CN112646783B - Expressed gene, construction method of fertilized egg modified by gene and construction method of mouse model - Google Patents

Abstract

The invention relates to a construction method of expressed genes, genetically modified fertilized eggs and a construction method of a mouse model, wherein the construction method of the genetically modified fertilized eggs comprises the following steps: and knocking in an expression gene for expressing a human interferon receptor on an interferon receptor gene of a mouse by using a CRISPR/Cas9 mediated homologous recombination technology to prepare a fertilized egg modified by the gene. The mouse model prepared from fertilized eggs prepared by the construction method can identify human interferon and normally respond to the stimulation of human interferon.

Description

Expressed gene, construction method of fertilized egg modified by gene and construction method of mouse model

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a construction method of a fertilized egg with expressed genes and modified genes and a construction method of a mouse model.

Background

According to the data published by the World Health Organization (WHO), 20 million people worldwide are infected with Hepatitis B Virus (HBV), 2.4 hundred million people among them are chronically HBV infected, HBV infection can cause various acute and chronic liver diseases, and about 60 ten thousand people worldwide die each year from HBV-related liver failure, cirrhosis and hepatocellular carcinoma.

For antiviral treatment of chronic hepatitis b, two major classes of drugs are currently approved for clinical use: a class of small molecule Nucleotide Analogs (NA) targeting HBV reverse transcription from pregenomic RNA (pgRNA) to the progeny viral DNA link, which can completely inhibit the production of new viruses in a short time, but cannot clear cccDNA, and has limited effect of reducing HBsAg; the other type of drugs is interferon-alpha (IFN-alpha) with direct antivirus and immunoregulation and its polyethylene glycol form ((PegIFN-alpha). A research shows that the interferon can increase antivirus immune response, inhibit virus from entering liver cells, induce partial degradation of cccDNA of HBV, inhibit cccDNA transcription and inhibit secretion of posttranscriptional virus through epigenetic science, and has important regulation and control functions on a plurality of links of HBV infected liver cells, so that the interferon becomes one of potential drugs capable of realizing an ideal end point of curing hepatitis B, but the action mechanism of the interferon in vivo is not clear, and the action mechanism of the interferon needs to be further researched on animal models.

The animal models of interferon-cured HBV currently available for research can be divided into the following classes: the first is naturally occurring animal models for HBV infection replication, such as gorilla, maolis monkey, tree shrew, woodchuck, etc., but the HBV infection animal models have high experimental cost, and the interferon receptor of the HBV infection animal models is far away from the interferon receptor of human from the evolutionary point of view, so that the action effect of the interferon in the animal models is a certain difference from the action effect of the interferon in the human body; the second type is a liver humanized mouse model, which can support high-level HBV replication, can test drugs directly acting on HBV (DAA drugs like hepatitis c), but has fundamental defects for evaluating the effect of interferon on HBV: 1) The construction of double-humanized mice with both liver and immune system is costly; 2) Except the liver and immune system, other organs only express murine interferon receptors and cannot normally respond to the stimulation of human interferon.

Disclosure of Invention

Based on this, it is necessary to provide a construction method of a genetically modified fertilized egg, by which a mouse model prepared from the fertilized egg can correctly recognize human interferon and normally respond to the stimulation of human interferon.

In addition, it is also necessary to provide a method for constructing an expressed gene, a mouse model, and a kit for constructing a mouse model, which has low construction cost and which can correctly recognize human interferon in response to the stimulation of human interferon.

A method for constructing a genetically modified fertilized egg, comprising the steps of:

knocking in an expression gene on a wild type mouse type I interferon receptor gene by using a CRISPR/Cas9 mediated homologous recombination technology to prepare a genetically modified fertilized egg, wherein the expression gene comprises:

a first functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

A junction region for connecting the first functional region and the second functional region, the junction region comprising a nucleic acid fragment encoding a protein having a self-cleaving function.

The construction method of the genetic modification has lower cost, and the obtained fertilized ovum can identify the human interferon when being used for preparing a mouse model, and normally responds to the stimulation of the human interferon.

A method for constructing a genetically modified fertilized egg, comprising the steps of:

in one embodiment, the step of preparing a genetically modified fertilized egg comprises:

constructing a homologous recombination vector containing an expressed gene according to a target site, wherein the target site is positioned on an I-type interferon receptor gene of the mouse;

designing a gRNA from the target site; and

Introducing the homologous recombination vector, the gRNA and Cas9 protein into a fertilized egg of the wild-type mouse, preparing a genetically modified fertilized egg.

In one embodiment, the target site is located in a region following the start codon of the second exon on the IFNAR2 gene of the mouse.

In one embodiment, the nucleotide sequence of the target site is shown in SEQ ID NO. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homology arm of the homologous recombination vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

In one embodiment, the wild-type mouse is a C57BL/6J mouse.

An expressed gene comprising:

a first functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

A junction region for connecting the first functional region and the second functional region, the junction region comprising a nucleic acid fragment encoding a protein having a self-cleaving function.

