JP6855066B2 - Transgenic animals of non-human mammals that express human EGFR in a lung-specific manner - Google Patents

Description

In the present invention, human epidermal growth factor receptor (human epidermal growth factor receptor; also referred to as epidermal growth factor receptor, epidermal growth factor receptor, epidermal growth factor receptor); It relates to a disease control experimental animal represented by a transgenic (TG) mouse expressing (referred to as "human EGFR" or "h-EGFR").

Lung cancer is a malignant tumor that begins in the lungs. By the time lung cancer is discovered, it is often already fairly advanced and is known as an intractable disease. In Japan, the number of deaths from lung cancer is the highest among males and the third highest among females among various tumor patients.
As for the types of lung cancer, 20% are small cell lung cancer and 80% are non-small cell lung cancer. Of these, as a treatment method for small cell lung cancer, surgery or radiotherapy is used in the case of stage Ia, and chemotherapy (anticancer drug) is used in the subsequent cases. In addition, as a treatment method for non-small cell lung cancer, surgical treatment is used up to stage III, and chemotherapy is used after that. In recent years, molecular-targeted therapeutic agents (eg, EGFR tyrosine kinase inhibitors, antivascular endothelial cell growth factors, etc.) have been used as chemotherapeutic agents for non-small cell lung cancer. Molecular-targeted therapeutic agents are highly effective at the initial stage of administration. However, with long-term use, resistance develops in cancer cells, and the effect is not recognized. Currently, research on drug resistance of cancer cells is underway, but the mechanism has not been fully elucidated.

About half of all lung cancers have h-EGFR gene mutations. h-EGFR is a protein that penetrates the lipid bilayer and is a receptor that recognizes and signals epidermal growth factor (EGF). Normally, when h-EGFR is activated by EGF, the tyrosine kinase activity of h-EGFR increases, and by phosphorylating itself, it exerts various actions including cell proliferation as a normal reaction. However, mutations are often found in the h-EGFR gene of lung cancer patients. The h-EGFR gene mutations found in patients include L858R (leucine at position 858 mutated to arginine), T790M (sleoinin at position 790 mutated to methionine), and ΔE749-A750 (glutamic acid at position 749 and 750). Those lacking alanine in the rank) are known. Of these, L858R derived from the mutation located in the 21st exon (Ex21) is found in about 40% to 45% of lung cancer patients. H-EGFR ([L858R] -h-EGFR) with this mutation has increased tyrosine kinase activity, autophosphorylation, and excessive cell proliferation even in the absence of EGF. Therefore, a TG mouse having [L858R] -h-EGFR has been prepared and research on its properties has been conducted (Non-Patent Documents 1 and 2).
In addition, h-EGFR with the [T790M] mutation in the 20th exon (Ex20) has resistance to EGFR tyrosine kinase inhibitors.

Politi K et al. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down0-regulation of the receptors. Genes & Development 20: 1496-1510, 2006 Ji H, et al. The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cell. 2006 Jun; 9 (6): 485-95.

However, since the conventional [L858R] -h-EGFR TG mouse uses cDNA prepared from mRNA expressing the protein, it cannot be said that it sufficiently reflects the clinical condition. Therefore, a TG mouse showing an aspect closer to that of a human patient with lung cancer has been desired.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a TG mouse that expresses h-EGFR in a lung-specific manner and spontaneously develops lung cancer.

The transgenic animal according to the invention for achieving the above object is a human [L858R] epidermal growth factor receptor containing 28 exons (Ex) and 27 introns in the entire gene region downstream of the expression promoter region. It is a non-human mammal in which the body ([L858R] -h-EGFR) gene has been integrated.
In addition, the transgenic animal according to another invention is a human [L858R + T790M] epidermal growth factor receptor ([L858R)) containing the entire gene region of 28 exons and 27 introns downstream of the promoter region for expression. It is a non-human mammal in which the + T790M] -h-EGFR) gene has been integrated.
At this time, the non-human mammal is preferably at least one selected from the group consisting of mice, rats, pigs, sheep, horses, cows, dogs, cats, and monkeys.
In addition, the expression promoter region contains a tetracycline response element (TRE), and the non-human transgenic mammal further regulates reverse tetracycline downstream of the expression promoter of the organ-specific expression gene in the mammal. It is preferable that the sex trans activator (rtTA) gene is integrated.
The organ is the lung, and the organ-specific expression gene is a group consisting of Clara cell secretory protein (CCSP), surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC). It is preferably at least one selected from. Since these genes have been confirmed to be specifically expressed in the lung, at least one of these genes has been selected, and by using the expression promoter region, the rtTA gene incorporated downstream can be incorporated into the lung. Can be specifically expressed in.

Further, it is preferable that the non-human mammal is a mouse and administration of doxycycline causes lung cancer. At this time, the mouse strain is preferably C57BL / 6J.
The method for producing a transgenic mouse according to another invention is as follows: (1) Ex21 of the h-EGFR gene in BAC containing the h-EGFR gene containing the entire gene region of 28 exons (Ex) and 27 introns. Selection gene recombination step to obtain the selection gene-introduced h-EGFR gene by incorporating the selection cassette into (2) The genome sequence before and after Ex21 of the selection gene-introduced h-EGFR gene is used as a homologous sequence [L858R]. A modification step of placing at both ends of the mutation to prepare an EGFR transfer construct, (3) a replacement step of substituting the Ex21 selection cassette with a [L858R] mutant repair fragment of the EGFR transfer construct, (4) [L858R] -h. -Receptor expression gene preparation step as a receptor expression gene that links the expression promoter region to the 5'side of EGFR, (5) Purification of the receptor expression gene and m-SSCPrtTA RecBAC expression gene It is preferable to include a step of microinjecting a mouse embryo to obtain a transgenic mouse.

