mice.

[0060] Phenotype of Mettl3 knockout mice in pancreatic beta cells related to glucose metabolism

[0061] First, the glucose metabolism-related phenotypes of the two Mettl3 knockout mice were observed. Compared with the control group, the fasting blood glucose level of MKO-RIP mice was significantly higher from 8 weeks of age ( Figure 2 A), while there was no significant difference in the fasting blood glucose of MKO-MIP mice ( Figure 2 B). Glucose tolerance test found that the glucose tolerance of MKO-RIP and MKO-MIP mice was significantly impaired ( Figure 2 C and D). Insufficient insulin secretion and insulin resistance can both lead to impaired glucose tolerance. To further explore the main reason for impaired glucose tolerance after Mettl3 knockout, whether there is peripheral insulin resistance was determined by insulin tolerance test, and the results showed that the insulin sensitivity did not change significantly ( Figure 2 E and F). It is indicated that the phenotype of hyperglycemia and impaired glucose tolerance caused by Mettl3 knockout is more likely to be caused by insufficient insulin secretion due to islet dysfunction or reduced islet beta cell volume, rather than peripheral insulin resistance.

[0062] Beta cell-specific knockout of Mettl3 reduces glucose-stimulated insulin secretion and insulin content in islets

[0063] To determine the specific reason for hyperglycemia and impaired glucose tolerance in Mettl3 knockout mice, glucose-stimulated insulin secretion (IRT) was used to find that the serum insulin level of MKO-RIP and MKO-MIP mice was significantly lower than that of the control group after glucose stimulation at the in vivo level ( Figure 3 A and 3B). In addition to the in vivo level, the insulin secretion capacity after glucose stimulation was also evaluated at the in vitro level. The primary islets of MKO-MIP mice were isolated, and in vitro glucose-stimulated insulin secretion (GSIS) was performed on the primary islets to evaluate the insulin release capacity. The results showed that after correcting the DNA concentration of islets, the insulin secretion of MKO-MIP mouse islets was significantly reduced under low and high sugar stimulation compared with the control group ( Figure 3 C). At the same time, the insulin content in the islets of MKO-MIP mice was also determined, and the experimental results showed that the insulin content in the islets of knockout mice was significantly reduced compared with the control group ( Figure 3 D). In addition, the levels of Ins1 and Ins2 in the islets of MKO-MIP and MKO-RIP mice were also significantly lower than those in the control group ( Figure 3 E and 3F). The above experimental results show that the hyperglycemia and impaired glucose tolerance in Mettl3 knockout mice are caused by reduced insulin content and secretion in islet beta cells.

[0064] Beta cell-specific knockout of Mettl3 reduces the proliferation, apoptosis and differentiation of islet beta cells

[0065] The changes of pancreatic islet β cell volume in MKO-RIP mice were detected. By pancreatic section immunofluorescence staining, it can be seen that compared with the control mice, the number of knockout mice islet is significantly less, the size of islet is significantly smaller, the percentage of Ki67 positive cells in insulin positive cells in pancreatic section is significantly reduced Figure 4 A and B); the percentage of TUNEL positive cells in insulin positive cells is significantly increased Figure 4 C). Among them, Ki67 is a cell proliferation marker, TUNEL is an apoptosis marker. In addition, the proportion of multiple hormone cells, that is, cells expressing other hormones in addition to insulin, is significantly increased in knockout mice Figure 5 A and 5B), which suggests that the islet β cells have a tendency of transdifferentiation. The above results show that specific knockout of Mettl3 in mature islet β cells not only reduces the proliferation of islet β cells but also promotes the apoptosis of islet β cells, and makes islet β cells appear transdifferentiation.

[0066] Specific knockout of Mettl3 in islet β cells inhibits insulin transcription and expression of β cell identity genes:

[0067] The results of pancreatic section immunofluorescence staining suggest that islet β cells appear transdifferentiation, and the change of β cell identity gene expression can lead to β cell transdifferentiation, dedifferentiation, etc. The expression level of β cell identity gene was observed, and it was found by qPCR Figure 6 A and 6B) and pancreatic section immunofluorescence staining Figure 6 C) and found that the expression levels of key identity genes such as Pdx1 that maintain the identity and function of β cells in the islets of knockout mice were significantly lower than those in the control group. In addition, the identity marker genes of other hormone cells such as α cells and pp cells were increased in the knockout mice Figure 6 D). These experimental results show that the reason for the transdifferentiation of β cells after knockout of Mettl3 is the down-regulation of key genes that maintain the identity and function of β cells, and the up-regulation of identity marker genes of other hormone cells.