The construction method of the mouse model comprises the following steps:

constructing a genetically modified fertilized egg by adopting the construction method of the genetically modified fertilized egg;

and transplanting the genetically modified fertilized eggs into a surrogate mouse to prepare a mouse model.

In one embodiment, after the step of transplanting the genetically modified fertilized egg into a surrogate mouse, further comprising:

raising the surrogate mice to obtain F0 surrogate mice; and

And (3) carrying out homozygote screening on the F0-generation mice to obtain a mouse model.

A kit for constructing a mouse model, comprising:

a homologous recombination vector comprising two homology arms and an expression gene located between the two homology arms, the homology arms being capable of homologous recombination with a target site, the expression gene comprising a first functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1 and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1, a second functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2 and a transcription terminator, and a junction region for connecting the first functional region to the second functional region, the junction region comprising a nucleic acid fragment encoding a protein with self-cleaving function; and

gRNA, designed from the target site.

In one embodiment, the Cas9 protein is also included.

In one embodiment, the nucleotide sequence of the target site is shown in SEQ ID NO. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homology arm of the homologous recombination vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

Drawings

FIG. 1 is a schematic diagram of a transgenic strategy of a mouse model according to an embodiment;

FIG. 2 is a schematic diagram of the structure of a chimeric receptor of human murine type I interferon formed after expression of a expressed gene in the embodiment shown in FIG. 1;

FIG. 3 is a schematic diagram of the structure of a wild-type mouse type I interferon receptor;

FIG. 4 is a schematic diagram of the primer design in step 2 of test verification;

FIG. 5 is a gel electrophoresis diagram of the PCR products in step 2 of the test verification;

FIG. 6 shows the results of Knockin at the 5' end of homozygous mice in step 3 of sequencing validation;

FIG. 7 shows the results of Knockin at the 3' end of homozygous mice in step 3 of sequencing validation;

FIG. 8 is an expression profile of the extracellular domain of human IFNAR2 in IFNAR2 mice;

FIG. 9 shows expression of MX1 gene in liver, blood and spleen of IFNAR2 mice after stimulation with human interferon;

FIG. 10 shows the expression of the ISG15 gene in liver, blood and spleen of IFNAR2 mice after stimulation with human interferon.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate the understanding of the invention, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Interferons are a class of active proteins with broad-spectrum antiviral functions on homogeneous cells, whose activity is regulated and controlled by cellular genes. Interferons include type I interferons and type II interferons. Type I interferons IFN- α and IFN- β, wherein IFN- α has twenty subtypes and IFN- β has only one subtype. In vivo, the receptor that binds IFN-a and IFN- β is the type I interferon a/β receptor (IFNAR). IFNAR contains two subunits, a and β, known as type I interferon receptor 1 (interferon alpha receptor 1, IFNAR 1) and type I interferon receptor2 (IFNAR 2), respectively, and the structures of IFNAR1 and IFNAR2 each include domains for extracellular, transmembrane and intracellular use.

Referring to fig. 1, an embodiment of the present invention provides a method for constructing a mouse model, which includes steps a to b:

step a: and knocking in the expressed gene on the I-type interferon receptor gene of the wild mouse by using a CRISPR/Cas9 mediated homologous recombination technology to prepare the fertilized ovum modified by the gene.

Specifically, the expressed gene includes a first functional region, a second functional region, and a junction region.

The first functional region comprises a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, and a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1, the first functional region being for expressing the extracellular domain of human type I interferon receptor 1, the transmembrane domain of mouse type I interferon receptor 1, and the intracellular domain of mouse type I interferon receptor 1.

Alternatively, in the first functional region, a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1 are sequentially linked.

In this embodiment, the nucleotide sequence of the nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1 is shown in SEQ ID NO. 5; the nucleotide sequence of the nucleic acid fragment encoding the transmembrane domain of the mouse type I interferon receptor 1 is shown in SEQ ID NO. 6; the nucleotide sequence of the nucleic acid fragment encoding the intracellular domain of the mouse type I interferon receptor 1 is shown in SEQ ID NO. 7.

The second functional region comprises a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2, and a transcription terminator, and is useful for expressing the extracellular domain of human type I interferon receptor2, the transmembrane domain of mouse type I interferon receptor2, and the intracellular domain of mouse type I interferon receptor 2.

Optionally, in the second functional region, a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2, and a transcription terminator are sequentially linked. The terminator serves to stop transcription. Alternatively, the terminator is polyA.

In this embodiment, the nucleotide sequence of the nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2 is shown in SEQ ID NO. 8; the nucleotide sequence of the nucleic acid fragment encoding the transmembrane domain of the mouse type I interferon receptor2 is shown in SEQ ID NO. 9; the nucleotide sequence of the nucleic acid fragment encoding the intracellular domain of the mouse type I interferon receptor2 is shown in SEQ ID NO. 10; the nucleotide sequence of the terminator is shown as SEQ ID NO. 11.