Further, the method for producing a transgenic mouse according to another invention is as follows: (1) In BAC containing the h-EGFR gene containing the entire gene region of 28 exons and 27 introns, the h-EGFR gene Ex21 was used. Selection gene recombination step of incorporating a selection cassette to obtain a selection gene-introduced h-EGFR gene, (2) [L858R] mutation using the genome sequences before and after Ex21 of the selection gene-introduced h-EGFR gene as homologous sequences Modification step of arranging at both ends of the gene to prepare an EGFR transfer construct, (3) Substitution step of replacing the selection cassette of Ex21 with the [L858R] mutant repair fragment of the EGFR transfer construct, (4) [L858R] -h- A gene preparation step for receptor expression, which is a gene for receptor expression that links the expression promoter region to the 5'side of EGFR, (4-1) Obtained in the gene preparation step for receptor expression [L858R]- Incorporating a selection cassette into Ex20 of h-EGFR and introducing a gene for selection [L858R]-Second-select gene recombination step to obtain the h-EGFR gene, (4-2) Introduction of the second-select gene [L858R] -The genome sequences before and after Ex20 of the h-EGFR gene are placed at both ends of the [T790M] mutation as homologous sequences, and the second modification step to prepare an EGFR transfer construct, (4-3) The cassette for selecting Ex20 is EGFR. By substituting with the [T790M] mutant repair fragment of the transfer construct (second substitution step), the gene for expression of the second receptor having the promoter region for expression on the 5'side of EGFR can be used for the expression of the second receptor. It is preferable to include a gene preparation step, (5) a step of purifying the second receptor expression gene and the m-SSCPrtTA RecBAC expression gene and microinjecting them into a mouse embryo to obtain a transgenic mouse.
At this time, the number of bases of the pair of primers for the PCR reaction used when preparing the selection gene-introduced h-EGFR gene is preferably 50 to 60 bases.

According to the present invention, it is possible to provide a TG mouse that expresses h-EGFR in a lung-specific manner and spontaneously develops lung cancer. Compared with the conventional model mouse, this TG mouse has a higher degree of malignancy and shows a state close to the clinical state of human lung cancer, so that research on lung cancer can be dramatically developed.

It is a conceptual diagram which shows the gene structure of h-EGFR. It is a figure which shows the mechanism that TG mouse develops lung cancer. It is a figure (1) explaining the method of making a lung-specific [L858R] -h-EGFR expression TG mouse. FIG. 2 is a diagram (2) illustrating a method for producing a lung-specific [L858R] -h-EGFR-expressing TG mouse. It is a figure which shows the result of having confirmed Ex21 in the BAC genome clone (RP11-815K24) containing the h-EGFR locus by the PCR method. The upper side is an image of the h-EGFR gene, and the lower side is a gel electrophoresis photograph after PCR. It is a figure which shows the progress and result when Ex21 of the WT-h-EGFR locus was deleted. [L858R] It is a figure which shows the construction method and result of the mutant gene donor construct. It is a figure which shows the construction method and result of the recombinant BAC type h-EGFR-tetO expression construct. It is a figure which shows the purification process and result of a high-purity BAC DNA fragment. It is a figure which shows the construction of CCSP_rtTA RecBAC using Red / ET Recombination Technology (Red / ET homologous recombination method). It is a figure which shows the sequence analysis of the construction of CCSP_rtTA RecBAC. It is a photographic figure which shows the result of making the lung-specific [L858R] -h-EGFR RecBAC gene-induced expression transgenic mouse founder, and performing Southern screening. It is a photographic figure which shows the result of genotyping of TG mouse. It is a photographic figure which shows the lung-specific expression of h-EGFR of TG mouse. It is a photographic figure which shows the result of having examined the lung of a TG mouse by CT. The arrow T in the figure indicates the site of occurrence / progression of lung cancer (the same applies to FIGS. 14 and 15). It is a photograph figure which shows one in FIG. 13 enlarged. It is a micrograph when the lung tissue of a TG mouse was stained by H & E. It is a micrograph of the lung tissue of a TG mouse when H & E staining is performed (showing infiltration into blood vessels by cancer). It is a micrograph when the lung tissue of TG mouse was stained with H & E (showing epithelial-mesenchymal transition of cancer cells and overexpression of mucin by cancer cells). It is a photographic figure which shows the lymph node metastasis by cancer. It is a graph confirming that PD-1 and PD-L1 are highly expressed in lung cancer tissue of TG mice. It is a figure explaining the production method of the lung-specific [L858R + T790M] -h-EGFR expression TG mouse. It is a figure which shows the progress and result when Ex20 of the [L858R] -h-EGFR locus was deleted. It is a figure which shows the result of the recombinant BAC type [L858R + T790M] -h-EGFR-tetO expression construct. It is a figure which shows the purification process and result of a high-purity BAC DNA fragment. It is a photographic figure which shows the result of making the lung-specific [L858R + T790M] -h-EGFR RecBAC gene-induced expression transgenic mouse founder, and performing Southern screening.

Next, embodiments of the present invention will be described with reference to figures and tables, but the technical scope of the present invention is not limited to these embodiments, and various embodiments without changing the gist of the invention. Can be carried out at.
A. Preparation and evaluation of [L858R] -h-EGFR-expressing TG mouse First, a TG mouse expressing [L858R] -h-EGFR in a lung-specific manner was prepared and its characteristics were investigated.
<Preparation of lung-specific h-EGFR-expressing TG mice>
1. 1. Method for producing TG mouse FIG. 1 shows the state of the gene structure of h-EGFR (particularly, the exon and intron regions). The h-EGFR gene contains 28 exons and 27 introns. According to a conventional research report, when producing a TG mouse expressing h-EGFR as a protein, a DNA to which only exons are bound may be prepared from a cDNA library and used (for example). , Non-Patent Documents 1 and 2 disclose TG mice produced by this method). On the other hand, it is being elucidated that RNA transcribed from an intron portion, which was conventionally thought to be junk, has an epigenetic effect (for example, action as siRNA or microRNA). Focusing on this point, the h-EGFR gene used in the TG mouse of the present invention contains all (28) exons and all (27) introns, and thus alternative splicing. Can occur. Alternative splicing is the production of multiple types of mature mRNA by changing the site / combination of gene splicing. That is, it means that many products (proteins) are produced from one gene. There are various types of alternative splicing, such as selection of splicing sites within exons, cassette exons, and intron retention. This alternative splicing produces mutant proteins called splice variants. It is said that 95% of genes with multiple exons undergo alternative splicing. Therefore, unlike the h-EGFR TG mouse prepared from normal h-EGFR cDNA, the TG mouse of the present invention may synthesize a mutant protein of h-EGFR that increases the malignancy of cancer. Is high. For this reason, it is possible that h-EGFR TG mice prepared from conventional cDNA do not fully represent the clinical status of human lung cancer.
Therefore, the present inventor used a method for producing a TG mouse using all exon introns of h-EGFR. FIGS. 2 to 4 show an outline of a method for producing a TG mouse that expresses [L858R] -h-EGFR in a lung-specific manner and develops lung cancer.

