CN115777626A - Construction method and application of Ift140 gene rod-like cell conditional knockout mouse model - Google Patents

Abstract

The invention discloses a construction method of an Ift140 gene rod-like cell conditional knockout mouse model, which comprises the following steps: knock out the Ift140 gene in the mouse retinal rod cell genome. The mice with the knocked-out Ift140 gene in the retinal rod cells constructed by the invention can be used as a retinal degeneration disease model, and a new model is provided for research of the disease, such as pathogenesis, mechanism and screening of related drugs.

Description

Construction method and application of Ift140 gene rod-like cell conditional knockout mouse model

Technical Field

The invention relates to the technical field of medical engineering, in particular to a construction method and application of an Ift140 gene rod cell conditional knockout mouse model.

Background

Hereditary retinal degeneration (IRD) is a large group of diseases with retinal damage caused by different gene defects, has high clinical heterogeneity, has obvious difference between the onset age of patients and the severity of visual disturbance, and the like, has no effective treatment method at present, is the most main reason of irreversible blindness-causing eye diseases, and is a scientific problem and a great public health problem which need to be overcome urgently. IRDs can occur individually or in combination with other organ manifestations and constitute a variety of clinical syndromes. IRDs have high genetic heterogeneity, and the genetic modes mainly include autosomal recessive, dominant and X-linked recessive, with autosomal recessive being the most inherited. According to the current report, the pathogenic genes of IRD are hundreds.

Cilia internal transport protein (IFT) 140 is a member of the intracellular cilia transporter family. Cilia and flagella of eukaryotic cells, including the protocilia and the motile cilia, are distributed on the surface of almost all cells and are well-conserved cellular structures. It has a bidirectional transport function and a special set of devices including IFT movement protein, IFT transport unit and transported molecules. IFT transport units include forward transported complex a and reverse transported complex B, where IFT140 belongs to complex a.

IFT140 gene variation leads to both syndromic and non-syndromic IRDs. IRD phenotypes include Leber Congenital Amaurosis (LCA), early Onset Severe Retinal Degeneration (EOSRD) and autosomal recessive retinitis pigmentosa (arRP). LCA/EOSRD patients have serious visual impairment at birth or in the early childhood, and RP patients have diseases from teenagers or adults, gradually progress to complete blindness, seriously affect the life quality of the patients and cause heavy burden to families and society of the patients. In 2015, the applicant's topic group first reported that IFT140 variation resulted in non-syndromic IRDs, and subsequently several reports further confirmed that IFT140 was one of the causative genes of IRDs. However, only 1 mechanism research aiming at IFT 140-related IRD exists at present, and the research adopts a cone cell-specific Ift140 condition to knock out a model mouse, so that the effect of the Ift140 in a photoreceptor cell cannot be well simulated (most of the mouse retina photoreceptor cells are known to be rod cells, and the cone cells only account for 3%); the clinical phenotype and natural course of disease of the model mouse were not identified and described; the location and expression of other IFT proteins and retinal-specific cilia proteins in model mice were not studied, and the mechanism of IRD caused by IFT140 variation was not thoroughly discussed. Therefore, in order to better study the function of IFT140 protein in retina, a better disease model needs to be constructed.

In view of this, the invention is particularly proposed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a construction method and application of an Ift140 gene rod cell conditional knockout mouse model.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a construction method of an Ift140 gene rod cell conditional knockout mouse model comprises the following steps: knock out the Ift140 gene in mouse retina rod cell genome.

IFT140 is one of the core components of IFT-A. IFT-A consists of 6 proteins and IFT-B consists of 16 proteins, mediating intra-ciliary reversal and forward direction, respectively. The retinal photoreceptor outer segment is a specialized cilium structure, and the membrane disc is updated quickly, so that the requirement on the transport rate in cilium is higher. The inventor speculates that the IFT140 gene variation may change the expression and distribution of an IFT protein network, influence the transport of inner and outer segment substances and cause the progressive death of photoreceptor cells. Based on the above, the inventor constructs an Ift140 gene rod cell conditional knockout mouse model, knocks out the Ift140 gene in a retinal rod cell of a mouse, namely silences the expression of the Ift140 gene in the rod cell, and verifies that the conditional knockout rod cell Ift140 gene can enable the mouse to show the characteristics related to retinal degenerative diseases, such as obviously thinned retina, abnormal pigment, obviously atrophied outer layer, seriously reduced cone rod function, atrophied outer layer photoreceptor cell of retina and the like. Therefore, the mouse with the knock-out Ift140 gene in the retinal rod cells can be used as a retinal degeneration disease model and used in the fields of research of retinal degeneration pathological diseases and the like, and provides a new model for the research of the diseases, such as pathogenesis, mechanism and screening of related drugs.

