CN112243953B

CN112243953B - TfR1 lung tissue specific knockout mouse model and construction method and application thereof

Info

Publication number: CN112243953B
Application number: CN202011147609.3A
Authority: CN
Inventors: 郗艳丽; 徐斌; 左丽丽; 许娜
Original assignee: Beihua University
Current assignee: Beihua University
Priority date: 2020-07-15
Filing date: 2020-10-23
Publication date: 2022-02-11
Anticipated expiration: 2040-10-23
Also published as: CN112243953A

Abstract

TfR1 lung tissue-specific knockout mouse model, construction method and application thereof, belong to the field of mouse model construction, including: crossbreeding TfR1 ^‑/‑ and Sftpc‑CreERT2 mice; F1 generation TfR1 ^+/‑ ; Sftpc‑CreERT2 Transgenic mice and F1 generation TfR1 ^+/- transgenic mice are crossed to obtain F2 generation, and the genotypes are respectively TfR1 ^+/- ; Sftpc-CreERT2, TfR1 ^+/+ ; Sftpc-CreERT2 and TfR1 ^+/+ ; F2 generation TfR1 ^+/- ; Sftpc-CreERT2 transgenic mice were crossed to obtain F3 generation TfR1 ^-/- ; Sftpc-CreERT2 homozygous knockout mice. The invention provides a solid research basis for in-depth research on the role of TfR1 in lung tissue diseases and the pathogenesis of lung cancer.

Description

TfR1 lung tissue specific knockout mouse model and construction method and application thereof

Technical Field

The invention belongs to the technical field of mouse model construction, and particularly relates to a TfR1 lung tissue specificity knockout mouse model and a construction method and application thereof.

Background

Transferrin receptor 1(TfR1), also known as CD71, is a membrane protein that is essential for the uptake of iron by some cells, but is not required for the uptake of iron by all cell types. Transferrin (Tf) is a ligand of TfR1, and carries extracellular iron. Fe²⁺-Tf/TfR1 is internalized by receptor-mediated endocytosis and transported to the endosome, where iron is released from Tf and leaves the endosome through transmembrane proteins in a low pH environment. This transport is mediated by a divalent metal transporter (DMT1/SLC11A 2). The ingested iron can be directly utilized by heme or iron-sulfur clusters, and can also be stored in ferritin. Transferrin receptor 1(TfR1) is involved in the regulation of iron content in lung cancer cells. The research shows that the expression level of TfR1 is obviously increased in the lung cancer cells compared with the normal cells. TfR 1-expressing water is detected in human sarcoidosis, allergic pneumonia and interstitial lung diseaseA flat anomaly. In a pulmonary fibrosis model in mice, it was also confirmed that there was high expression of TfR1 in lung tissue. However, the regulatory mechanisms of TfR1 in the pathogenesis of lung disease, especially lung cancer, are not fully understood. Current functional studies on TfR1 in lung tissue have focused mainly on cellular experiments, which may be associated with the lack of stable animal models. Therefore, it is urgently needed to construct a stable animal model so as to better study the role and mechanism of TfR1 in lung tissues and the pathogenesis of lung cancer.

Disclosure of Invention

The invention aims to provide a TfR1 lung tissue specific knockout mouse model, a construction method and application thereof, so as to deeply research the action and mechanism of TfR1 in lung tissues and in the pathogenesis of lung cancer.

The technical scheme adopted by the invention for solving the technical problem is as follows:

the invention discloses a construction method of a TfR1 lung tissue specific knockout mouse model, which comprises the following steps:

step one, TfR1 is processed^-/-Hybridizing the transgenic mouse with a Sftpc-CreERT2 transgenic mouse to obtain F1 generations, and identifying that the genotypes are TfR1 respectively^+/-(ii) a Sftpc-CreERT2 and TfR1^+/-；

Step two, F1 is substituted for TfR1^+/-(ii) a Sftpc-CreERT2 transgenic mice and F1 generation TfR1^+/-Hybridizing the transgenic mice to obtain F2 generations, and identifying that the genotypes are TfR1^+/-；Sftpc-CreERT2、TfR1^+/+(ii) a Sftpc-CreERT2 and TfR1^+/+；

Step three, F2 is substituted into TfR1^+/-(ii) a Sftpc-CreERT2 transgenic mice are hybridized and identified to obtain F3 generation TfR1^-/-(ii) a Sftpc-CreERT2 homozygote gene knockout mice, i.e., TfR1 lung tissue-specific knockout mice models.