The TG mouse produced by the method of this embodiment incorporates two types of foreign genes. As shown in FIG. 2, one of the foreign genes ((A) in the lower part of the figure) is a predetermined substance (doxycycline (doxycycline), following the promoter of the gene (CCSP) specifically expressed in the mouse lung. Doxycycline): Has a reverse tetracycline-regulated transactivator (rtTA protein) that binds Dox) and a polyA tail (polyA). CCSP is a Clara cell secretory protein promoter. rtTA is a fusion protein of the tetracycline repressor (TetR) and the VP16 transactivation domain. In this fusion protein, four amino acids at the DNA binding site in TetR are altered, and in the presence of Dox, the tetO sequence in the TRE (tetracycine response element) of the target gene is recognized. In this system, the TRE-regulated target gene is expressed by rtTA only in the presence of Dox.
The other foreign gene ((B) in the upper part of the figure) has the [L858R] -h-EGFR gene and polyA downstream of the TRE. The TRE contains seven contiguous tetO operators (including a 10-base bacterial tet-O sequence and a spacer sequence) and a CMV (cytomegalovirus) promoter sequence (minimal promoter).
In TG mice in which two types of foreign genes have been integrated, the rtTA protein is constitutively expressed in the lung, and Dox is present in the body as shown by the broken lines (A) and (B) in FIG. Only if you do, dox-rtTA complex is generated. When it binds to tetO, it induces the expression of the [L858R] -h-EGFR gene. Thus, the presence of Dox provides a system that induces the expression of [L858R] -h-EGFR.
3 and 4 show a method of incorporating two kinds of foreign genes into mice. In FIG. 3, Step A is a method for producing a gene expressing [L858R] -h-EGFR ((B) in FIG. 2), and Step B is a gene expressing rtTA specifically for mouse lungs (Fig. 3). The method for producing (A) in 2) and the outline of the method for purifying high-purity DNA after linearizing each gene are shown in Step C. In FIG. 4, Step D is a method for producing a sperm incorporating a mixture of two types of genes, Step E is a male TG founder incorporating two types of foreign genes, and Step F and Step G are male TG founders. The outline of the method for producing TG mice (male F1 and female F1) from sperms obtained from the above was shown respectively. Details of each step will be described below.

2. Preparation of h-EGFR gene genome clone
tetO- [L858R] -h-EGFR RecBAC TG We analyzed the h-EGFR gene information, which is a target gene for producing mice, and obtained a BAC genome clone (RP11-815K24) containing the human EGFR locus from RIKEN. did. When this clone was screened by the PCR method, a band showing a target gene marker was observed as shown in FIG. From this, it was confirmed that this clone is the target BAC genome clone containing the target gene.
In addition, FIG. 5 (A) shows the position and image of Ex21 in the human wild-type (WT) EGFR gene, and FIG. 5 (B) shows the result of confirming the band of Ex21 amplified by the PCR method from the clone by electrophoresis. showed that.
Based on the genomic DNA sequence of the EGFR gene, PCR primers (SEQ ID NO: 1 and SEQ ID NO: 2) of the sequences shown in Table 1 were set in exon 21 of the EGFR gene. With this primer combination, using the purchased BAC clone as a template, LA-Taq (manufactured by Takara Bio Co., Ltd.) was used to perform PCR to amplify the genomic DNA fragment of Exon 21 of the EGFR gene, 490 bp, and the purchased BAC clone was obtained. It was confirmed that the clone contains the DNA sequence of the EGFR locus required for the construction of tetO-h [L858R] EGFR RecBAC.

3. 3. Construction of h-EGFR gene recipient construct A recombinant BAC that serves as a recipient for incorporating the [L858R] mutant gene into a genomic clone (RP11-815K24) containing the human EGFR locus using Red / ET Recombination Technology. I built a clone. (i) Insert a selection marker into Ex21 of the EGFR gene of the human BAC clone (Fig. 6 (A)), (ii) Delete Ex21 from the genomic region encoding the EGFR gene product of the human BAC clone, and tetO- An intermediate of [L858R] -h-EGFR RecBAC was prepared (selective gene recombination step. FIG. 6 (B)). FIG. 6C shows the result of confirming that the intermediate was produced by electrophoresis.
Using Red / ET Recombination Technology, which is an active homologous recombination reaction in E. coli, a recombinant BAC clone (tetO-h [L858R] EGFR RecBAC) with a [L858R] mutation was constructed from a BAC clone containing the EGFR gene. ..
As shown in Table 2, the 5'and 3'flanking sequences of the 5'-UTR genomic DNA sequence of the human EGFR gene, and the 5'and 3'flanking sequences of the [L858R] mutant gene in Escherichia coli. The key sequence for the active homologous recombination reaction was used (SEQ ID NO: 3 to SEQ ID NO: 6).

First, in order to insert a positive / negative selection marker cassette (Rpsl-kan) into the exon 21 region of the EGFR locus, a DNA fragment in which the EGFR-H3 sequence, Rpsl-kan, and EGFR-H4 sequences are tandemly linked is inserted. , LA-Taq (manufactured by Takara Bio Co., Ltd.) constructed by PCR (EGFR Rpsl-Kan break-in fragment). A human BAC clone containing the EGFR locus and an EGFR Rpsl-Kan break-in fragment are introduced into an Escherichia coli strain capable of the Red / ET reaction, and the Red / ET reaction is induced in this host Escherichia coli to induce chloramphenicol. By picking up resistant and canamycin-resistant colonies, recombinant BAC clones in which the EGFR Rpslkan break-in fragment was inserted into EGFR locus exon 21 were screened (tetO-h [L858R] EGFR RecBAC Intermediate).