In a preferred embodiment of the present invention, the knocking out of the Ift140 gene in the mouse retinal rod cell genome refers to knocking out an exon sequence of the Ift140 gene in the mouse retinal rod cell genome.

The Ift140 gene knockout sequence can be an Ift140 gene knockout full-length sequence, an Ift140 gene knockout exon sequence, and can also be an Ift140 gene knockout partial sequence such as a partial exon sequence, and the Ift140 gene knockout sequence is of any type (partial or full-length) as long as the Ift140 gene expression can be silenced in rod cells, so that the animals show the corresponding characteristics of the retinal degeneration diseases, and the Ift140 gene knockout sequence is within the protection scope of the invention.

In a preferred embodiment of the present invention, the sequence of exon 7 of Ift140 gene in the target animal retina rod cell genome is knocked out.

In a preferred embodiment of the present invention, the constructing method includes: knocking out the Ift140 gene in the mouse retina rod cell genome by adopting a Cre-loxP knocking-out technology to knock out the Ift140 gene in the mouse retina rod cell genome.

In other embodiments, siRNA mediated gene silencing techniques are also contemplated. In addition, other means for realizing the knock-out of the Ift140 gene also belong to the protection scope of the invention.

In a preferred embodiment to which the present invention is applied, the target animal is a mouse, but is not limited to a mouse, and may be any one of a rat, dog, pig, monkey, rabbit, cow, horse, sheep, and ape.

No matter which animal is selected, as long as the animal has the Ift140 gene, the animal can be used as a target animal in the construction method of the invention, the Ift140 gene is knocked out in the rod cells of the animal, so that the rod cells of the animal show the characteristics of the retinal degeneration diseases, and the animal is used as a retinal degeneration disease model in the research field of the retinal degeneration diseases, and belongs to the protection scope of the invention.

In a preferred embodiment of the present invention, the construction method is to knock out the Ift140 gene in the mouse retinal rod cell genome by Cre-loxP knockout technology, and comprises: the Neo positive chimeric mouse and the flp mouse are bred to obtain an Ift140-loxp heterozygote mouse, then the Ift140-loxp heterozygote mouse is mutually bred to obtain an Ift140-loxp homozygote mouse, and an Ift140-loxp homozygote male mouse and a Rho-iCre heterozygote female mouse are bred to obtain a retinal rod cell Ift140 conditional knockout gene mouse.

In a preferred embodiment of the present invention, the chimeric mouse is constructed by the following steps: injecting the targeted Ift140 gene ES cell line into a blastocyst cavity of a wild mouse, transplanting the injected blastocyst into a pseudopregnant mouse uterus, and developing and maturing to obtain the chimeric mouse.

The ES cell line targeting the Ift140 gene is a KOMP-derived (www.komp.org) ES cell line EPD0073_5_F01 (genotype is Ift 140) ^neo ) The Flp mice were C57BI/6Flp1 mice (germ cells expressing FlpE).

In a preferred embodiment of the invention, the Ift140 gene of the initial mouse with conditional knock-out of the Ift140 gene has a loxP site.

In a preferred embodiment of the invention, after breeding the chimeric mice with flp mice, genotyping and screening to obtain Ift140-loxp heterozygote mice.

Rho-iCre heterozygote mice are rod-cell-specific Cre enzyme-expressing mice purchased from Setaria corporation. Homozygote mice were mated with Rho-cre gene mice to obtain retinal rod eye cells Ift140 conditional knockout mice. The modified Cre protein can enter the nucleus of a rod cell, and a LoxP site on a genome is identified, so that conditional knockout of the Ift140 gene is realized. Rho is a rod cell specific expression gene, and a Cre sequence is inserted after a Rho gene promoter can mediate the specific expression of Cre enzyme in the cell.

The rod cell conditional knockout Ift140 gene mouse model constructed by the construction method is applied to screening of drugs for preventing or treating retinal degeneration diseases.

The mouse model obtained by the construction method has characteristics of retinal degeneration and has very wide application prospect, for example, the mouse model is used for researching the pathogenesis process and pathogenesis of retinal degeneration diseases, and provides a basis for further understanding and researching the diseases. Or screening a drug for preventing or treating a retinal degeneration disease, evaluating the efficacy or prognosis of a drug, or the like.