In a preferred embodiment, in step one, the genotype identification process for the F1 mouse is as follows:

(1)PCR

TfR1^-/-the genotype identification primer sequence of the transgenic mouse is as follows:

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

the genotype identification primer sequence of the Sftpc-CreERT2 transgenic mouse is as follows:

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

20. mu.L of the amplification reaction system, wherein ddH₂O7. mu.L, 2 XTaq Plus MasterMix 10. mu. L, DNA template 1. mu.L, primers 1. mu.L each;

reaction conditions are as follows: 3 steps are repeated for 35 cycles at 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min, the temperature is kept at 72 ℃ for 5min and 12 ℃;

(2) agarose gel electrophoresis

Carrying out electrophoresis on the PCR amplification product by 1% agarose gel;

TfR1^-/-gene knockout result judgment indexes: the mouse with the about 165bp band is a wild mouse, and the mouse with the about 310bp band is a transgenic mouse;

Sftpc-CreERT2 gene knock-out result judgment indexes are as follows: the wild mouse with the strip of about 327bp appears, and the transgenic mouse with the strip of about 210bp appears;

(3) the TfR1 genotype identification result is as follows: the mice of the F1 generation all show double bands of 165bp and 310bp, the mice of the F1 generation all are heterozygote mice, and the genotype is TfR1^+/-；

The identification result of the Sftpc-CreERT2 genotype is as follows: a part of F1 generation mice show double bands of 210bp and 327bp, are heterozygote mice, and have the genotype of TfR1^+/-(ii) a Sftpc-CreERT 2; the other part of F1 mouse shows 327bp band, is wild mouse, and has TfR1 genotype^+/-。

In a preferred embodiment, in step one, the genotype identification process for the F2 mouse is as follows:

(1)PCR

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

(2) agarose gel electrophoresis

(3) the identification result is as follows: the F2 generation mice comprise Sftpc-CreERT2 gene mice, and the genotype is TfR1^+/-(ii) a Sftpc-CreERT2 and TfR1^+/+(ii) a Sftpc-CreERT 2; the F2 generation mice also comprise wild mice, and the genotype is TfR1^+/+。

In a preferred embodiment, in step one, the genotype identification process for the F3 mouse is as follows:

(1)PCR

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

(2) agarose gel electrophoresis

(3) the identification result is as follows: f3 mouse includes TfR1^-/-Homozygote mouse with the genotype of TfR1^-/-(ii) a Sftpc-CreERT 2; f3 mouse includes TfR1^+/-Heterozygote mouse, genotype TfR1^+/-(ii) a Sftpc-CreERT 2; the F3 generation mice include heterozygote mice with the genotype of TfR1^+/-(ii) a The F3 mouse also includes TfR1^+/+(ii) a Sftpc-CreERT2 genotype mouse, TfR1^+/+Genotype mouse and TfR1^-/-A genotypic mouse.

The TfR1 lung tissue specific knockout mouse model is obtained by the construction method of the TfR1 lung tissue specific knockout mouse model.

The TfR1 lung tissue specificity knockout mouse model constructed by the construction method of the TfR1 lung tissue specificity knockout mouse model is applied to screening and preparing drugs for detecting and/or treating lung cancer.

The invention has the beneficial effects that:

the invention aims to construct TfR1 of TfR1 lung tissue specific knockout^-/-(ii) a Sftpc-CreERT2 transgenic mouse model. The genotype of the mouse is identified by a PCR method, the expression levels of TfR1 and Sftpc in the lung tissue of the mouse are detected by a western blotting method, and the distribution and the expression level of TfR1 in the lung tissue are detected by an immunohistochemical method. The results show that: the PCR detection result of the mouse shows that TfR1 is obtained^-/-(ii) a Sftpc-CreERT2 homozygous mouse; the Westernblotting detection result shows that TfR1^-/-(ii) a Sftpc-CreERT2 homozygote mouse lung tissue TfR1 expression level is significantly lower than wild type (WT, TfR1)^+/+)(P<0.05), while the expression level of Sftpc is significantly higher than that of WT (P)<0.05); immunohistochemical staining detection results show that the TfR1 of the WT mice is mainly distributed in type II alveolar epithelial cells and bronchial epithelial cells; at TfR1^-/-(ii) a Sftpc-CreERT2 homozygote mice have lung tissues which are mainly distributed in II type alveolar epithelial cells, and the expression level is lower than that of WT mice.

The invention successfully constructs TfR1 specifically expressed by TfR1 lung tissue^-/-(ii) a The Sftpc-CreERT2 homozygote mouse model provides a solid research foundation for the deep research of the role of TfR1 in the lung tissue diseases and the lung cancer pathogenesis.

Drawings

FIG. 1 shows the hybridization process of transgenic mice.

FIG. 2 shows the identification result of TfR1 genotype of F1 mouse.

FIG. 3 shows the identification result of the Sftpc-CreERT2 genotype of the F1 mouse.