4. [L858R] Construction of mutant gene donor construct
The genomic sequences before and after Ex21 at the EGFR locus were placed at both ends of the [L858R] mutation as homologous sequences to prepare an EGFR transfer construct for inserting the [L858R] mutation (modification step). 7 (A) and 7 (B) show a method for constructing a [L858R] transfer plasmid using TA cloning, and FIGS. 7 (C) and 7 (D) show electrophoresis of [L858R]. The result confirmed in is shown.
Using Red / ET Recombination Technology, which is an active homologous recombination reaction in E. coli, a tetO-h [L858R] EGFR RecBAC gene transfer plasmid was constructed from a DNA clone containing the tetO gene.
As shown in Table 2, the 5'and 3'flanking sequences of the 5'-UTR genomic DNA sequence of the human EGFR gene were used as key sequences for the active homologous recombination reaction in Escherichia coli.
First, in order to subclon the [L858R] mutant gene into a plasmid vector, the EGFR-H3 sequence, [L858R] mutant-H1, [L858R] mutant gene, [L858R] mutant-H2, and EGFR-H4 sequence are tandem. The DNA fragment connected to was amplified by PCR. The [L858R] mutant transfer plasmid was screened by simultaneously introducing a PCR fragment and a pGEM-T easy plasmid into a transforming E. coli strain and picking up ampicillin-resistant colonies.

5. Construction of recombinant BAC-type EGFR tetO expression construct
Using Red / ET Recombination Technology, a recombinant BAC clone in which the [L858R] mutant gene was inserted at the human EGFR locus was constructed. The selection marker inserted into Ex21 of the human EGFR recipient construct described above was accurately replaced and removed with the [L858R] mutant repair fragment (substitution step. FIGS. 8 (A) to 8 (C)). That is, at this stage, in order to insert the [L858R] mutant gene into the exon 21 region of the human EGFR locus, the previously constructed human EGFR recipient construct was applied to the Escherichia coli strain capable of the Red / ET reaction. [L858R] Mutant repair fragment was introduced. By inducing the Red / ET reaction in this host E. coli, picking up chloramphenicol and streptomycin-resistant colonies, and screening by colony PCR, the selection marker inserted into exon 21 at the human EGFR locus was expressed in [L858R. ] A tetO-h [L858R] EGFR RecBAC expression vector exactly substituted with a mutant repair fragment was identified.
When the DNA sequence at the junction of tetO- [L858R] -h-EGFR RecBAC was examined by sequence analysis, it was confirmed that the [L858R] mutant gene was inserted into the human EGFR locus as intended (receptor). Gene preparation step for expression. FIGS. 8 (D) and 8 (E).

6. Purification of high-purity BAC DNA fragments
The tetO- [L858R] -h-EGFR RecBAC recombinant BAC clone was purified, linearized with a restriction enzyme, and then subjected to pulsed field gel electrophoresis to purify a high-purity BAC DNA fragment. When the purity and concentration of the expression construct purified by agarose gel electrophoresis for analysis were examined, it was found that the pure long-chain expression construct could be recovered. The DNA concentration was 215.3 ng / μl, which was sufficient for the production of transgenic mice. In FIG. 9, (A) is a guide marker for analytical agarose gel electrophoresis, (B) is a band of linearized DNA fragments, and (C) is the result of examining the DNA concentration by HPLC. Indicated.

7. Construction of m-CCSP rtTA RecBAC expression construct As shown in Fig. 10, a genomic clone encoding the mouse CCSP locus was obtained using Red / ET Recombination Technology so that it could be in-framed in the coding sequence. A recombinant BAC clone with the rtTA_SV40 polyA sequence inserted was constructed. Mouse CCSP gene information was analyzed, and a BAC genome clone containing the mouse CCSP locus, RP23-223J21, was obtained from RIKEN. A selection marker is inserted into the translation initiation codon present in exon 1 of the CCSP gene of the mouse BAC clone, and (ii) the genomic DNA of the translation initiation codon in the genomic region encoding the CCSP gene product of the mouse BAC clone. The sequence was deleted to prepare an intermediate for CCSP_rtTA RecBAC. Next, (iii) the flaking sequence of the selection marker insertion site of the mouse CCSP gene is ligated at both ends of the rtTA_SV40 polyA sequence to prepare a CCSP_rtTA RecBAC repair fragment, and (iv) finally the translation existing in exon 1 of the mouse CCSP gene. The selection marker inserted at the start codon was removed by exactly replacing it with a CCSP_rtTA RecBAC repair fragment.
In other words, a recombinant BAC clone (CCSP_rtTA RecBAC) of the rtTA_SV40 polyA sequence was constructed from a BAC clone containing the CCSP gene using Red / ET Recombination Technology, which is an active homologous recombination reaction in Escherichia coli. As shown in Table 3, active homologous recombination of the genomic DNA sequence 5'and 3'flanking sequence of the translation initiation codon of the mouse CCSP gene and the 5'and 3'flanking sequence of the rtTA gene in E. coli. It was used as a key sequence for the reaction (SEQ ID NOs: 7 and 8).

As shown in FIG. 10, first, the CCSP-H3 sequence, Rpsl-kan, to insert a positive / negative selection marker cassette (Rpsl-kan) into the region of the translation initiation codon present at exon 1 of the CCSP locus, A DNA fragment in which the CCSP-H4 sequence was tandemly linked was constructed by PCR using LATaq (manufactured by Takara Bio) (CCSP Rpsl-Kan break-in fragment). A BAC clone containing the CCSP locus and a CCSP Rpsl-Kan break-in fragment are introduced into an Escherichia coli strain capable of the Red / ET reaction, and the Red / ET reaction is induced in this host Escherichia coli to induce chloramphenicol resistance. , And by picking up canamycin-resistant colonies, recombinant BAC clones in which the CCSP Rpsl-kan break-in fragment was inserted into the translation initiation codon present at exon 1 at the CCSP locus were screened (CCSP_rtTA RecBAC Intermediate). Then, in order to introduce the DNA sequence of the rtTA gene into this RecBAC Intermediate, a DNA cassette was constructed by connecting the flaking sequence of the selection marker insertion site of the mouse CCSP gene to both ends of the rtTA_SV40 polyA sequence in tandem (rtTA_SV40 polyA sequence repair fragment). ). In the same manner as above, an rtTA repair fragment is introduced into an E. coli strain capable of Red / ET reaction together with CCSP_rtTA RecBAC Intermediate, and the Red / ET reaction is induced in this host E. coli to generate ampicillin and streptomycin resistant colonies. By picking up, a recombinant BAC clone (CCSP_rtTA RecBAC) was screened in which the selection marker inserted into the translation initiation codon present in exon 1 of the mouse CCSP gene was accurately replaced by the CCSP_rtTA RecBAC repair fragment.
By the above four-step Red / ET reaction, CCSP_rtTA RecBAC could be constructed without damaging the exon / intron structure of the mouse CCSP locus and the expression control sequence of the CCSP locus. When the DNA sequence at the junction of the recombinant BAC clone of CCSP_rtTA RecBAC was examined by sequence analysis, it was confirmed that the rtTA_SV40 polyA sequence was inserted into the mouse CCSP locus as intended (Fig. 11).
Next, the cyclic recombinant BAC clone was linearized with a specific restriction enzyme, and the long-chain DNA was purified by pulsed-field gel electrophoresis.