In preferred embodiments of the invention, the use includes early onset severe retinal degeneration.

According to the results of the study, the rod conditioned knockout Ift140 gene mouse model constructed by the construction method showed significant retinal degeneration symptoms at 1 month of age, and thus it was found that the mouse model was applicable to the study of early severe retinal degeneration.

In some embodiments, the rod cell conditional knockout Ift140 gene mouse model constructed by the construction method can also be applied to screening of drugs for preventing or treating multiple organ-affected complex cilia diseases.

The invention has the following beneficial effects:

the invention provides a construction method and application of an Ift140 gene rod cell conditional knockout mouse model, and the invention discovers for the first time that the Ift140 gene knockout mouse model is used for silencing expression of the Ift140 gene in a rod cell of a retina of a mouse, so that the mouse can show characteristics related to retinal degenerative diseases, such as obviously thinned retina, abnormal pigment, obviously atrophied outer layer, seriously reduced cone rod function, atrophied photoreceptor cell of the outer layer of the retina and the like. Therefore, the mice with the knock-out gene Ift140 in the retinal rod cells constructed by the invention can be used as a retinal degeneration disease model, and a new model is provided for research of the disease, such as pathogenesis, mechanism and screening of related drugs.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the design and fabrication of a model mouse;

FIG. 2 shows the results of the rat tail identification in Experimental example 1;

FIG. 3 shows the results of constructing and identifying a model mouse in Experimental example 2, wherein A is a gene knockout route and B is a sequence of a knockout gene; c is the genotype identification result;

FIG. 4 shows the results of OCT scan of the model mouse in Experimental example 3;

FIG. 5 shows the result of ERG detection of the model mouse in Experimental example 3;

FIG. 6 shows the results of HE staining of the model mouse in Experimental example 3;

FIG. 7 shows the results of OCT scan, HE staining and thickness change of each layer of retina in the model mouse in Experimental example 4;

FIG. 8 shows the results of ERG detection in the model mouse in Experimental example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents and the like used are commercially available.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a method for constructing an Ift140 gene rod cell conditional knockout mouse model. The Ift140 gene knockout route is shown in FIG. 1, and the specific operation is as follows:

1) KOMP derived (www. KOMP. Org) targeted Ift140 gene ES cell line EPD0073_5_F01 (genotype is Ift 140) ^neo ) Injecting the blastocyst into the blastocyst cavity of a C57BI/6J mouse, and transplanting the blastocyst after injection into the uterus of a pseudopregnant mouse to obtain a first-built mouse;

2) Mating and breeding the initial knockout mouse obtained in the step 1) with an Flp mouse (C57 BI/6Flp 1) to obtain an Ift140 gene flox heterozygote mouse (Ift 140) ^flox/+ )；

3) The mice with the Ift140 gene flox heterozygotes obtained in the step 2) are mutually mated and bred to obtain mice with the Ift140 gene flox homozygotes (Ift 140) ^flox/flox )；

4) Mating the male mice of the flox homozygote of the Ift140 gene obtained in the step 3) with the female mice of Rho-iCre to obtain mice (Rho-iCre, ift140 gene conditionally knocked-out of retinal rod cells) ^flox/flox )。

Experimental example 1

In this experimental example, the rat tail of the mice with conditional knockout of Ift140 gene of retinal rod cells obtained in example 1 was identified by the following specific method:

1) Cutting a little tissue sample from the tail tip of the mouse, and placing the cut tissue sample in a clean 1.5ml centrifuge tube;

2) Extracting DNA of a mouse;

3) PCR amplification; a PCR reaction system is configured according to the following system:

the preformed Taq polymerase mix in the above table is from Vazyme, P222-C2.

Wherein the primer sequences are as follows:

ift140-loxP-F1 sequence: 5 'TCAGCCCTCCTATGCCACTCT-3', SEQ ID NO.1;

ift140-loxp-R1 sequence: 5 'CTTCCCTATGCCTTCAGCAG-doped 3', SEQ ID NO.2;

ift140-neo-F2 sequence: 5 'TCAGCCCTCCTATGCCACTCT-3', SEQ ID NO.3;

ift140-neo-R2 sequence: 5 'TGGTTTGTCCAAACTCATCA-A-3', SEQ ID NO.4;

Rho-iCre-F3 sequence of 5 'TCAGTGCCTGGAGTTGCGCTGTGG-3', SEQ ID NO.5;

Rho-iCre-R3 sequence of 5 'CTTAAAGGCCAGGGCCTGCTTGGC-TTGGC-3', SEQ ID NO.6.