FIG. 4 shows the identification result of TfR1 genotype in F2 mouse.

FIG. 5 shows the identification result of the Sftpc-CreERT2 genotype of the F2 mouse.

FIG. 6 shows the identification result of TfR1 genotype in F3 mouse.

FIG. 7 shows the identification result of the Sftpc-CreERT2 genotype of the F3 mouse.

FIG. 8 shows the results of western blotting assay for a given mouse.

Fig. 9 shows the results of immunohistochemical staining detection of TfR1 in a given mouse (200 ×).

FIG. 10 shows the effect of Bap on mouse longevity, gross observation of lung tissue and HE staining (400X).

FIG. 11Results of comparisons of total iron, TfR1, TfR2, Tf, Fn and hepcidin in the sera of groups of mice were obtained. Tfr1^-/-(ii) a Sftpc-CreERT2 transgenic mice compared to WT, P<0.05。

FIG. 12 shows the results of comparing the iron content in lung tissues of mice in each group. Tfr1^-/-(ii) a Sftpc-CreERT2 transgenic mice compared to WT, P<0.05。

FIG. 13 shows the results of Pierce staining (200X) of the indicated mice.

Figure 14 shows the identification of immunohistochemical staining for TfR1, Tf, TfR2, and Fn in the indicated mice (400 ×).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.

Materials (I) and (II)

1. Experimental mouse and breeding environment

Tfr1 for use in the invention^-/-(028363) and Sftpc-CreERT2(028054) transgenic mice were purchased from Jackson laboratories. Tfr1^-/-Mice have LoxP sites flanking exons 3-6 of the transferrin receptor 1(TfR1) gene. TfR1 is a transmembrane glycoprotein that can be involved in iron uptake by cells via receptor-mediated endocytosis. Sftpc-CreERT2 mice were able to express Cre recombinase from the endogenous promoter/enhancer element of the Sftpc locus, which enzyme could be induced to be expressed by tamoxifen. Cre activity was observed in type ii alveolar epithelial cells when induced by tamoxifen. The Sftpc gene encodes lung-associated surfactant protein C, which is highly expressed in type II alveolar epithelial cells. These Sftpc-CreERT2 transgenic mice harbored IRES-Cre/ERT2 in the 3' UTR of the Sftpc gene. When crossed with mice containing LoxP site flanking sequences, progeny can be induced with tamoxifen, resulting in targeted deletion of the flox sequence in Cre-regulated type ii alveolar epithelial cells. Tamoxifen treated adultsCre recombinase activity is detectable in approximately 85% of lung epithelial cells in mice.

The mice are raised in an SPF-level clean environment, the temperature is controlled to be 18-22 ℃, the humidity is 50-70%, and the automatic light control is carried out (12h light/12 h dark). The rat cage box, the feed and the padding are sterilized, and the drinking water is sterilized at high temperature and high pressure. The feeding chamber is disinfected every day to ensure that the feeding environment meets the environmental requirements of SPF animals, and the padding is replaced every 3 days.

2. Instrument and reagent

An Epoch multifunctional microplate reader (BioTek, usa); mastercyclernexus gradient PCR instrument (Eppendorf, germany); agarose gel electrophoresis apparatus (six instruments, Beijing); Mini-Protean electrophoresis tank, Mini Trans-Blot transfer tank and Gel Doc XR Gel imaging system (Bio-Rad, USA); niu inverted microscope (nikon, japan); DNA amplification Taq enzyme (Biotech, Inc., Cunningchu-Cheng-Koch); PCR primers (Shanghai Producer, Ltd.); rabbit Anti-TfR 1 monoclonal antibody, Rabbit Anti-Sftpc monoclonal antibody, Rabbit Anti-beta-actin, HRP-Goat Anti-Rabbit IgG, HRP-Goat Anti-Mouse IgG (Abcam USA); ECL luminescence (martian Proteintech); DAB color development liquid and immunohistochemical detection reagent (Beijing China fir gold bridge).

Second, construction method of TfR1 lung tissue specific knockout mouse model

1. The mouse hybridization protocol is shown in FIG. 1, taking 2 female TfR1^-/-The transgenic mouse and 1 male Sftpc-CreERT2 transgenic mouse are respectively hybridized to obtain F1 generation gene type TfR1^+/-(ii) a Sftpc-CreERT2 and TfR1^+/-Two kinds. Hybridizing the two genotype mice with 1 female mouse and 1 male mouse respectively to obtain the F2 generation genotype TfR1^+/-；Sftpc-CreERT2、TfR1^+/+(ii) a Sftpc-CreERT2 and TfR1^+/+Three kinds of the components are adopted. Taking the genotype of the F2 generation as TfR1^+/-(ii) a 1 female mouse and 1 male mouse of Sftpc-CreERT2 continue mating to obtain F3 generation genotype TfR1^-/-；Sftpc-CreERT2、TfR1^+/-；Sftpc-CreERT2、TfR1^+/+；Sftpc-CreERT2、TfR1^+/+、TfR1^-/-And TfR1^+/-A mouse.