8. Gene microinjection into C57BL / 6J mice Next, as described in detail, fertilized eggs were collected from C57BL / 6J mice, and two types of foreign genes (tetO- [L858R] -h-EGFR RecBAC / CCSP_rtTA RecBAC expression construct) was introduced. That is, pregnant horse serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were administered to female C57BL / 6J mice to induce hyperovulation, and after mating with male mice of the same strain, fertilized eggs were collected. Purified tetO- [L858R] -h-EGFR RecBAC / CCSP-rtTA RecBAC expression constructs were directly injected into the male nuclei of pronuclear stage embryos of C57BL / 6J mice using a micromanipulator. This DNA-injected embryo was transplanted into the oviduct of a pseudopregnant-induced female mouse (Fig. 4).
A fertilized egg into which an expression construct was introduced was transplanted into the oviduct of a mouse treated with pseudopregnancy. A total of 322 fertilized eggs were collected from C57BL / 6J female mice that were crossed by inducing hyperovulation. When the fertilized mouse eggs subjected to microinjection were observed under a microscope, only a few fertilized eggs died due to the damage caused by the injection operation. Of these fertilized eggs, 300 fertilized eggs that could be injected with the expression construct without damage could be transplanted into pseudopregnant mice.
Table 4 shows data on fertilized mouse eggs and finder that were microinjected, and Table 5 shows a list of pregnant mice transplanted with embryos.

9. Pulmonary-specific [L858R] -h-EGFR RecBAC gene-induced expression TG mouse founder's Southern screening fertilized eggs of C57BL / 6J mice introduced with tetO- [L858R] -h-EGFR RecBAC / CCSP_rtTA RecBAC expression construct by microinjection method From this, we obtained offspring that are candidates for the founder of TG mice, performed genotyping by Southern analysis, and identified individual TG mouse founders. When the expression construct was injected into a total of 322 fertilized eggs and a total of 300 injected embryos were transplanted into pseudopregnant mice for spontaneous delivery, a total of 43 mouse offspring were produced from the fertilized eggs injected with these genes. Got All 43 of these individuals were able to be raised until weaning. The number of offspring and weaning from the early embryo into which the tetO- [L858R] -h-EGFR RecBAC / CCSP-rtTA RecBAC expression construct was introduced was good. From this, it was considered that the injection of the expression construct did not adversely affect the development and differentiation of fertilized mouse eggs.
As shown in FIG. 12, the genomic DNA fragment extracted from the offspring, which is a candidate for the founder of TG mice, was hybridized with the [32P] labeled probe. 12 (A) and 12 (B) show the results of tetO- [L858R] -h-EGFR, and FIGS. 12 (C) and 12 (D) show the results of RecBAC / CCSP-rtTA, respectively. From this result, 5 TG mouse founder individuals (Nos. 11, 25, 27, 36 and 41 in FIG. 12) into which the tetO- [L858R] -h-EGFR RecBAC / CCSP-rtTA RecBAC expression construct was introduced were introduced. Identified. The copy numbers of the expression constructs introduced into these TG mouse founder individuals ranged from 1 to 3 copies.

10. Genotyping of TG mice
Genotyping of TG mouse founder individuals was performed using the PCR method. Table 6 shows the primers used (SEQ ID NO: 9 to SEQ ID NO: 12). As shown in FIG. 13, tetO- [L858R] -h-EGFR RecBAC / CCSP-rtTA RecBAC expression construct was observed in each of the TG mouse founder individuals.

11. Lung-specific [L858R] -h-EGFR expression (RT-PCR)
Table 7 shows the sequences of the primers used for RT-PCR (SEQ ID NOs: 13 and 14). Fertilized eggs were prepared from sperms obtained from the epididymis of TG mouse founder individuals (male) and unfertilized eggs obtained from C57BL / 6J mice (female). This fertilized egg was transplanted into the oviduct of a mouse treated with pseudopregnancy. TG mice (male and female first generation (F1)) were obtained from each mouse.

TG mice were fed a diet containing Dox to express [L858R] -h-EGFR in a lung-specific manner. It was confirmed by reverse transcriptase PCR (RT-PCR) that [L858R] -h-EGFR was expressed lung-specifically in TG mice. Total RNA was extracted from each tissue using TRIZOL (TRIZOL (Invitrogen, Carlsbad, CA)), and then RT-PCR was performed. The PCR reaction product was electrophoresed on a 2% agarose gel, the band was stained with ethidium bromide, and then observed with ultraviolet light. The results are shown in FIG. In the figure, A549 indicates a control cell having [L858R] -h-EGFR, and Gapdh indicates a housekeeping gene marker. In the wild type (WT), expression of [L858R] -h-EGFR was not observed, and in TG mice, expression of [L858R] -h-EGFR was observed in a lung-specific manner.

12. Expression of PD-1 and PD-L1 in lung cancer tissues In recent years, it has been found that cancer cells suppress the function of immunity and prevent the attack of immune cells. Therefore, a new treatment method has been considered in which the function of immune cells is reactivated and the cancer cells can be attacked by releasing the brake by the cancer cells. Among them, "immune checkpoint inhibitors" that block the braking part (the binding of PD-L1 and PD-1) called immune checkpoints are currently used in treatment.
In lung cancer tissues, a link has been pointed out between EGFR gene mutation and immune checkpoint molecule expression. So far, there have been reports of examining both in human biopsy tissues and surgical specimens, but no reports have been made in lung cancer model mice incorporating the full length of the human mutant EGFR gene.
Therefore, using the TG mouse of the present embodiment, the expression of immune checkpoint molecules (PD-1 and PD-L1) in the lung cancer tissue of the mouse was examined by fluorescence microscopy and RT-PCR. Total RNA was extracted from lung cancer tissue using TRIZOL (TRIZOL (Invitrogen, Carlsbad, CA)), and then RT-PCR was performed. The PCR reaction product was electrophoresed on a 2% agarose gel, the band was stained with ethidium bromide, and then observed with ultraviolet light.