The junction region includes a nucleic acid fragment encoding a protein having a self-cleaving function for linking the first functional region to the second functional region. Alternatively, the junction region comprises a nucleic acid fragment encoding a2A peptide. In one specific example, the nucleotide sequence of the nucleic acid fragment encoding the 2A peptide is shown in SEQ ID NO.12

More specifically, steps a1 to a3 of preparing a genetically modified fertilized egg:

step a1: constructing homologous recombination vector containing expressed genes according to target sites.

Specifically, the target site is located on a mouse type I interferon receptor gene. Alternatively, the target site is located on a type I interferon 2 gene.

In this embodiment, the target site is located in a region following the start codon of the second exon on the type I interferon receptor2 (IFNAR 2) gene. In one specific example, the nucleotide sequence of the target site is shown as SEQ ID NO. 1. Of course, in other embodiments, the target site is not limited to the above, but may be located at other positions of the mouse type I interferon receptor gene. So long as it is capable of allowing expression of a human murine type I interferon chimeric receptor (human type I interferon receptor2 extracellular domain-mouse type I interferon receptor2 transmembrane domain-mouse type I interferon receptor2 intracellular domain, and human type I interferon receptor 1 extracellular domain-mouse type I interferon receptor 1 transmembrane domain-mouse type I interferon receptor 1 intracellular domain, resulting in the human murine type I interferon chimeric receptor) after expression of the gene knock-in, and does not express the mouse type I interferon receptor.

Specifically, the homologous recombination vector containing the expressed gene includes two homology arms and the above expressed gene located between the homology arms. Homology arms are used for homologous recombination with the target site. Homology arms are designed according to the target site. In this embodiment, the nucleotide sequence of the homology arm is shown in SEQ ID NO. 3-SEQ ID NO. 4.

The step of constructing a homologous recombination vector containing an expressed gene includes: inserting the homology arm and the expression gene into empty vector to prepare the homologous recombination vector containing the expression gene.

Step a2: gRNA was designed according to the target site.

In this embodiment, the target site is adjacent to the second exon of the IFNAR2 gene, and the nucleotide sequence of the target site is shown in SEQ ID NO. 1. Therefore, the gRNA in this embodiment is designed based on the target site with the sequence shown as SEQ ID NO.1, wherein the PAM sequence is 5'-CGG-3'. Alternatively, the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2.

Of course, in other embodiments, the nucleotide sequence of the gRNA is not limited to the above, but may be other.

Step a3: introducing the homologous recombination vector, the gRNA and the Cas9 protein into fertilized eggs of a type I interferon receptor-deficient mouse to prepare fertilized eggs containing expressed genes.

Specifically, under the guidance of the gRNA, the Cas9 protein reaches the target site and breaks the double-stranded DNA of the target site, and simultaneously, the repair mechanism of the cell makes the homologous arm on the homologous recombination vector and two fragments formed after the target site is broken undergo homologous recombination, so that the expressed gene is connected to the connection part of the target site, and the expressed gene is knocked into the target site.

In this embodiment, the wild type mice were C57BL/6 mice. The C57BL/6 mouse strain is stable and easy to reproduce, is also the first mouse strain for finishing genome sequencing, is genetically pure, and can ensure high stability on genetic background when being applied to gene knockout.

Referring to fig. 2 and 3, the expression gene expression results in a chimeric human murine type I interferon receptor (see fig. 2), whose extracellular domain is the extracellular domain of a human interferon receptor, compared to the structure of a wild-type murine type I interferon receptor (see fig. 3).

Step b: fertilized eggs containing the expressed genes were cultured to prepare a mouse model.

Specifically, the step of preparing a mouse model by culturing genetically modified fertilized eggs comprises the steps of b1 to b2: step b1: culturing fertilized eggs containing expressed genes to obtain F0 generation mice.

Transferring fertilized eggs into a surrogate mouse body, and culturing to obtain an F0 generation mouse.

Step b2: homozygous selection was performed on F0 mice to prepare a mouse model.

Specifically, homozygous mice capable of inheriting a stable shape were obtained by homozygous selection of F0 mice.

Alternatively, screening for homozygotes is performed using classical breeding in combination with PCR identification. Specifically, the F0 mice were bred with wild-type mice to produce F1 mice, and positive mice among the F1 mice were identified by PCR. The F2 mice were then generated by sibling mating of positive mice in the F1 generation. The F3 mice were generated by selecting pairs of positive mice from F2 mice and mating them. Based on the integration rate of each mouse and the PCR sequencing result, a homozygote mouse is selected.

The construction method of the mouse model has at least the following advantages:

the CRISPR/Cas9 mediated homologous recombination technology enables the expression of the extracellular domain of the human type I interferon receptor in mice, and the transmembrane region domain and the intracellular domain of the mouse type I interferon receptor are reserved, so that the constructed mice can correctly recognize human interferon and induce the response of downstream genes of the mice. Compared with the traditional liver humanized mouse model, the construction method of the mouse model is low in cost, and the constructed mouse model can respond to the stimulation of human interferon; compared with the traditional transgenic mouse model, the mouse model constructed by the mouse model has the advantages that the I-type interferon receptor is inactivated, the recognition of the human interferon by the human I-type interferon receptor is not interfered, and the natural expression mode of the human I-type interferon receptor can be reflected. Compared with other traditional animal models with far avidity, the mouse model has closer avidity with human, and can better reflect the action effect of human interferon in vivo.