2. Mouse genotype identification

(1) Toe genomic DNA extraction

The mouse toes were clipped, placed into numbered 1.5mL centrifuge tubes and the caps were closed. 0.5mL of lysis solution and 50. mu.L of proteinase K stock solution were added to each tube and the lid was closed tightly. The centrifuge tubes were placed in a thermostatted metal bath at 55 ℃ overnight. The next day the samples were placed at room temperature and centrifuged at 12000rpm for 10 min. The supernatant was poured into a 1.5mL Eppendorf tube, 1mL absolute ethanol (about 2 times the volume of the supernatant) was added, and after the cap was closed, the tube was shaken gently to see a flocculent precipitate. Centrifuge at 13000rpm for 15min, discard the supernatant. Adding 1mL of 70% ethanol, washing, centrifuging at 13000rpm for 10-15 min, removing supernatant, collecting precipitate, and airing at room temperature for 15 min. Adding 80-100 mu L of sterilized water into each tube, covering, fully dissolving DNA at 37 ℃ for 1h, and storing at-20 ℃ after the DNA is completely dissolved.

(2)PCR

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT。

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA。

20. mu.L of the amplification reaction was used.

Wherein ddH₂O7. mu.L, 2 XTaq Plus Master Mix 10. mu. L, DNA template 1. mu.L, primers 1. mu.L each.

Reaction conditions are as follows: 3 cycles of 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 1min, and 3 steps are repeated for 35 cycles, 72 ℃ for 5min, and 12 ℃ is maintained.

(3) Agarose gel electrophoresis

The PCR amplification product was electrophoresed through 1% agarose gel.

TfR1^-/-Gene knockout result judgment indexes: a wild-type mouse showed a band of about 165bp, and a transgenic mouse showed a band of about 310 bp.

Sftpc-CreERT2 gene knock-out result judgment indexes are as follows: a wild-type mouse showed a band of about 327bp, and a transgenic mouse showed a band of about 210 bp.

3. As a result:

(1) genotype identification of F1 mouse

F0 co-produced 6 offspring F1 under natural mating conditions, numbered 1-6(F1), and the corresponding genotype identification was performed on F1 generations, and the TfR1 genotype identification results are shown in FIG. 2. The identification result shows that the 1-6(F1) mice all show double bands of 165bp and 310bp, namely the F1 generation is all heterozygote mice (the genotype is TfR1)^+/-)。

The genotype identification of Sftpc-CreERT2 is carried out on the F1 generation, and the identification result is shown in figure 3. The identification result shows that in the mice No. 1-6(F1), the mice No. 1-3 (F1) all show double bands of 210bp and 327bp, and combine with the identification result of TfR1 genotype, namely the mice No. 1-3 (F1) are heterozygote mice (the genotype is TfR1)^+/-(ii) a Sftpc-CreERT2), while the 4-6 (F1) mice only show 327bp bands, i.e. the 4-6 (F1) mice are wild type mice (the genotype is TfR1)^+/-)。

Combining the identification results of FIGS. 2 and 3, the method continues to use No. 2-3 (F1) (genotype is TfR1)^+/-(ii) a Sftpc-CreERT2) and No. 5 (F1) (genotype is TfR1)^+/-) The mice were crossed and bred to obtain F2.

(2) Genotype identification of F2 mouse

After mice No. 2 and 3 in the F1 generation were freely mated with mice No. 5, 9 mice No. 7-15(F2) in F2 generation were symbiotically produced, and the F2 generation was genotyped, wherein the TfR1 genotyped result is shown in FIG. 4. The identification results showed that 8(F2), 12(F2) and 13(F2) all showed double bands of 165bp and 310bp, i.e., mice No. 8(F2), 12(F2) and 13(F2) were all heterozygote mice, and the remaining mice all showed bands of 165bp, indicating that mice No. 7 (F2), 9 (F2), 10 (F2), 11 (F2), 14 (F2) and 15(F2) were all wild-type mice.

The genotype of the F2 generation was identified by Sftpc-CreERT2, and the identification result is shown in FIG. 5. The identification results showed that mice nos. 7 (F2), 8(F2), 12(F2) and 13(F2) all showed double bands of 210bp and 327bp, i.e., mice nos. 7 (F2), 8(F2), 12(F2) and 13(F2) were Sftpc-CreERT2 gene mice, and mice nos. 9 (F2), 10 (F2), 11 (F2), 14 (F2) and 15(F2) did not show bands of 210bp, i.e., mice nos. 9 (F2), 10 (F2), 11 (F2), 14 (F2) and 15(F2) were all wild-type mice.