<Evaluation of lung-specific [L858R] -h-EGFR-expressing TG mice>
1. 1. Onset of lung cancer in TG mice
Lung CT images were taken before the start of Dox administration and on the 7th and 13th days from the day when the diet containing Dox was started. FIG. 15 shows a CT image of one TG mouse (horizontally showing the same slide level of CT of the same mouse; vertically showing different slide levels of CT of the same mouse). Further, FIG. 16 shows an enlarged image of the TG mouse (3) on the 13th day.
Lung cancer was observed 7 days after Dox administration, and the onset was earlier than in the conventional model. The lung cancer was also advanced and increased as the number of days after Dox administration increased.

2. Lung tissue and other findings of [L858R] -h-EGFR TG mice The lung tissue of [L858R] -h-EGFR TG mice that developed lung cancer was observed under a microscope. The lung tissue of TG mice was stained with hematoxylin and eosin (H & E staining), and the results observed under a microscope (A → × 40; B → × 100; C, D → × 200) are shown in FIG. Tumor cells (arrow T in the figure) were found in each image. When the morphology of the tumor cells was confirmed, epithelial-mesenchymal transition (EMT) (Fig. 18A) and overexpression of mucin (Fig. 18B) exhibited by undifferentiated and highly malignant tumors were observed. Was done. The metastasis of the mesenteric lymph node (Fig. 18A) and the diaphragm lymph node (Fig. 20B) was shown.
Close examination of [L858R] -h-EGFR TG mice that developed lung cancer revealed infiltration of cancer cells into blood vessels (Fig. 19A) and distant metastasis of mesenteric lymph nodes (Fig. 20A) and diaphragmatic lymph nodes (Fig. 20B). Was observed, and it was confirmed that the malignancy was high. No reports on vascular infiltration or distant metastasis of cancer cells have been observed in conventional TG mice (using cDNA). In addition, the number of days from the start of feeding Dox-containing food to the onset of lung cancer was as short as 7 days, and it was recognized that the malignancy was high.
According to WHO, the lung cancer histological types are classified as shown in Table 8.

[L858R] -h-EGFR TG mouse lung cancer includes various lung cancer histological types such as alveolar replacement pattern, papillary pattern, solid pattern, and mucinous lung adenocarcinoma (high expression of mucin). It was found that it can be used as a clinical model for invasive lung cancer. Infiltration into blood vessels and distant metastasis to mesenteric and diaphragmatic lymph nodes were also observed. From this, it was considered that it was closer to the human clinical model.
In addition, it was confirmed by reverse transcriptase PCR (RT-PCR) that PD-1 and PD-L1 were expressed lung-specifically in TG mice. A summary of the results is shown in FIG. 21 (A) and 21 (B) are gel photographs and graphs examining the expression of PD-L1, and FIGS. 21 (C) and 21 (D) are gel photographs and graphs examining the expression of PD-1. Are shown respectively. In the figure, GAPDH indicates a housekeeping gene marker. Expression of PD-1 and PD-L1 was observed in both wild type and TG mice (EGFR-TG), but higher expression was observed in TG mice than in WT mice. In particular, PD-L1 was significantly (P <0.05) highly expressed in TG mice. Therefore, the use of the TG mouse of the present embodiment is useful for the development of a new therapeutic method such as the usefulness of the combined therapy of the EGFR tyrosine kinase inhibitor (TKI) and the immune checkpoint inhibitor.

B. Preparation and evaluation of [L858R + T790M] -h-EGFR-expressing TG mouse Next, a TG mouse expressing h-EGFR with two types of mutations [L858R] and [T790M] in a lung-specific manner was prepared. The characteristics were investigated.
<Preparation of lung-specific h-EGFR-expressing TG mice>
1. 1. Method for producing TG mouse FIG. 22 shows the structure of a gene for producing a TG mouse that expresses [L858R + T790M] -h-EGFR in a lung-specific manner. In StepA', the above <A. Preparation and evaluation of [L858R] -h-EGFR-expressing TG mouse> Using the tetO- [L858R] -h-EGFR prepared in, tetO- [L858R + T790M] -h-EGFR was prepared.
For other steps (StepB to StepG), see <A. Preparation and evaluation of [L858R] -h-EGFR-expressing TG mouse> was carried out in the same manner. In order to eliminate duplicate descriptions, only the data considered important for the production of TG mice are described below.

2. Construction of h-EGFR gene recipient construct
For tetO- [L858R] -h-EGFR, Red / ET Recombination Technology was used to construct a BAC clone as a recipient for incorporating the [T790M] mutation. (i) Insert a selection marker into Ex20 of the EGFR gene of the human BAC clone (Fig. 23 (A)), (ii) Delete Ex20, and remove the intermediate of tetO- [L858R + T790M] -h-EGFR Rec BAC. Prepared (second selection gene recombination step; FIG. 23 (B)). FIG. 23 (C) shows the result of confirming that the intermediate was produced by electrophoresis.
Using Red / ET Recombination Technology, a recombinant BAC clone of the [L858R + T790M] mutation (tetO-h [L858R + T790M] EGFR RecBAC) was constructed.
As shown in Table 9, active homologous recombination reactions of the 5'and 3'flanking sequences of the [T790M] mutant gene of the human EGFR gene and the 5'and 3'flanking sequences of the exon 20 genomic DNA sequence in Escherichia coli. (SEQ ID NO: 15 to SEQ ID NO: 18).

3. 3. Construction of recombinant BAC-type EGFR tetO expression construct
The genomic sequences before and after Ex20 at the EGFR locus were placed at both ends of the [T790M] mutation as homologous sequences to prepare an EGFR transfer construct for inserting the [T790M] mutation (second modification step). Then, using Red / ET Recombination Technology, a recombinant BAC clone in which the [T790M] mutant gene was inserted at the human EGFR locus was constructed. The selection marker inserted into Ex20 of the human EGFR recipient construct was accurately replaced and removed by the [T790M] mutation repair fragment (second substitution step). The tetO-h [L858R + T790M] EGFR RecBAC expression vector was then identified by exactly substituting the selection marker inserted at Ex20 of the human EGFR locus with the [T790M] mutant repair fragment.
The DNA sequence at the junction of tetO- [L858R + T790M] -h-EGFR RecBAC was confirmed by sequence analysis, and it was confirmed that the [T790M] mutant gene was inserted (gene preparation step for second receptor expression). FIG. 24).