Primers Ift140-loxp-F1 and Ift140-loxp-R1 were used to identify loxp insertion (wildtype: 220bp, loxp; primers Ift140-neo-F2 and Ift140-neo-R2 were used to identify the neo condition (wildtype: 0, neo; the primers Rho-iCre-F3 and Rho-iCre-R3 were used to identify the Cre status (wildtype: 0, cre.

The amplification procedure was:

amplification ofThe results are shown in FIG. 2: the offspring mice all have loxp insertion and wild bands, neo detects no band, and prompts to obtain an Ift140-loxp heterozygote mouse [ Ift140 ^flox/+ ]. The results show that the model mouse is homozygous for loxp and positive for Cre.

Experimental example 2

In this example, the genotype of the mice with conditional Ift140 gene knockout of retinal rod cells obtained in example 1 was identified as follows: taking the retina of the rod cell conditional knockout Ift140 gene mouse in example 1, extracting DNA, using the DNA as a template, performing PCR amplification, and configuring a PCR reaction system according to the following system:

the preformed Taq polymerase mix in the above table is from Vazyme, P222-C2.

The primer sequences are as follows:

BF4：5’-TGGCCGCAACTACTAACGAACTG-3’，SEQ ID NO.7；

BR2:5’-TCAGCCCTCTATGCCACTCTTAA-3’，SEQ ID NO.8。

BF4 and BR2 were used to identify knock-out genotypes (wildtype: 948bp, targeted.

The amplification procedure was:

the amplification results are shown in FIG. 3: it can be seen from FIG. 3 that Rho-iCre functions with the presence of a KO band (373 bp).

Experimental example 3

In the experimental example, the phenotype of the mice with conditional Ift140 gene knockout of retinal rod cells obtained in the example 1 is identified, and the phenotype comprises fundus color photography, OCT and ERG examination of the model mice, and HE staining of paraffin sections of eyeball rows of the mice.

The steps of fundus color photography and OCT inspection are as follows:

1) The compound tropicamide eye drops are used for mydriasis;

2) 1.25% tribromoethanol is used for abdominal anesthesia;

3) After the mouse is completely anesthetized, placing the mouse on an operation table, and applying a proper amount of unique information to the cornea to keep the cornea moist and transparent;

4) The operating platform is moved to slightly stick one eye to the lens of the Micron IV camera, parameters are adjusted to enable the fundus picture and the OCT picture to be clear after the fundus picture appears in a computer screen, the position of the operating platform is moved to enable the lens and the mouse eye to form different angles, and photographing and OCT scanning are respectively carried out on all quadrants of the mouse fundus.

5) The same procedure was performed for the lateral eye.

The ERG examination procedure was as follows:

1) Dark adaptation was performed on mice one night before the experiment;

2) Mydriasis is performed three times by using compound tropicamide eye drops under dark red light, and the compound tropicamide eye drops are used once every 5 minutes;

3) 1.25% tribromoethanol is used for abdominal anesthesia;

4) After the mice are completely anesthetized, placing the mice on an operating table (constant temperature of 37 ℃), and dripping oxybuprocaine hydrochloride eye drops to the cornea to keep the cornea moist and increase the conductivity;

5) Contacting the cornea stimulator with mouse cornea, recording dark adaptation ERG with different light intensities when the impedance of each electrode is less than 10 kilo-ohm, recording the light adaptation ERG with different light intensities for 10 minutes after the recording of the dark adaptation ERG is finished, and recording the light adaptation ERG with different light intensities.

Retinal paraffin sections H & E staining:

the retinas of 1-month-old mice were subjected to paraffin sectioning and staining by hematoxylin-eosin staining (H & E staining method), and the specific procedures were as follows:

1) Quickly taking eyeball tissues of the mouse, and placing the eyeball tissues in a stationary liquid for fixation for 24 hours;

2) Embedding in paraffin, and slicing to obtain slices with a thickness of 4 μm;

3) The slices were dewaxed conventionally with xylene, washed with multi-stage ethanol to water: xylene (I) 5min → xylene (II) 5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing 2min;

4) Hematoxylin staining for 5 minutes and washing with tap water;

5) Ethanol hydrochloride is differentiated for 30 seconds;

6) Soaking in tap water for 15 minutes;

7) Placing in eosin solution for 2 minutes.

8) Conventional dehydration, transparency, mounting: 95% ethanol (I) 1min → 95% ethanol (II) 1min → 100% ethanol (I) 1min → 100% ethanol (II) 1min → xylenesulfonic acid (3) 1min → xylene (I) 1min → xylene (II) 1min → neutral resin blocking.