[0068] Specific knockout of Mettl3 in islet β cells causes endoplasmic reticulum stress:

[0069] By determining the transcriptome level of endoplasmic reticulum stress related genes in the islets of MKO-MIP mice, it was found that compared with the control group, the transcriptome level of endoplasmic reticulum stress related genes in the knockout mice was significantly increased Figure 7 A). Further verified by pancreatic section immunohistochemical staining, Xbp1 and Ire1α staining was significantly deeper Figure 7B). Ire1a is one of the three stress sensing proteins of ERS, and Xbp1 is downstream of it. The above experimental results show that the mouse islet appears endoplasmic reticulum stress after Mettl3 knockout.

[0070] Mettl3 affects the expression of Ire1a in an m6A-dependent manner to cause endoplasmic reticulum stress

[0071] Endoplasmic reticulum stress occurs in a mouse model of Mettl3 knockout in pancreatic beta cells, and there are many reasons for causing endoplasmic reticulum stress. To determine whether it is due to the change of m6A modification mediated by Mettl3 and then affecting the corresponding gene to cause ERS. First of all, it is clear that compared with control mice, the expression level of Ire1a in the islets of knockout mice is increased Figure 7 A). At the same time, the MeRIP-qPCR experiment results show that the m6A methylation modification of Ire1a is reduced Figure 8 ). The above results show that Mettl3 knockout reduces the m6A level of Ire1a, which further leads to the decrease of the expression level of Ire1a, that is, Mettl3 affects the expression of Ire1a in an m6A-dependent manner.

[0072] Thapsigargin is a commonly used endoplasmic reticulum stress inducer. By treating primary islets with thapsigargin to induce endoplasmic reticulum stress, an in vitro ERS model is constructed, and the expression of endoplasmic reticulum stress related genes is verified to be increased Figure 9 ).

[0073] On this basis, 4μ8c, a specific inhibitor of Ire1a, was added to the culture medium, and it was found that inhibiting Ire1a could partially alleviate endoplasmic reticulum stress Figure 10 A), and can improve the ability of islet insulin secretion after glucose stimulation Figure 10 B). The above experiments show that by regulating the expression of Ire1a, the level of endoplasmic reticulum stress can be regulated, and the ability of insulin secretion after glucose stimulation of islets can be improved. Therefore, it can be preliminarily concluded that after Mettl3 knockout, the expression of Ire1a is increased in an m6A-dependent manner, causing ERS, and further damaging the insulin secretion ability of islet beta cells.

[0074] Overexpression of Mettl3 can alleviate ERS

[0075] If overexpression of Mettl3 can alleviate endoplasmic reticulum stress, it can be further proved that it is due to the endoplasmic reticulum stress caused by Mettl3 knockout. On the in vitro ERS model, overexpression of Mettl3 with adenovirus was found that compared with the control virus infection, the transcriptome levels of Xbp1 and Ire1a in the Mettl3 overexpression group were decreased, while the transcription level of insulin in the overexpression group was increased Figure 11 ). The above experiment shows that overexpression of Mettl3 can alleviate the changes caused by ERS, further proving that it is due to the endoplasmic reticulum stress caused by Mettl3 knockout, which is one of the reasons for the impairment of insulin synthesis and secretion function of pancreatic beta cells.

Claims

1. A method for constructing a mouse model of endoplasmic reticulum stress in pancreatic beta cells, characterized by, After knocking out Mettl3 specifically in pancreatic beta cells, the expression of Ire1α is increased through m6A-dependent manner, thereby causing endoplasmic reticulum stress; The construction method is: mating MIP-Cre mice with Mettl3 flox / flox mice to obtain heterozygous Mettl3 flox / + MIP-Cre mice; and then mating the heterozygous Mettl3 flox / + MIP-Cre mice with Mettl3 flox / flox mice to obtain MKO-MIP mice; the primer sequence used for PCR identification of the MKO-MIP mice is shown as SEQ ID NO. 1-4.

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