Construction and application of Ift140 gene rod photoreceptor conditional knockout mouse model

Abstract

The present invention discloses a method for constructing a rod photoreceptor conditional knockout mouse model of the Ift140 gene, comprising knocking out the Ift140 gene in the mouse retinal rod photoreceptor genome. The rod photoreceptor conditional knockout mouse model constructed by the present invention can be used as a model for retinal degenerative diseases, providing a novel model for studying the disease, including its pathogenesis and mechanisms, and for screening related drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical engineering technology, and in particular to a method for constructing and applying an Ift140 gene rod photoreceptor conditional knockout mouse model. Background Art

[0002] Inherited retinal degeneration (IRD) is a large class of diseases caused by various genetic defects that lead to retinal damage. They are highly clinically heterogeneous, with significant differences in the age of onset and severity of visual impairment among patients. Currently, there is no effective treatment. IRD is the leading cause of irreversible blindness, a scientific challenge and a major public health issue that urgently needs to be addressed. IRD can occur alone or in combination with manifestations in other organs, forming a variety of clinical syndromes. IRD has high genetic heterogeneity, with three main modes of inheritance: autosomal recessive, dominant, and X-linked recessive, with autosomal recessive inheritance being the most common. Current reports suggest that there are hundreds of genes responsible for IRD.

[0003] Intraflagellar transport (IFT) 140 is a member of the intraciliary transport protein family. Cilia and flagella, including primary and motile cilia, are found on the surface of nearly all eukaryotic cells and are highly conserved cellular structures. They possess bidirectional transport capabilities, utilizing a specialized machinery comprising IFT motor proteins, IFT transport units, and transported molecules. The IFT transport unit comprises Complex A for forward transport and Complex B for reverse transport, with IFT140 belonging to Complex A.

[0004] Mutations in the IFT140 gene cause syndromic and nonsyndromic IRDs. IRD phenotypes include Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), and autosomal recessive retinitis pigmentosa (arRP). LCA / EOSRD patients experience severe visual impairment from birth or in childhood, while RP patients develop symptoms in adolescence or adulthood and gradually progress to total blindness, severely impacting their quality of life and placing a heavy burden on their families and society. In 2015, the applicant's research group first reported that IFT140 mutations cause nonsyndromic IRDs, and subsequent reports further confirmed that IFT140 is a causative gene for IRDs. However, only one study has yet to address the mechanisms of IFT140-related IRDs. This study, which used a cone-specific Ift140 conditional knockout mouse model, fails to adequately simulate the role of Ift140 in photoreceptors (it is known that rods make up the majority of photoreceptors in the mouse retina, with cones accounting for only 3%). The clinical phenotype and natural course of the disease in this model mouse model have not been identified or described. Furthermore, the location and expression of other IFT proteins and retinal-specific ciliary proteins in this model mouse model have not been studied, resulting in an incomplete and intricate exploration of the mechanisms by which Ift140 mutations lead to IRDs. Therefore, to better investigate the function of the IFT140 protein in the retina, a better disease model is needed.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a method for constructing a rod photoreceptor conditional knockout mouse model of the Ift140 gene and its application.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] A method for constructing an Ift140 gene rod photoreceptor conditional knockout mouse model comprises: knocking out the Ift140 gene in the mouse retinal rod photoreceptor genome.

[0009] IFT140 is one of the core components of IFT-A. IFT-A is composed of 6 proteins, and IFT-B is composed of 16 proteins, which mediate the reverse and forward directions in the cilia respectively. The outer segment of retinal photoreceptor cells is a specialized ciliary structure with a fast membrane disk renewal rate, so a higher transport rate is required in the cilia. The inventors speculate that mutations in the Ift140 gene may change the expression and distribution of the IFT protein network, affect the transport of substances in the inner and outer segments, and cause progressive death of photoreceptor cells. Based on this, the inventors constructed a rod cell conditional knockout mouse model of the Ift140 gene. Knocking out the Ift140 gene in the rod cells of the mouse retina silenced its expression in the rod cells, and verified that conditional knockout of the rod cell Ift140 gene can make mice show characteristics related to retinal degenerative diseases, such as significant thinning of the retina, abnormal pigmentation, obvious atrophy of the outer layer, severe reduction in cone and rod function, and atrophy of the outer retinal photoreceptor cells. Therefore, mice in which the Ift140 gene in retinal rod cells is knocked out can be used as a model of retinal degenerative diseases and in fields such as retinal degenerative disease research, providing a new model for the study of the disease, such as the pathogenesis, mechanism, and screening of related drugs.