One embodiment of the present invention provides an expressed gene for expression of a chimeric receptor for human murine type I interferon. That is, the expressed genes are used to express the extracellular domain of human type I interferon receptor 1, the transmembrane domain of mouse type I interferon receptor 1 and the intracellular domain of mouse type I interferon receptor 1, and to express the extracellular domain of human type I interferon receptor2, the transmembrane domain of mouse type I interferon receptor2 and the intracellular domain of mouse type I interferon receptor2, thereby forming a human type I interferon chimeric receptor. The specific composition of the expressed gene is as described above.

In addition, an embodiment of the present invention also provides a kit for constructing a mouse model, which corresponds to the method for constructing a mouse model described above, that is, the kit can be used for constructing a mouse model described above. Specifically, the kit comprises: homologous recombination vectors and grnas.

Specifically, the homologous recombination vector includes two homology arms and an expressed gene located between the two homology arms. The design and expression of the homology arms are as described above and will not be described in detail here.

In some embodiments, the above-described kits further comprise a Cas9 protein.

The kit for constructing the mouse model is simple and convenient, and the mouse model prepared by using the kit can truly reflect the natural expression mode of the human interferon receptor and normally respond to the stimulation of human interferon.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.

Example 1

The transgene strategy of Hu-IFNAR2 mice is schematically shown in FIG. 1, and comprises the following steps.

(1) Constructing a homologous recombination vector: a homologous recombination vector is prepared by inserting an expression gene comprising two homology arms and located between the two homology arms into an empty vector. Wherein: the empty vector is a conventional T vector (pUC-57), the nucleotide sequence of the homology arm is shown as SEQ ID NO. 3-SEQ ID NO.4, the expression gene is composed of a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 5) which codes for the extracellular domain of the human type I interferon receptor 1, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 6) which codes for the transmembrane domain of the mouse type I interferon receptor 1, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 7) which codes for the intracellular domain of the mouse type I interferon receptor 1, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 12) which codes for the 2A peptide, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 8) which codes for the extracellular domain of the human type I interferon receptor2, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 9) which codes for the transmembrane domain of the mouse type I interferon receptor2, a nucleic acid fragment (the nucleotide sequence is shown as SEQ ID NO. 10) which codes for the intracellular domain of the mouse type I interferon receptor2, and a nucleotide terminator (nucleotide sequence is shown as SEQ ID NO. 11).

(2) In vitro, the homologous recombination vector, gRNA (nucleotide sequence shown as SEQ ID NO. 2) and Cas9 protein are introduced into fertilized eggs of mice C57BL/6J to prepare fertilized eggs containing expressed genes.

(3) Transferring fertilized eggs into a body, and culturing to obtain F0 generation mice.

(4) The F0 mice were bred with wild type mice to produce F1 mice, and positive mice among the F1 mice were identified by PCR. The F2 mice were then generated by sibling mating of positive mice in the F1 generation. The F3 mice were generated by selecting pairs of positive mice from F2 mice and mating them. And selecting homozygous mice according to the integration rate of each mouse and the PCR sequencing result.

Test verification

1. DNA was extracted from homozygous mice (hereinafter, or IFNAR2 mice), heterozygous mice and wild type mice (WT mice), and the knock-in position of the homozygous mice was confirmed:

step 1: the DNA of homozygous mice was extracted using a tissue genome DNA extraction kit (Tiangen biochemical technology), and the operations of the three types of mice all included: 1) Taking 20mg of mouse muscle tissue, processing the mouse muscle tissue into a cell suspension by using a grinder, centrifuging the cell suspension at 10000rpm (11200 Xg) for 1min, pouring out the supernatant, and adding 200 mu L of buffer GA; oscillating until thoroughly suspending; 2) Adding 20uL of protease K, mixing, and standing at 56 ℃ until the tissue is dissolved; 3) Adding 200 mu L of buffer solution GB, fully reversing and uniformly mixing, and standing at 70 ℃ for 10min to clear the solution; 4) Adding 200 mu L of absolute ethyl alcohol, fully oscillating and uniformly mixing for 15s, and removing water drops on the inner wall of the tube cover after short centrifugation when flocculent precipitate appears in the tube; 5) Adding the solution obtained in the last step and flocculent precipitate into an adsorption column, centrifuging at 12000rpm (13400 Xg) for 30s, pouring out waste liquid, and placing the adsorption column into a collecting pipe; 6) Adding 500 mu L of buffer GD into an adsorption column, centrifuging at 12000rpm for 30s, pouring out waste liquid, and placing the adsorption column into a collecting pipe; 7) Adding 600 μl of rinse solution PW (anhydrous ethanol is added before use) into the adsorption column, centrifuging at 12000rpm for 30s, pouring out the waste liquid, and placing the adsorption column into a collecting tube; 8) Repeating the operation step 7); 9) The column was put back into the collection tube, centrifuged at 12000rpm for 2min, and the waste liquid was discarded. Placing the attached column at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material; 10 Transferring the adsorption column into a clean centrifuge tube, suspending and dripping 50 μl of RNase-free water into the middle part of the adsorption membrane, standing at room temperature for 3min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.