Combining the identification results of FIG. 4 and FIG. 5, the No. 8(F2), No. 12(F2) and No. 13(F2) mice are all Sftpc-CreERT2 gene mice (genotype is TfR1)^+/-(ii) a Sftpc-CreERT 2). The No. 7 (F2) mouse is an Sftpc-CreERT2 gene mouse (the genotype is TfR1)^+/+(ii) a Sftpc-CreERT 2). No. 9 (F2), No. 10 (F2), No. 11 (F2), No. 14 (F2) and No. 15(F2) were all wild type mice (genotype is TfR1)^+/+)。

(3) Genotype identification of F3 mouse

The genotype of TfR1 was selected in the F2 generation^+/-(ii) a The mice of Sftpc-CreERT2 continued to be hybridized to produce 48F 3 mice, numbered 16-48 (F3). Genotyping was performed for the F3 generation, with TfR1 genotyping results as shown in fig. 6. The identification results showed that 49 (F3), 51 (F3), 60 (F3) and 61 (F3) all showed a band of 310bp, i.e., these mice were all TfR1^-/-Homozygous mice. No. 50 (F3), No. 62 (F3) and No. 63 (F3) all showed double bands of 165bp and 310bp, i.e., these mice were all TfR1^+/-Heterozygote mice.

The genotype of the F3 generation was identified by Sftpc-CreERT2, and the identification result is shown in FIG. 7. The identification results show that No. 49 (F3), No. 51 (F3), No. 60 (F3) and No. 62 (F3) all show double bands of 210bp and 327bp, namely that No. 49 (F3), No. 51 (F3), No. 60 (F3) and No. 62 (F3) mice are Sftpc-CreERT2 gene mice. No. 50 (F3), No. 61 (F3) and No. 63 (F3) did not show a 210bp band, i.e., these mice were all wild type mice.

Combining the results of FIG. 6 and FIG. 7, the mice No. 49 (F3), No. 51 (F3) and No. 60 (F3) were all TfR1^-/-Homozygote mouse (genotype is TfR1^-/-(ii) a Sftpc-CreERT 2). No. 62 (F3) mouse is TfR1^+/-Heterozygote mice (genotype TfR1^+/-(ii) a Sftpc-CreERT 2). Both the No. 50 (F3) and No. 63 (F3) mice were heterozygote mice (genotype is TfR1)^+/-). Mouse No. 61 (F3) is a homozygote mouseThe factor type is TfR1^-/-)。

In addition, TfR1 was also identified in F3 generation mice^+/+；Sftpc-CreERT2、TfR1^+/+(WT) and TfR1^-/-A genotypic mouse.

Third, Western blotting detection

49 (F3) and 51 (F3) mice (both of which are TfR1 in genotype are selected^-/-(ii) a Sftpc-CreERT2), the mice are given 75mg/kg tamoxifen corn oil solution for intraperitoneal injection, 1 time is given every day, and 3 days and WT (the genotype is TfR1)^+/+) Mice served as a control group and were given the same dose of tamoxifen i.p. corn oil solution. The mice were weighed daily and did not change significantly in body weight. And 4d, after the mice are anesthetized by urethane, opening the chest cavity, picking lung tissues, extracting lung tissue proteins by a conventional molecular biology method, determining the protein content by a BCA method, separating protein samples by SDS-PAGE, transferring the protein samples to a PVDF membrane, and sealing by 5% skimmed milk powder. Adding TfR1(1:5000), Sftpc (1:5000) and beta-actin (1:10000) antibodies respectively, incubating overnight at 4 ℃, adding secondary antibody of corresponding species, and incubating for 1h at room temperature. Washing the membrane for 3 times by TBST, 10min each time, detecting protein bands by ECL luminous liquid, photographing by a gel imaging system and carrying out gray level analysis.

The Westernblotting identification result is shown in FIG. 8, under the condition of consistent beta-actin, the genotype of the 49 (F3) mouse and the 51 (F3) mouse is TfR1^-/-(ii) a Sftpc-CreERT2, the expression level of mouse TfR1 protein was significantly reduced (as shown in fig. 8A and 8B), and it was evident that the knockout genotype remained intact in 4 generations of mice compared to WT mice. As shown in fig. 8C and 8D, results for Sftpc showed that significant Sftpc bands were observed in mice # 49 (F3) and 51 (F3), which were evident in comparison to WT mice, demonstrating that the Sftpc gene remained stably inherited in 4 generations of mice. Through western blotting verification, the 4-generation mouse screening and mating can be determined, and Sftpc-CreERT2 and TfR1 are obtained^-/-In the parental mouse of (1), the target genotype of TfR1 was obtained^-/-(ii) a Sftpc-CreERT2, namely 49 (F3) mice and 51 good (F3) mice.