[0010] In a preferred embodiment of the present invention, the knockout of the Ift140 gene in the genome of the mouse retinal rod photoreceptor cells refers to the knockout of the exon sequence of the Ift140 gene in the genome of the mouse retinal rod photoreceptor cells.

[0011] Knocking out the Ift140 gene sequence can be knocking out the full-length sequence of the Ift140 gene, knocking out the exon sequence of the Ift140 gene, or knocking out a partial sequence of the Ift140 gene, such as a partial exon sequence. Regardless of the type of sequence knocked out (partial or full-length), as long as it can silence the expression of the Ift140 gene in rod photoreceptors and cause animals to exhibit corresponding characteristics of retinal degeneration diseases, it falls within the scope of protection of the present invention.

[0012] In a preferred embodiment of the present invention, the knockout target animal retinal rod cell genome is the exon 7 sequence of the Ift140 gene.

[0013] In a preferred embodiment of the present invention, the construction method includes: using Cre-loxP knockout technology to knock out the Ift140 gene in the genome of mouse retinal rod cells.

[0014] In other embodiments, siRNA-mediated gene silencing technology may also be used. In addition, the use of other means to achieve knockout of the Ift140 gene also falls within the scope of protection of the present invention.

[0015] In a preferred embodiment of the present invention, the target animal is a mouse, but is not limited to a mouse, and may also be any one of rats, dogs, pigs, monkeys, rabbits, cows, horses, sheep and apes.

[0016] Regardless of the type of animal selected, as long as it is an animal with the Ift140 gene, it can be used as the target animal in the construction method described in the present invention. The Ift140 gene can be knocked out in its rod photoreceptor cells to make it exhibit the characteristics of retinal degeneration diseases. It can be used as a retinal degeneration disease model in the field of retinal degeneration disease research and is within the scope of protection of the present invention.

[0017] In a preferred embodiment of the present invention, the above-mentioned construction method is to use Cre-loxP knockout technology to knock out the Ift140 gene in the mouse retinal rod cell genome, which includes: mating Neo-positive chimeric mice with flp mice to obtain Ift140-loxp heterozygous mice, then mating the Ift140-loxp heterozygous mice with each other to obtain Ift140-loxp homozygous mice, and mating the Ift140-loxp homozygous male mice with Rho-iCre heterozygous female mice to obtain retinal rod cell Ift140 conditional knockout gene mice.

[0018] In a preferred embodiment of the present invention, the method for constructing chimeric mice is as follows: the ES cell line targeting the Ift140 gene is injected into the blastocyst cavity of wild-type mice, the injected blastocyst is transplanted into the uterus of pseudo-pregnant mice, and the chimeric mice are obtained after they develop to maturity.

[0019] The ES cell line targeted with the Ift140 gene was the ES cell line EPD0073_5_F01 (genotype Ift140) from KOMP (www.komp.org). neo ), flp mice were C57BI / 6Flp1 mice (germ cells expressed FlpE).

[0020] In a preferred embodiment of the present invention, the Ift140 gene of the founder mouse for conditional knockout of the Ift140 gene carries a loxP site.

[0021] In a preferred embodiment of the present invention, chimeric mice are mated with flp mice, and then genotype identification is performed to screen and obtain Ift140-loxp heterozygous mice.

[0022] Rho-iCre heterozygous mice, purchased from Cytochrome Plc, express the Cre enzyme specifically in rod photoreceptors. Mating homozygous mice with Rho-iCre mice yielded mice expressing the conditional knockout gene for Ift140 in retinal rod photoreceptors. The engineered Cre protein can enter the nucleus of rod photoreceptors, recognize LoxP sites on the genome, and conditionally knock out the Ift140 gene. Rho is a gene specifically expressed in rod photoreceptors, and inserting the Cre sequence after the Rho gene promoter mediates the specific expression of the Cre enzyme in these cells.

[0023] The application of the rod photoreceptor cell conditional knockout Ift140 gene mouse model constructed by the above construction method in screening drugs for preventing or treating retinal degenerative diseases.

[0024] The mouse model constructed using the method of the present invention exhibits characteristics of retinal degeneration and has broad application prospects. For example, it can be used to study the pathogenesis and pathogenesis of retinal degenerative diseases, providing a foundation for in-depth research on such diseases. Alternatively, it can be used to screen for drugs to prevent or treat retinal degenerative diseases, and to evaluate drug efficacy and prognosis.

[0025] In a preferred embodiment of the present invention, the above application includes early-onset severe retinal degeneration.