Step 2: PCR amplification of DNA fragments to be sequenced:

first, various inserts (5 'end, 3' end and full sequence) were amplified from the extracted DNA using PCR technique, and the specific schematic diagram is shown in FIG. 4, wherein "Knock in fragment" in FIG. 4 indicates expressed genes), and PCR primer information is shown in Table 1 below.

TABLE 1

Then, utilizeThe Taq 2 Xmix kit (NEB, M0287S) amplified the corresponding DNA fragments. Wherein the amplification system is shown in Table 2, and the PCR amplification procedure is: pre-denaturation at 94 ℃ for 5min; denaturation at 94℃for 30 seconds; annealing at 65 ℃ for 30 seconds; extension of 72 ℃ (extension time of the reaction systems of primer number (1) and (2) is 3 minutes, extension time of the reaction system of primer number (3) is 5 minutes); the number of cycles was 30.

TABLE 2

Finally, the PCR products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 5. Wild Type (WT), homozygote (Homozygous, homo) and heterozygote (hetrozygous, heter) have been identified in the figure.

As can be seen from FIG. 5, the construction of homozygous mice was successful, and it was confirmed that the genetically modified homozygous mice could be bred normally.

Step 3: agarose gel DNA recovery: the PCR product is recovered by using a common agarose gel DNA recovery kit (Tiangen biochemical technology), and the specific operations comprise: 1) Column balance: adding 500 mu L of balance liquid BL into the adsorption column, centrifuging at 12000rpm for 1min, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again; 2) Cutting a single target DNA strip from agarose gel (cutting off redundant parts as much as possible), putting the cut target DNA strip into a clean centrifuge tube, and weighing; 3) Adding equal volume of PN sol solution (100 mu L of PN sol solution is added if the gel weight is 0.1g and the volume can be regarded as 100 mu L) into the gel block, placing in a water bath at 50 ℃, and continuously and gently turning the centrifuge tube up and down to ensure that the gel block is fully dissolved; 4) Adding the solution obtained in the last step into an adsorption column (the adsorption column is placed in a collecting pipe), standing for 2min at room temperature, centrifuging at 12000rpm for 60s, pouring out waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe; 5) Adding 600 mu L of rinsing liquid PW into the adsorption column, centrifuging at 12000rpm for 60s, pouring out waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe; 6) Repeating the operation step 5); 7) Placing the adsorption column into a collecting pipe, centrifuging at 12000rpm for 2min, and removing the rinse solution as much as possible. Placing the adsorption column at room temperature for several minutes, and thoroughly airing; 8) Placing the adsorption column into a clean centrifuge tube, suspending and dripping 50 mu L of RNase-free water into the middle position of the adsorption film, and standing at room temperature for 2min. The DNA solution was collected by centrifugation at 12000rpm for 2min.

Step 4: the recovered product was sent to the test, and the results are shown in FIGS. 6 to 7. FIG. 6 shows the Knock in result at the 5 'end of the homozygous mouse, and FIG. 7 shows the Knock in result at the 3' end of the homozygous mouse.

As can be seen from FIGS. 6 and 7, the Knock in of the homozygote was correct.

2. Expression of the extracellular domain of human IFNAR2 in homozygous mice (IFNAR 2 mice):

liver tissue RNA of homozygous mice and WT mice is extracted by using a tissue total RNA extraction kit (Tiangen biochemical technology), and the specific operation comprises the following steps:

1) The liver tissue of IFNAR2 mouse and WT mouse was taken at about 20g each, the tissue was thoroughly ground with a grinding pestle, 300. Mu.L of a lysate RL (containing 1% beta-mercaptoethanol) was added, and 590. Mu.L of RNase-Free ddH was then added to the homogenate ₂ O and 10. Mu.L of protease K,mixing, and treating at 56 deg.C for 20min; 2) Centrifuging at 12000rpm for 5min, collecting supernatant, slowly adding anhydrous ethanol with volume 0.5 times of that of the supernatant, mixing, transferring the obtained solution and precipitate into an adsorption column, centrifuging at 12000rpm for 60s, discarding the waste liquid in the collection pipe, and placing the adsorption column into the collection pipe; 3) Adding 350 mu L deproteinized liquid RW1 into the adsorption column, centrifuging at 12000rpm for 360s, discarding the waste liquid, and placing the adsorption column back into a collecting pipe; 4) To the center of the adsorption column, 80. Mu.L of DNase I working solution (preparation of DNase I working solution: taking 10 mu L of DNase I storage solution, placing the DNase I storage solution into a new RNase-Free centrifuge tube, adding 70 mu L of RDD buffer solution, gently mixing the DNase I storage solution and the RDD buffer solution, and standing the DNase I storage solution at room temperature for 15min; 5) Adding 350 mu L deproteinized liquid RW1 into the adsorption column, centrifuging at 12000rpm for 60s, discarding the waste liquid, and placing the adsorption column back into a collecting pipe; 6) Adding 500 mu L of rinsing liquid RW into the adsorption column, standing for 2min at room temperature, centrifuging at 12000rpm for 60s, discarding the waste liquid, and placing the adsorption column back into a collecting pipe; 7) Repeating step 6); 8) Centrifuge at 12000rpm for 2min, and discard the waste liquid. Placing the adsorption column at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material; 9) Transferring the adsorption column into a new RNase-Free centrifuge tube, suspending and dripping 50 μl of RNase-Free ddH2O into the middle part of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain RNA solution.

2) The detection of the level of human IFNAR2 RNA in homozygous mice (IFNAR 2 mice) comprises the following specific steps:

a. extracting IFNAR2 and WT mouse liver RNA by using an RNA extraction kit;

b. reverse transcribing each RNA into cDNA;

c. the detection was performed using Bio-rad SYBR detection reagents. Wherein the primer sequence of the human IFNAR2 is F: CACAAGCCTGAGATCAAG (SEQ ID NO. 19), R: TAGACAGAGACACAGTAGTT (SEQ ID NO. 20). The results are shown in FIG. 8, and in FIG. 8, the ordinate represents the multiple of human IFNAR2 gene expression.

As can be seen from FIG. 8, human IFNAR2 was expressed in homozygous mice.

3. Functional identification of human IFNAR2 receptor in homozygous mice (IFNAR 2 mice):

(1) Homozygous mice were injected subcutaneously with interferon PEG-IFNa2a (trade name: perroxen, roche company) at a dose of 10ug/kg, and after 16 hours of injection, the mice were euthanized and blood and individual organs were collected.

(2) And (3) extracting RNA of blood, liver and spleen of the IFNAR2 mouse by using an RNA extraction kit.

(3) Each RNA was reverse transcribed into cDNA, respectively.

(4) The detection was performed using Bio-rad SYBR detection reagents. Wherein: the MX1 gene and the ISG15 gene are target genes, and the upstream primer of the MX1 gene is as follows: AAGATGGTCCAAACTGCCTTCG (SEQ ID NO. 21); downstream primer of MX1 gene: GCCTTGGTCTTCTCTTTCTCAGC (SEQ ID NO. 22); upstream primer of ISG15 gene: AACTGCAGCGAGCCTCTGA (SEQ ID NO. 23); downstream primer of ISG15 gene: CACCTTCTTCTTAAGCGTGTCTACAG (SEQ ID NO. 24), GAPDH (upstream primer: AGGTCGGTGTGAACGGATTTG (SEQ ID NO. 25)) and downstream primer: TGTAGACCATGTAGTTGAGGTCA (SEQ ID NO. 26)) are internal genes. The reaction liquid of RT-qPCR was as follows:

the RT-qPCR reaction system is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 60 ℃;30s; extending at 65 ℃ for 30s;35 cycles.

The results are shown in FIGS. 9 and 10, where FIG. 9 shows the expression of the MX1 gene in the liver (liver), blood (blood) and spleen (spleen) of IFNAR2 mice after human interferon stimulation, and FIG. 10 shows the expression of the ISG15 gene in the liver (liver), blood (blood) and spleen (spleen) of IFNAR2 mice after human interferon stimulation. In FIG. 9, the ordinate represents the fold expression of MX1 gene, and in FIG. 10, the ordinate represents the fold expression of ISG15 gene.

As can be seen from FIGS. 9 and 10, the IFNAR2 mice constructed in example 1 can activate the response of the downstream genes of the mice under the stimulation of human interferon, that is, can normally respond to the stimulation of human interferon.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> Guangzhou City eighth people's hospital

GUANGZHOU FURONG BIOTECHNOLOGY Co.,Ltd.

<120> expressed gene, method for constructing genetically modified fertilized egg and method for constructing mouse model