4. Purification of high-purity BAC DNA fragments
The tetO- [L858R + T790M] -h-EGFR RecBAC recombinant BAC clone was purified, linearized with a restriction enzyme, and then subjected to pulsed field gel electrophoresis to purify a high-purity BAC DNA fragment. When the purity and concentration of the expression construct purified by agarose gel electrophoresis for analysis were examined, it was found that the pure long-chain expression construct could be recovered. The DNA concentration was 202.2 ng / μl, which was sufficient for the production of transgenic mice. In FIG. 25, (A) shows a state in which a gel containing an expression construct separated in an agarose gel was cut out, and (B) shows a band of DNA fragments obtained by pulsed-field gel electrophoresis of a DNA fragment purified by electroelution and dialysis. , (C) show the results of examining the DNA concentration with a NanoDrop spectrophotometer.
The m-CCSP rtTA RecBAC expression construct is described in <A. Preparation and evaluation of [L858R] -h-EGFR-expressing TG mouse> prepared according to the method described.

5. Gene microinjection into C57BL / 6J mice Next, fertilized eggs were collected from C57BL / 6J mice, and two types of foreign genes (tetO- [L858R + T790M] -h-EGFR RecBAC / CCSP_rtTA RecBAC expression construct) were collected by the microinjection method. ) Was introduced. For details of the method, refer to <A. [L858R] -Preparation and evaluation of h-EGFR-expressing TG mice>.
A total of 627 fertilized eggs were collected from C57BL / 6J female mice that were crossed by inducing hyperovulation. Of these, 355 fertilized eggs were microinjected with two types of foreign genes. Few fertilized eggs were killed by the damage caused by this operation, and a total of 308 fertilized eggs remained stable. Of these, 300 fertilized eggs were transplanted into pseudopregnant mice.
Table 10 shows data on fertilized mouse eggs and finder (childbirth) subjected to microinjection, and Table 11 shows a list of pregnant mice transplanted with embryos.

6. Southern screening of lung-specific [L858R + T790M] -h-EGFR RecBAC gene-induced expression TG mouse founder In addition to the microinjection data shown in Tables 10 and 11, 365 new embryos were obtained on different experimental days. Three embryos were collected and 300 embryos were transplanted into pseudopregnant mice for spontaneous delivery, resulting in a total of 52 mouse offspring. Of the total of 108 offspring, 105 were raised to weaning.
tetO- [L858R + T790M] -h-EGFR RecBAC / CCSP-rtTA The number of offspring and weaning from the early embryos into which the RecBAC expression construct was introduced was good. It was considered that there was no adverse effect on development and differentiation.
As shown in FIG. 26, the genomic DNA fragment extracted from the offspring, which is a candidate for the founder of TG mice, was hybridized with the [32P] labeled probe. 26 (A) and 26 (B) show the results of tetO- [L858R + T790M] -h-EGFR, and FIGS. 26 (C) and 26 (D) show the results of RecBAC / CCSP-rtTA. .. From this result, 5 mice were identified as TG mouse founder individuals into which the tetO- [L858R + T790M] -h-EGFR RecBAC / CCSP-rtTA RecBAC expression construct was introduced. The copy numbers of the expression constructs introduced into these TG mouse founder individuals ranged from 1 to 10 copies.

<Evaluation of lung-specific [L858R + T790M] -h-EGFR-expressing TG mice>
The above <A. [L858R] -Preparation and evaluation of h-EGFR-expressing TG mice> Lung cancer is observed from about 7 days after starting feeding with Dox-containing food according to the method described in>.
In addition, the TG mouse of the present embodiment has a [T790M] mutation in addition to [L858R]. Having this mutation makes the lung cancer that develops resistant to EGFR tyrosine kinase inhibitors. Clinically, [T790M] mutations are often detected by mutations in lung cancer patients with [L858R] -h-EGFR while receiving EGFR tyrosine kinase inhibitors. Therefore, [L858R + T790M] -h-EGFR with both mutations is highly effective as a human clinical model.
Therefore, the TG mouse of the present embodiment can be used for screening a new drug for lung cancer to which a conventional EGFR tyrosine kinase inhibitor is ineffective.

<Application to other tests>
(1) [L858R] -h-EGFR TG mouse and [L858R + T790M] -h-EGFR TG mouse can be used for the next test.
-Can be used for research and development of preventive and / or therapeutic agents for the onset of lung cancer.
・ Check for the development of lung cancer by introducing cigarette smoke. At this time, the state of exacerbation of the developed lung cancer can be investigated in chronological order.
-Can be used for research and development of new anticancer agents targeting immune checkpoint inhibitors.
-In particular, TG mice with the [T790M] mutation can be used for research and development of new anticancer agents for lung cancer resistant to EGFR-TKI.
(2) The following developments can be considered by applying the technical idea of the present invention.
-When performing the induced expression of [L858R] -h-EGFR, in addition to the induced expression system of TRE and rtTA, an induced expression system using Cre enzyme and loxP sequence can also be used.
-There is a possibility that it can be applied to TG mice that develop organ-specific cancers in organs other than the lung.
From these facts, the lung cancer that develops in the [L858R] -h-EGFR TG mouse and the [L858R + T790M] -h-EGFR TG mouse of the present embodiment is more malignant than the conventional model mouse. It showed a clinical condition of human lung cancer. By using this TG mouse, research on lung cancer can be dramatically developed.