[0026] According to the research results, the rod photoreceptor conditional knockout Ift140 gene mouse model constructed by the above construction method showed obvious retinal degeneration symptoms at one month of age. Therefore, it can be seen that the above mouse model can be used in the study of early-onset severe retinal degeneration.

[0027] In some embodiments, the rod photoreceptor cell conditional knockout Ift140 gene mouse model constructed by the above construction method can also be used in screening drugs for preventing or treating syndromic ciliopathy with multi-organ involvement.

[0028] The present invention has the following beneficial effects:

[0029] The present invention provides a method for constructing a rod photoreceptor cell conditional knockout mouse model for the Ift140 gene, and its application. The present invention first discovered that knocking out the Ift140 gene in rod photoreceptor cells in mice, i.e., silencing its expression in rod photoreceptor cells, can cause the mice to exhibit characteristics associated with retinal degenerative diseases, such as significant retinal thinning, pigmentary abnormalities, significant atrophy of the outer layer, severely reduced cone and rod function, and atrophy of the outer retinal photoreceptor cells. Therefore, the Ift140 gene knockout mice constructed by the present invention in retinal rod photoreceptor cells can be used as a model for retinal degenerative diseases, providing a new model for studying the disease, such as the pathogenesis and mechanism, and screening for related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 Design and preparation of model mice;

[0032] Figure 2 This is the rat tail identification result of Experimental Example 1;

[0033] Figure 3 The results of the construction and identification of the model mice in Experimental Example 2, where A is the gene knockout route, B is the sequence of the knockout gene; C is the genotype identification result;

[0034] Figure 4 This is the OCT scan result of the model mouse in Experimental Example 3;

[0035] Figure 5 This is the ERG test result of the model mouse in Experimental Example 3;

[0036] Figure 6 HE staining results of the model mice in Experimental Example 3;

[0037] Figure 7 The OCT scan results, HE staining results, and thickness changes of each retinal layer of the model mice in Experimental Example 4 are shown;

[0038] Figure 8 This is the ERG test result of the model mice in Experimental Example 4. DETAILED DESCRIPTION

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described clearly and completely below. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the materials, reagents, etc. used are all commercially available.

[0040] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0041] Example 1

[0042] This example provides a method for constructing a rod photoreceptor conditional knockout mouse model of the Ift140 gene. Figure 1 The specific operations are as follows:

[0043] 1) The ES cell line EPD0073_5_F01 (genotype Ift140) targeted to the Ift140 gene from KOMP (www.komp.org) was used to generate neo ) were injected into the blastocyst cavity of C57BI / 6J mice, and the injected blastocysts were transplanted into the uterus of pseudo-pregnant mice to obtain founder mice;

[0044] 2) The first knockout mice obtained in step 1) were mated with flp mice (C57BI / 6Flp1) to obtain Ift140 gene flox heterozygous mice (Ift140 flox / + );

[0045] 3) The Ift140 gene flox heterozygous mice obtained in step 2) were mated and bred to obtain Ift140 gene flox homozygous mice (Ift140 flox / flox );

[0046] 4) The Ift140 gene flox homozygous male mice obtained in step 3) were mated with Rho-iCre female mice to obtain retinal rod cell conditional knockout Ift140 gene mice (Rho-iCre, Ift140 flox / flox ).

[0047] Experimental Example 1

[0048] This experimental example is to identify the tail of the mouse with conditional knockout of Ift140 gene of retinal rod photoreceptor cells obtained in Example 1. The specific method is as follows:

[0049] 1) Cut a small amount of tissue sample from the mouse tail and place it in a clean 1.5ml centrifuge tube;

[0050] 2) Mouse DNA extraction;

[0051] 3) PCR amplification: Configure the PCR reaction system as follows:

[0052]

[0053]

[0054] The pre-made Taq polymerase mixture in the above table is from Vazyme, P222-C2.

[0055] The primer sequences are as follows:

[0056] Ift140-loxP-F1 sequence: 5′-TCAGCCCTCTATGCCACTCT-3′, SEQ ID NO. 1;

[0057] Ift140-loxp-R1 sequence: 5′-CTTCCCTATGCCTTCAGCAG-3′, SEQ ID NO. 2;

[0058] Ift140-neo-F2 sequence: 5'-TCAGCCCTCTATGCCACTCT-3', SEQ ID NO.3;

[0059] Ift140-neo-R2 sequence: 5′-TGGTTTGTCCAAACTCATCAA-3′, SEQ ID NO. 4;

[0060] Rho-iCre-F3 sequence: 5'-TCAGTGCCTGGAGTTGCGCTGTGG-3', SEQ ID NO.5;

[0061] Rho-iCre-R3 sequence: 5'-CTTAAAGGCCAGGGCCTGCTTGGC-3', SEQ ID NO. 6.