In conclusion, the Westernblotting detection result shows that TfR1^-/-(ii) a Sftpc-CreERT2 homozygote mouse lung tissue TfR1 expressionThe level was significantly lower than wild type (WT, TfR1)^+/+)(P<0.05), while the expression level of Sftpc gene is significantly higher than that of wild-type WT (P)<0.05)。

In the detection process, all data are processed by a statistical method, namely, SPSS16.0 software is used for data processing, all data are represented by means of a mean value plus or minus standard deviation (x plus or minus s), differences among groups are compared by One-way ANOVA, and a detection level alpha is 0.05.

Detection of TfR1 immunohistochemical staining

49 (F3) and 51 (F3) mice (both of which are TfR1 in genotype are selected^-/-(ii) a Sftpc-CreERT2), the lung tissue of the right upper lung of the mouse is taken and put in 4% paraformaldehyde overnight, and then conventional dehydration, embedding and slicing are carried out. Immunohistochemical staining protocol was performed according to kit instructions, a primary anti-dilution concentration of TfR1(1: 400). The distribution and expression level of the TfR1 in the lung tissue are observed under a microscope.

The result of TfR1 immunohistochemical staining is shown in FIG. 9, and the genotype is TfR1^-/-(ii) a In Sftpc-CreERT2 mouse No. 49 (F3), TfR1 protein expression in lung tissue was significantly weaker than in WT mice, and was observed in lung tissue sections of WT mice, with TfR1 distributed mainly in bronchial epithelial cells and alveolar cells. But at TfR1^-/-(ii) a In lung tissue of Sftpc-CreERT2 homozygote mice, TfR1 is mainly distributed in bronchial epithelial cells, and TfR1 in most II-type alveolar epithelial cells is knocked out.

Taken together, immunohistochemical results showed that TfR1 in WT mice was predominantly distributed in type ii alveolar and bronchial epithelial cells; at TfR1^-/-(ii) a In lung tissue of Sftpc-CreERT2 homozygote mice, TfR1 is mainly distributed in type II alveolar epithelial cells, and the expression level is lower than that of WT mice.

In conclusion, the invention successfully constructs TfR1^-/-(ii) a The Sftpc-CreERT2 transgenic mouse model provides a new animal model for clarifying the action mechanism of TfR1 in lung tissues, provides a reference model for researching the action mechanism of TfR1 in the lung cancer pathogenesis process, and provides a solid experimental foundation for searching a novel treatment target of lung cancer.

Application of TfR1 lung tissue specific knockout mouse model in lung cancer

1. Experimental materials and methods

(1) Lung cancer model construction and pathological histology detection

Bred TfR1^-/-(ii) a 10 Sftpc-CreERT2 homozygous male mice were given an intraperitoneal injection of tamoxifen at a dose of 75mg/kg, once a day, for 3 consecutive days. Further 10mg/kg of benzo [ a ]]Pyrene corn oil solution for thoracic injection, named as TfR1^-/-(ii) a Sftpc-CreERT2 (BaP). WT Male mice were given the same dose of tamoxifen intraperitoneal injection and benzo [ a ]]Pyrene corn oil solution was injected intrathoracic and designated WT (BaP). Injections were given 1 time per week for 7 weeks. Mice status and death were observed periodically. At the end of the experiment, the eyeballs were bled and the mice sacrificed, 3 mice per group were harvested for right upper lung tissue, fixed with 4% paraformaldehyde, embedded, sectioned, HE stained, and pathological changes of the mouse lung tissue were observed.

The results of the histopathological determination are shown in FIG. 10, and TfR1 after administration of Bap^-/-(ii) a Sftpc-CreERT2(BaP) and WT (BaP) mice had no significant difference in lifespan. This result indicates that Bap significantly affected mouse longevity (see fig. 10A). After administration of Bap, TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice had less pronounced lung tissue cancer masses than WT (BaP) mice (see FIG. 10B). Pathology detection shows that the lung tissues of WT (BaP) mice have obvious alveolar structure loss, a large amount of inflammatory cells infiltrate into the lung tissues, the lung is seriously congested, and obvious cancer cells can be seen in the lung tissues. Tfr1^-/-(ii) a Sftpc-CreERT2(BaP) mice had lung tissue with missing alveolar structure, but was not as severe as WT (BaP) mice, had numerous inflammatory cells also in lung tissue, had cancer cells in lung tissue, but was not as severe as WT (BaP) mice (see FIG. 10C).