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

agctgagcag gatgcgttca 20

<210> 2

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

agctgagcag gatgcgttca cgg 23

<210> 3

<211> 304

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

caaacttgct atgtagccga ggatgacctt gaatgcctga cctttccccc acctctctgc 60

ttagaggaca gatgtgacac gcacagtaaa ctcatgcaag tttaacccta atcctaacca 120

atccagggct accacggggc cgcatctgca gctaaatctg gctcgttctt actcgtctct 180

cgttagcgtg tatgtgtcta tcatgtaaat tacaatataa ttgggtgctt ctgagttttg 240

accaactcaa tattgatctc tttcaggtgt gagagcagaa aaacggactt aagagctgag 300

cagg 304

<210> 4

<211> 420

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ttgtgggtaa gggctacttc tcagcacagc ccttagagga gaaagcctct gtttctgtca 60

tcacagagag ccctggtgtg gagcagcaca ctgatgtcca tatctggaga acccagatca 120

gcacggccag catcaggcac cccacggggg tcttcccctt cattttagct aagccagaat 180

aatataggct acagccatat tcaggaaacg gccttgttta taattcaaag ggttgcggct 240

ctgcacaccc tgaatctcac gcccggtggc gtttagaagg tggccatccc tttatctctt 300

cccatataaa ctaacttgaa aaatccatcc ctacacattg atttatactc ttcctttctt 360

tttagccctt atcttttcta tatctgtatt tcttcacgtc ctttctgttc cttcctctga 420

<210> 5

<211> 1296

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atgatggtgg tgctgctggg cgccaccacc ctggtgctgg tggctgtggc cccatgggtg 60

ctgtctgctg ctgctggcgg caagaacctg aagtcccccc agaaggtgga ggtggacatc 120

attgatgaca acttcatcct gaggtggaac aggtctgatg agtctgtggg caatgtgacc 180

ttctcctttg actaccagaa gacaggcatg gacaactgga tcaagctgtc tggctgccag 240

aacatcacct ccaccaagtg caacttctcc tccctgaaac tgaatgtcta tgaggagatc 300

aagctgagga tcagggctga gaaggagaac acctcctcct ggtatgaggt ggactccttc 360

accccattcc gcaaggccca gattggcccc cctgaagtgc atctggaggc tgaggacaag 420

gccattgtga tccacatctc ccctggcacc aaggactctg tgatgtgggc tctggatggc 480

ctgtccttca cctactccct ggtgatctgg aagaactcct ctggcgtgga ggagaggatt 540

gagaacatct actccaggca caagatctac aagctgtccc ctgagaccac ctactgcctg 600

aaggtgaagg ctgccctgct gacctcctgg aagattggcg tctactcccc tgtgcactgc 660

atcaagacca cagtggagaa tgagctgccc ccccctgaga acattgaggt ctctgtgcag 720

aaccagaact atgtgctgaa gtgggactac acctatgcca acatgacctt ccaagtgcag 780

tggctgcatg ccttcctgaa gaggaaccct ggcaaccatc tgtacaagtg gaagcagatc 840

cctgactgtg agaatgtgaa gaccacccag tgtgtcttcc cccaaaatgt cttccagaag 900

ggcatctacc tgctgagggt gcaggcctct gatggcaaca acacctcctt ctggtctgag 960

gagatcaagt ttgacacaga gatccaggcc ttcctgctgc cccctgtctt caacatccgc 1020

tccctgtctg actccttcca catctacatt ggcgccccca agcagtctgg caacacccct 1080

gtgatccagg actaccccct gatctatgag atcatcttct gggagaacac ctccaatgct 1140

gagaggaaga tcattgagaa gaagacagat gtgacagtgc ccaacctgaa gcccctgaca 1200

gtctactgtg tgaaggccag ggctcacacc atggatgaga agctgaacaa gtcctctgtc 1260

ttctctgatg ctgtctgtga gaagaccaag cctggc 1296

<210> 6

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tccttctcca ccatctggat catcaccggc ctgggcgtgg tcttc 45

<210> 7

<211> 450

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ttctctgtga tggtgctgta tgccctgagg tctgtctgga agtacctgtg ccatgtctgc 60

ttcccccccc tgaaaccccc ccgctccatt gatgagttct tctctgagcc cccatccaag 120

aacctggtgc tgctgacagc tgaggagcac acagagcgct gcttcatcat tgagaacaca 180

gacacagtgg ctgtggaggt gaagcatgcc cctgaggagg acctgaggaa gtactcctcc 240

cagacctccc aagactctgg caactactcc aatgaggagg aggagtctgt gggcacagag 300

tctggccagg ctgtgctgtc caaggcccca tgtggcggcc catgctctgt gccatccccc 360

cctggcaccc tggaggatgg cacctgcttc ctgggcaatg agaagtacct gcagtcccct 420

gccctgagga cagagcctgc cctgctgtgc 450

<210> 8

<211> 693

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atgctgctgt cccagaatgc cttcatcttc cgctccctga acctggtgct gatggtctac 60

atctccctgg tctttggcat ctcctatgac tcccctgact acacagatga gtcctgcacc 120

ttcaagatct ccctgaggaa cttcaggtcc atcctgtcct gggagctgaa gaaccactcc 180

attgtgccca cccactacac cctgctgtac accatcatgt ccaagcctga ggacctgaag 240

gtggtgaaga actgtgccaa caccaccagg tccttctgtg acctgaccga tgagtggagg 300

tccacccatg aggcctatgt gacagtgctg gagggcttct ctggcaacac caccctgttc 360

tcctgctccc acaacttctg gctggccatt gacatgtcct ttgagccccc tgagtttgag 420

attgtgggct tcaccaacca catcaatgtg atggtgaagt tcccatccat tgtggaggag 480

gagctgcagt ttgacctgtc cctggtgatt gaggagcagt ctgagggcat tgtgaagaag 540

cacaagcctg agatcaaggg caacatgtct ggcaacttca cctacatcat tgacaagctg 600

atccccaaca ccaactactg tgtctctgtc tacctggagc actctgatga gcaggctgtg 660

atcaagtccc ccctgaagtg caccctgctg ccc 693

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cctggccagg agtctgagtc tgctgagtct gccattgtgg gcatc 45