[0062] Primers Ift140-loxp-F1 and Ift140-loxp-R1 were used to identify loxp insertion (wildtype: 220bp, loxp: 269bp); primers Ift140-neo-F2 and Ift140-neo-R2 were used to identify neo (wildtype: 0, neo: 1099bp); primers Rho-iCre-F3 and Rho-iCre-R3 were used to identify Cre (wildtype: 0, Cre: 550bp).

[0063] The amplification procedure is:

[0064]

[0065] Amplification results such as Figure 2 As shown: All offspring mice had loxp insertion and wild-type bands, while neo detection showed no bands, indicating that Ift140-loxp heterozygous mice were obtained [Ift140 flox / + The results showed that the model mice were homozygous for loxp and Cre positive.

[0066] Experimental Example 2

[0067] This experimental example identifies the genotype of the rod photoreceptor conditional knockout Ift140 gene mice obtained in Example 1. The method is as follows: DNA is extracted from the retina of the rod photoreceptor conditional knockout Ift140 gene mice in Example 1 and used as a template for PCR amplification. The PCR reaction system is configured as follows:

[0068]

[0069] The pre-made Taq polymerase mixture in the above table is from Vazyme, P222-C2.

[0070] The primer sequences are as follows:

[0071] BF4: 5'-TGGCCGCAACTACTAACGAACTG-3', SEQ ID NO.7;

[0072] BR2: 5'-TCAGCCCTCTATGCCACTCTTAA-3', SEQ ID NO. 8.

[0073] BF4 and BR2 were used to identify the knockout genotype (wildtype: 948 bp, targeted: 1149 bp, KO: 373 bp).

[0074] The amplification procedure is:

[0075]

[0076] Amplification results such as Figure 3 Shown: From Figure 3 It can be seen that Rho-iCre plays a role and a KO band (373bp) is present.

[0077] Experimental Example 3

[0078] This experimental example is to identify the phenotype of the retinal rod cell conditional knockout Ift140 gene mice obtained in Example 1, including fundus color photography, OCT and ERG examinations of the model mice, and HE staining of paraffin sections of the mouse eyeballs.

[0079] The steps for fundus color photography and OCT examination are as follows:

[0080] 1) Compound tropicamide eye drops dilate the pupil;

[0081] 2) 1.25% tribromoethanol intraperitoneal anesthesia;

[0082] 3) After the mouse is fully anesthetized, place it on the operating table and apply an appropriate amount of Vidisil to the cornea to keep it moist and transparent;

[0083] 4) Move the operating table and gently place one eye against the lens of the Micron IV camera. After the fundus image appears on the computer screen, adjust the parameters to make the fundus image and OCT image clear. Move the operating table to adjust the lens to different angles between the mouse eye and the mouse eye. Take photos and perform OCT scans of each quadrant of the mouse fundus.

[0084] 5) Repeat the same procedure for the other eye.

[0085] The steps of ERG examination are as follows:

[0086] 1) Dark-adapt the mice the night before the experiment;

[0087] 2) dilate the pupil three times with tropicamide eye drops under dim red light, once every 5 minutes;

[0088] 3) 1.25% tribromoethanol intraperitoneal anesthesia;

[0089] 4) After the mouse is fully anesthetized, place it on an operating table (constant temperature 37°C) and apply oxybuprocaine hydrochloride eye drops to the cornea to keep it moist and increase conductivity.

[0090] 5) Place the corneal stimulator in contact with the mouse cornea, with the impedance of each electrode less than 10 kilo-ohms, and record dark-adapted ERGs at different light intensities. After recording the dark-adapted ERGs, light-adapt the mouse for 10 minutes, and then record the light-adapted ERGs at different light intensities.

[0091] H&E staining of retinal paraffin sections:

[0092] The retinas of 1-month-old mice were paraffin-sectioned and stained with hematoxylin-eosin (H&E) staining as follows:

[0093] 1) Quickly remove mouse eyeball tissue and fix it in fixative for 24 hours;

[0094] 2) paraffin-embedded and sectioned at a thickness of 4 μm;

[0095] 3) Sections were routinely dewaxed with xylene and washed in multiple ethanol washes followed by water washes: xylene (I) for 5 min → xylene (II) for 5 min → 100% ethanol for 2 min → 95% ethanol for 1 min → 80% ethanol for 1 min → 75% ethanol for 1 min → distilled water for 2 min.