9) The photographs were taken under a microscope.

As shown in fig. 4-6, with fossa [ Ift140 ^flox/flox ]As a control, the results in FIG. 4 show [ Rho-iCre, ift140 ] ^flox/flox ]The retina of the mouse is obviously thinned, pigment is abnormal, OCT shows that the outer layer is obviously atrophied, ERG in figure 5 shows that the functions of the cone rods are all seriously reduced, and HE staining result in figure 6 shows that the photoreceptor cells on the outer layer of the retina are atrophied.

In addition, the mice used in the experimental example were 1 month old, and from the experimental results, the retinal degeneration symptoms were evident at 1 month old, and it was confirmed that the deletion of the Ift140 gene caused early severe retinal degeneration.

Experimental example 4

This example is an observation of the natural course of disease, including changes in retinal structure and function, of the mice with conditional knockdown of Ift140 gene in retinal rods obtained in example 1.

Model mice of different age groups (PN 10, PN15, PN20 and PN 25) and littermate control mice were taken, three mice per group.

a. OCT and ERG data acquisition were performed.

b. Taking the eyeball to carry out paraffin embedding HE section, and observing the change of the thickness of each layer of retina.

As shown in FIGS. 7-8, with the same socket [ Ift140 ^flox/flox ]As a control, the mouse showed [ Rho-iCre, ift140 ^flox/flox ]Progressive degeneration of mouse retina structure and function. FIG. 7 shows OCT results showing significant atrophy of the outer layer from postnatal day 15 to postnatal day 25 of mice (FIG. 7-A), HE staining results showing significant atrophy disappearance of photoreceptor cells in the outer retina (FIG. 7-B), and FIG. 7-CThe thickness variation of each layer of the retina is shown. In fig. 8 it is shown that the ERG showed a severe reduction of cone rod function to flat waves after 30 days of age.

The observation recorded the natural course of disease, and the result further demonstrated that the deletion of the Ift140 gene caused early severe retinal degeneration.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for constructing an Ift140 gene rod-conditioned knockout mouse model is characterized by comprising the following steps: knock out the Ift140 gene in the mouse retinal rod cell genome.

2. The method for constructing according to claim 1, wherein the knock-out of the Ift140 gene in the mouse retinal rod cell genome is an exon sequence of the Ift140 gene in the mouse retinal rod cell genome.

3. The method for constructing a human rod eye model according to claim 2, wherein the Ift140 gene in the genome of the knockout mouse retinal rod eye cell is the exon 7 sequence of the Ift140 gene in the genome of the knockout mouse retinal rod eye cell.

4. The construction method according to any one of claims 1 to 3, characterized by comprising: knocking out the Ift140 gene in the mouse retina rod cell genome by using Cre-loxP knocking-out technology.

5. The building method according to claim 4, characterized in that the building method comprises: a Neo positive mouse targeted by the first built Ift140 gene is hybridized with a flp mouse to obtain a flox heterozygote mouse (flox/+) which removes the Neo gene of a targeting vector and contains two loxP sites, and then the flox heterozygote mouse (flox/flox) is obtained by mutual mating of the flox heterozygote mouse (flox/+) and then is mated with a Rho-iCre mouse to obtain a conditional rod cell Ift140 gene knockout mouse.

6. The construction method according to claim 5, wherein the Ift140 gene of the initial Ift140 gene targeted Neo positive mouse carries Neo and loxP sites;

preferably, the construction method of the first Neo positive mouse targeted by Ift140 gene comprises the following steps: injecting the targeted Ift140 gene ES cell line into a blastocyst cavity of a wild mouse, transplanting the injected blastocyst into a false pregnant mouse uterus, and obtaining the first established Neo positive mouse targeted by the Ift140 gene after the embryo grows to be mature.

7. The construction method of claim 6, wherein the initial Neo positive mouse targeted by Ift140 gene is hybridized with flp mouse, genotype identification is carried out, and flox heterozygote mouse (flox/+) containing two loxP sites is obtained by screening.

8. An Ift140 gene rod conditioned knockout mouse model, which is the rod conditioned knockout Ift140 gene mouse model constructed by the construction method according to any one of claims 1 to 7.

9. Use of the rod conditioned knockout Ift140 gene mouse model of claim 8 in screening for a medicament for preventing or treating a retinal degenerative disease.

10. The use of claim 9, wherein the retinal degenerative disease comprises early onset severe retinal degeneration.

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