(2) Determination of iron, Tf, Fn, hepcidin, TfR1 and TfR2 in mouse serum

The results are shown in FIG. 11, and TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice had significantly reduced levels of total iron, TfR1, Tf, and hepcidin in the serum as compared to WT (BaP) mice (P1)<0.05)。TfR1^-/-；Sftpc-CreERT2(BaThe Fn level in serum of P) mice is significantly lower than that of WT (BaP) mice (P)<0.05)。TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice have higher levels of TfR2 in serum than WT (BaP) mice, but the comparison difference is not statistically significant (P)>0.05)。

(3) Determination of total iron content in mouse lung tissue

The results are shown in FIG. 12, and TfR1^-/-(ii) a The iron content in the lung tissue of Sftpc-CreERT2(BaP) mice is significantly lower than that of WT (BaP) mice (P)<0.05)。

(4) Determination of TfR1, Tf, TfR2 and Fn in mouse lung tissue

The results are shown in FIG. 14, and TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice with TfR1 and Tf levels lower than those of WT (BaP) mice, TfR1^-/-(ii) a Fn levels in Sftpc-CreERT2(BaP) mice were significantly higher than those in WT (BaP) mice, TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice have TfR2 levels slightly lower than WT (BaP) mice.

(5) Pierss staining

The results are shown in FIG. 13, and TfR1^-/-(ii) a The non-heme iron content in lung tissue of Sftpc-CreERT2(BaP) mice was significantly lower than that of WT (BaP) mice. This result is consistent with the determination of iron content in lung tissue.

In summary, the following conclusions are drawn:

(1) BaP significantly affected mouse longevity. After administration of BaP, TfR1^-/-(ii) a Sftpc-CreERT2(BaP) mice had less pronounced lung tissue cancer masses than WT (BaP) mice. TfR1 plays an important role in inhibiting the canceration of mouse lung tissue.

(2)TfR1^-/-(ii) a The levels of total iron, TfR1, Tf and hepcidin in the serum of Sftpc-CreERT2(BaP) mice are significantly higher than those of WT (BaP) mice, and the level of Fn is significantly lower than that of WT (BaP) mice (P)<0.05). The result shows that the long-term inhibition of the expression of TfR1 by mouse lung tissues can cause iron metabolism disorder in the body. In addition, disturbances of iron metabolism in the mouse body may also be associated with BaP administration.

(3)TfR1^-/-(ii) a The iron content in lung tissue of Sftpc-CreERT2(BaP) mice was significantly lower than that of WT (BaP) type mice, which is consistent with the results of Pierce staining.

(5) TfR1 significantly decreased Tf and TfR2 elevation by BaP and decreased Fn levels by increased BaP following lung knockout.

The invention discloses a TfR1 lung tissue specificity knockout mouse model, a construction method and application thereof, and a person skilled in the art can realize the model by appropriately improving process parameters by referring to the contents in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.

Sequence listing

<110> Jilin medical college

<120> TfR1 lung tissue specific knockout mouse model and construction method and application thereof

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Claims

A method for constructing a TfR1 lung tissue specific knockout mouse model, which is characterized by comprising the following steps:

step one, TfR1 is processed^-/-Hybridizing the transgenic mouse with a Sftpc-CreERT2 transgenic mouse to obtain F1 generations, and identifying that the genotypes are TfR1 respectively^+/-(ii) a Sftpc-CreERT2 and TfR1^+/-；

Step two, F1 is substituted for TfR1^+/-(ii) a Sftpc-CreERT2 transgenic mice and F1 generation TfR1^+/-Hybridizing the transgenic mice to obtain F2 generations, and identifying that the genotypes are TfR1^+/-；Sftpc-CreERT2、TfR1^+/+(ii) a Sftpc-CreERT2 and TfR1^+/+；

Step three, F2 is substituted into TfR1^+/-(ii) a Sftpc-CreERT2 transgenic mice are hybridized and identified to obtain F3 generation TfR1^-/-(ii) a Sftpc-CreERT2 homozygote gene knockout mice, i.e., TfR1 lung tissue-specific knockout mice models.
2. The construction method according to claim 1, wherein in the first step, the genotype identification process of the F1 mouse is as follows:

(1)PCR

TfR1^-/-the genotype identification primer sequence of the transgenic mouse is as follows:

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

the genotype identification primer sequence of the Sftpc-CreERT2 transgenic mouse is as follows:

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

20. mu.L of the amplification reaction system, wherein ddH₂O7. mu.L, 2 XTaq Plus Master Mix 10. mu. L, DNA template 1. mu.L, primers 1. mu.L each;

reaction conditions are as follows: 3 steps are repeated for 35 cycles at 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min, the temperature is kept at 72 ℃ for 5min and 12 ℃;