Claims (14)

A transgenic mouse in which the human [L858R] epithelial growth factor receptor ([L858R] -h-EGFR) gene containing the entire gene region of 28 exons and 27 introns is integrated downstream of the expression promoter region. The expression promoter region contains a tetracycline response element (TRE), and the reverse tetracycline-regulated trans activator (rtTA) gene is located downstream of the expression promoter of the lung-specific expression gene in mice. , PD-1 and PD-L1 are expressed in a lung-specific manner, and a transgenic mouse that develops lung cancer by administration of doxycycline and has the property of metastasizing this lung cancer. Downstream of the expression promoter region, the human [L858R + T790M] epithelial growth factor receptor ([L858R + T790M] -h-EGFR) gene, which contains the entire gene region of 28 exons and 27 introns, is integrated. In the transgenic mouse , the expression promoter region contains a tetracycline response element (TRE), and a reverse tetracycline-regulated trans activator is located downstream of the expression promoter of the lung-specific expression gene in the mouse. The (rtTA) gene is integrated , PD-1 and PD-L1 are expressed in a lung-specific manner, and administration of doxicycline causes lung cancer, which is metastatic and anticancer drug resistance. Transgenic mouse with . The lung-specific expression gene is at least one selected from the group consisting of Clara cell secretory protein (CCSP), surfactant protein A (SPA), surfactant protein B (SPB), and surfactant protein C (SPC). The transgenic mouse according to claim 1 or 2. The transgenic mouse according to any one of claims 1 to 3, wherein the mouse strain is C57BL / 6J. (1) In a BAC containing the h-EGFR gene containing the entire gene region of 28 exons and 27 introns, a selection cassette was incorporated into Ex21 of the h-EGFR gene, and the selection gene-introduced h-EGFR gene was introduced. Obtaining selection gene recombination step, (2) Modification step of arranging the genome sequences before and after Ex21 of the selection gene introduction h-EGFR gene as homologous sequences at both ends of the [L858R] mutation to prepare an EGFR transfer construct. (3) Substitution step of substituting the selection cassette of Ex21 with the [L858R] mutant repair fragment of the EGFR transfer construct, (4) Acceptance linking the expression promoter region to the 5'side of [L858R] -h-EGFR. Receptor expression gene preparation step as a body expression gene, (5) Purification of the receptor expression gene and m-SSCPrtTA RecBAC expression gene and microinjection into mouse embryos to obtain transgenic mice the provided, the transgenic mice, lungs specifically PD-1 and PD-L1 are expressed by administration of doxycycline, developed lung cancer, and wherein Rukoto to have a characteristic that the lung cancer is metastatic How to make a transgenic mouse. The fifth aspect of claim 5, wherein in the selective gene recombination step, a DNA is used in which the sequences set forth in SEQ ID NOs: 3 to 6 are used as key sequences for an active homologous recombination reaction in Escherichia coli. Method for producing transgenic mice. According to claim 5 or 6, for the founder individual obtained through the step of obtaining the transgenic mouse, DNA having the sequences set forth in SEQ ID NO: 9 to SEQ ID NO: 12 is used as a PCR primer for genotyping. The method for producing a transgenic mouse according to the above. A sperm obtained from the upper body of a transgenic mouse (male) obtained by the production method according to any one of claims 5 to 7, and an unfertilized egg obtained from a C57B1 / 6J mouse (female). A method for producing a transgenic mouse, which comprises producing a fertilized egg from a mouse and transplanting the fertilized egg into the oviduct of a mouse treated with pseudopregnancy to obtain a transgenic mouse (first generation). With respect to the transgenic mouse obtained according to claim 8, the presence or absence of lung-specific expression of [L858R] -h-EGFR is confirmed by RT-PCR using the DNA having the sequences shown in SEQ ID NOs: 13 and 14. A method for confirming a transgenic mouse. (1) In a BAC containing the h-EGFR gene containing the entire gene region of 28 exons and 27 introns, a selection cassette was incorporated into Ex21 of the h-EGFR gene, and the selection gene-introduced h-EGFR gene was introduced. Obtaining selection gene recombination step, (2) Modification step of arranging the genome sequences before and after Ex21 of the selection gene introduction h-EGFR gene as homologous sequences at both ends of the [L858R] mutation to prepare an EGFR transfer construct. (3) Substitution step of substituting the selection cassette of Ex21 with the [L858R] mutant repair fragment of the EGFR transfer construct, (4) Acceptance linking the expression promoter region to the 5'side of [L858R] -h-EGFR. Receptor expression gene preparation step as a body expression gene, (4-1) Selection gene by incorporating a selection cassette into Ex20 of [L858R] -h-EGFR obtained in the receptor expression gene preparation step. Second-selection gene recombination step to obtain the introduction [L858R] -h-EGFR gene, (4-2) Homologous sequence of the genome sequences before and after Ex20 of the second-selection gene introduction [L858R] -h-EGFR gene In the second modification step of preparing an EGFR transfer construct by arranging it at both ends of the [T790M] mutation, (4-3) by replacing the Ex20 selection cassette with the [T790M] mutation repair fragment of the EGFR transfer construct. Second receptor expression gene preparation step as a second receptor expression gene having the expression promoter region on the 5'side of EGFR, (5) Second receptor expression gene and m-SSCPrtTA RecBAC expression The gene is purified and microinjected into a mouse embryo to obtain a transgenic mouse. The transgenic mouse expresses PD-1 and PD-L1 in a lung-specific manner, and administration of doxicycline. by, developed lung cancer, a method for manufacturing a transgenic mouse characterized by Rukoto to have a characteristic that the lung cancer is metastatic. 10. The method according to claim 10, wherein in the selective gene recombination step, DNA is used in which the sequences set forth in SEQ ID NOs: 15 to 18 are used as key sequences for an active homologous recombination reaction in Escherichia coli. Method for producing transgenic mice. The method according to claim 10 or 11, wherein the DNA having the sequences set forth in SEQ ID NO: 9 to SEQ ID NO: 12 is used as a PCR primer for genotyping in the founder individual obtained through the step of obtaining the transgenic mouse. The method for producing a transgenic mouse according to the above. A sperm obtained from the upper body of a transgenic mouse (male) obtained by the production method according to any one of claims 10 to 12, and an unfertilized egg obtained from a C57B1 / 6J mouse (female). A method for producing a transgenic mouse, which comprises producing a fertilized egg from a mouse and transplanting the fertilized egg into the oviduct of a mouse treated with pseudopregnancy to obtain a transgenic mouse (first generation). With respect to the transgenic mouse obtained according to claim 13, the presence or absence of lung-specific expression of [L858R + T790M] -h-EGFR was determined by RT-PCR using the DNA having the sequences shown in SEQ ID NOs: 13 and 14. A method for confirming a transgenic mouse, which comprises confirming.

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