[0096] 4) Hematoxylin staining for 5 minutes, then rinse with tap water;

[0097] 5) Hydrochloric acid ethanol differentiation for 30 seconds;

[0098] 6) Soak in tap water for 15 minutes;

[0099] 7) Leave in eosin solution for 2 minutes.

[0100] 8) Conventional dehydration, transparency, and sealing: 95% ethanol (I) 1 min → 95% ethanol (II) 1 min → 100% ethanol (I) 1 min → 100% ethanol (II) 1 min → xylene carbolic acid (3:1) 1 min → xylene (I) 1 min → xylene (II) 1 min → neutral resin sealing.

[0101] 9) Take photos under a microscope.

[0102] like Figure 4-6 As shown, the littermates [Ift140 flox / flox ] mice served as controls, Figure 4 The results showed that [Rho-iCre, Ift140 flox / flox The mouse retina was significantly thinner and had abnormal pigmentation, and OCT showed significant atrophy of the outer layer. Figure 5 The ERG showed that the cone and rod functions were severely reduced. Figure 6 HE staining results showed atrophy of the outer retinal photoreceptor cells.

[0103] In addition, the mice used in this experiment were one month old. Judging from the experimental results, the obvious symptoms of retinal degeneration at one month old can prove that the deletion of the Ift140 gene can cause early-onset severe retinal degeneration.

[0104] Experimental Example 4

[0105] This experimental example is an observation of the natural course of the retinal rod photoreceptor conditional knockout Ift140 gene mice obtained in Example 1, including changes in retinal structure and function.

[0106] Model mice of different age groups (PN10, PN15, PN20 and PN25) and control mice from the same litter were selected, with three mice in each group.

[0107] a. Perform OCT and ERG data acquisition.

[0108] b. Take the eyeballs and perform paraffin-embedded HE sections to observe the changes in the thickness of each retinal layer.

[0109] like Figure 7-8 As shown, the littermates [Ift140 flox / flox ] mice were used as controls, and the results showed that [Rho-iCre, Ift140 flox / flox ] Progressive degeneration of mouse retinal structure and function. Figure 7 The OCT image shows that the outer layer of the mouse is significantly atrophied from 15 days to 25 days after birth ( Figure 7 -A), HE staining results showed that the outer layer of retinal photoreceptor cells significantly atrophied and disappeared ( Figure 7 -B), Figure 7 -C shows the changes in thickness of each retinal layer. Figure 8 The middle shows that ERG 30 days after birth showed that the cone and rod functions were severely reduced to flat waves.

[0110] The natural course of the disease was observed and recorded, and the results further demonstrated that Ift140 gene deletion caused early-onset severe retinal degeneration.

[0111] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for constructing an early-onset severe retinal degenerative disease model, characterized in that: It includes: Knockout of exon 7 of the Ift140 gene in the genome of mouse retinal rod cells.

2. The construction method according to claim 1, characterized in that The construction method comprises: using Cre-loxP knockout technology to knock out the Ift140 gene in the genome of mouse retinal rod cells.

3. The construction method according to claim 2, characterized in that The construction method includes: hybridizing Neo-positive mice with Ift140 gene targeting first constructed with flp mice to obtain flox heterozygous mice (flox / +) with the Neo gene of the targeting vector removed and containing two loxP sites, and then mating the flox heterozygous mice (flox / +) with each other to obtain flox homozygous mice (flox / flox), and then mating the flox heterozygous mice with Rho-iCre mice to obtain conditional rod cell Ift140 gene knockout mice.

4. The construction method according to claim 3, characterized in that The Ift140 gene of the first Ift140 gene-targeted Neo-positive mouse carries Neo and loxP sites.

5. The construction method according to claim 4, characterized in that The method for constructing the first Ift140 gene-targeted Neo-positive mouse is as follows: the ES cell line targeted to the Ift140 gene is injected into the blastocyst cavity of a wild-type mouse, the injected blastocyst is transplanted into the uterus of a pseudo-pregnant mouse, and the first Ift140 gene-targeted Neo-positive mouse is obtained after development and maturity.

6. The construction method according to claim 5, characterized in that: Neo-positive mice with Ift140 gene targeting were first constructed and crossed with flp mice for genotyping, and flox heterozygous mice (flox / +) containing two loxP sites were obtained through screening.

7. Use of the early-onset severe retinal degenerative disease model constructed by the construction method according to any one of claims 1 to 6 in screening drugs for preventing or treating early-onset severe retinal degenerative diseases.

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