(2) agarose gel electrophoresis

Carrying out electrophoresis on the PCR amplification product by 1% agarose gel;

TfR1^-/-gene knockout result judgment indexes: the mouse with the about 165bp band is a wild mouse, and the mouse with the about 310bp band is a transgenic mouse;

Sftpc-CreERT2 gene knock-out result judgment indexes are as follows: the wild mouse with the strip of about 327bp appears, and the transgenic mouse with the strip of about 210bp appears;

(3) the TfR1 genotype identification result is as follows: the mice of the F1 generation all show double bands of 165bp and 310bp, the mice of the F1 generation all are heterozygote mice, and the genotype is TfR1^+/-；

The identification result of the Sftpc-CreERT2 genotype is as follows: a part of F1 generation mice show double bands of 210bp and 327bp, are heterozygote mice, and have the genotype of TfR1^+/-(ii) a Sftpc-CreERT 2; the other part of F1 mouse shows 327bp band, is wild mouse, and has TfR1 genotype^+/-。
3. The construction method according to claim 1, wherein in the first step, the genotype identification process of the F2 mouse is as follows:

(1)PCR

TfR1^-/-the genotype identification primer sequence of the transgenic mouse is as follows:

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

the genotype identification primer sequence of the Sftpc-CreERT2 transgenic mouse is as follows:

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

20. mu.L of the amplification reaction system, wherein ddH₂O7. mu.L, 2 XTaq Plus Master Mix 10. mu. L, DNA template 1. mu.L, primers 1. mu.L each;

reaction conditions are as follows: 3 steps are repeated for 35 cycles at 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min, the temperature is kept at 72 ℃ for 5min and 12 ℃;

(2) agarose gel electrophoresis

Carrying out electrophoresis on the PCR amplification product by 1% agarose gel;

TfR1^-/-gene knockout result judgment indexes: the mouse with the about 165bp band is a wild mouse, and the mouse with the about 310bp band is a transgenic mouse;

Sftpc-CreERT2 gene knock-out result judgment indexes are as follows: the wild mouse with the strip of about 327bp appears, and the transgenic mouse with the strip of about 210bp appears;

(3) the identification result is as follows: the F2 generation mice comprise Sftpc-CreERT2 gene mice, and the genotype is TfR1^+/-(ii) a Sftpc-CreERT2 and TfR1^+/+(ii) a Sftpc-CreERT 2; the F2 generation mice also comprise wild mice, and the genotype is TfR1^+/+。
4. The construction method according to claim 1, wherein in the first step, the genotype identification process of the F3 mouse is as follows:

(1)PCR

TfR1^-/-the genotype identification primer sequence of the transgenic mouse is as follows:

TfR1^-/-F：TTCAGTTCCCAGTGACCACA；

TfR1^-/-R：TCCTTTCTGTGCCCAGTTCT；

the genotype identification primer sequence of the Sftpc-CreERT2 transgenic mouse is as follows:

Sftpc-CreERT2(1)：ACACCGGCCTTATTCCAAG；

Sftpc-CreERT2(2)：TGCTTCACAGGGTCGGTAG；

Sftpc-CreERT2(3)：CATTACCTGGGGTAGGACCA；

20. mu.L of the amplification reaction system, wherein ddH₂O7. mu.L, 2 XTaq Plus Master Mix 10. mu. L, DNA template 1. mu.L, primers 1. mu.L each;

reaction conditions are as follows: 3 steps are repeated for 35 cycles at 94 ℃ for 3min, 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min, the temperature is kept at 72 ℃ for 5min and 12 ℃;

(2) agarose gel electrophoresis

Carrying out electrophoresis on the PCR amplification product by 1% agarose gel;

TfR1^-/-gene knockout result judgment indexes: the mouse with the about 165bp band is a wild mouse, and the mouse with the about 310bp band is a transgenic mouse;

Sftpc-CreERT2 gene knock-out result judgment indexes are as follows: the wild mouse with the strip of about 327bp appears, and the transgenic mouse with the strip of about 210bp appears;

(3) the identification result is as follows: f3 mouse includes TfR1^-/-Homozygote mouse with the genotype of TfR1^-/-(ii) a Sftpc-CreERT 2; f3 mouse includes TfR1^+/-Heterozygote mouse, genotype TfR1^+/-(ii) a Sftpc-CreERT 2; the F3 generation mice include heterozygote mice with the genotype of TfR1^+/-(ii) a The F3 mouse also includes TfR1^+/+(ii) a Sftpc-CreERT2 genotype mouse, TfR1^+/+Genotype mouse and TfR1^-/-A genotypic mouse.
5. The construction method of claim 1, wherein the constructed TfR1 lung tissue specific knockout mouse model is applied to screening and preparing drugs for detecting and/or treating lung cancer.