<210> 10

<211> 801

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

accacctcct gcctggtggt gatggtcttt gtctccacca ttgtgatgct gaagaggatt 60

ggctacatct gcctgaagga caacctgccc aatgtgctga acttcaggca cttcctgacc 120

tggatcatcc ctgagaggtc cccatctgag gccattgaca ggctggagat catccccacc 180

aacaagaaga agaggctgtg gaactatgac tatgaggatg gctctgactc tgatgaggag 240

gtgcccacag cctctgtgac aggctacacc atgcatggcc tgacaggcaa gcccctgcag 300

cagacctctg acacctctgc ctcccctgag gaccccctgc atgaggagga ctctggcgct 360

gaggagtctg atgaggctgg cgctggcgct ggcgctgagc ctgagctgcc cacagaggct 420

ggcgctggcc catctgagga ccccacaggc ccatatgaga ggaggaagtc tgtgctggag 480

gactccttcc ccagggagga caactcctcc atggatgagc ctggcgacaa catcatcttc 540

aatgtgaacc tgaactctgt cttcctgagg gtgctgcatg atgaggatgc ctctgagacc 600

ctgtccctgg aggaggacac catcctgctg gatgagggtc cccagaggac agagtctgac 660

ctgaggattg ctggcggcga caggacccag ccccccctgc catccctgcc atcccaagac 720

ctgtggacag aggatggctc ctctgagaag tctgacacct ctgactctga tgctgatgtg 780

ggcgatggct acatcatgag g 801

<210> 11

<211> 537

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct 60

cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc 120

cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 180

aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag 240

aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa 300

ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc 360

catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt 420

tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc 480

tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcag 537

<210> 12

<211> 66

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggctctggcg ccaccaactt ctccctgctg aagcaggctg gcgatgtgga ggagaaccct 60

gggccc 66

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ggtggctacc gtaatgtcgg ta 22

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agcctccaga tgcacttcag gg 22

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tggacaactg gatcaagctg tc 22

<210> 16

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ggcccagact gtcaacatta ctct 24

<210> 17

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

cttagaggac agatgtgaca cgc 23

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gacatcagtg tgctgctcca cac 23

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

cacaagcctg agatcaag 18

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tagacagaga cacagtagtt 20

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

aagatggtcc aaactgcctt cg 22

<210> 22

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gccttggtct tctctttctc agc 23

<210> 23

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

aactgcagcg agcctctga 19

<210> 24

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

caccttcttc ttaagcgtgt ctacag 26

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

aggtcggtgt gaacggattt g 21

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

tgtagaccat gtagttgagg tca 23

Claims (5)

1. A method for constructing a genetically modified fertilized egg, comprising the steps of:

knocking in an expression gene on a type I interferon receptor gene of a wild mouse by using a CRISPR/Cas9 mediated homologous recombination technology to prepare a fertilized egg modified by the gene;

wherein the step of preparing a genetically modified fertilized egg comprises: constructing a homologous recombination vector containing an expressed gene according to a target site located in a region after the start codon of the second exon on the IFNAR2 gene of the wild-type mouse; designing a gRNA from the target site; and introducing the homologous recombination vector, the gRNA and Cas9 protein into a fertilized egg of the wild mouse, preparing a genetically modified fertilized egg;

the expressed genes include: a first functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1; a second functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and a junction region for connecting the first functional region and the second functional region, the junction region comprising a nucleic acid fragment encoding a protein having a self-cleaving function;

the nucleotide sequence of the target site is shown as SEQ ID NO. 1; the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2; the nucleotide sequence of the homology arm of the homologous recombination vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

2. The method of claim 1, wherein the wild-type mouse is a C57BL/6J mouse.

3. The construction method of the mouse model is characterized by comprising the following steps of:

constructing a genetically modified fertilized egg by adopting the construction method of the genetically modified fertilized egg of any one of claims 1-2;

and transplanting the genetically modified fertilized eggs into a surrogate mouse to prepare a mouse model.

4. The construction method according to claim 3, further comprising, after the step of transplanting the genetically modified fertilized egg into a surrogate mouse:

raising the surrogate mice to obtain F0 surrogate mice; and

And (3) carrying out homozygote screening on the F0-generation mice to obtain a mouse model.

5. A kit for constructing a mouse model, comprising:

a homologous recombination vector comprising two homology arms and an expression gene located between the two homology arms, the homology arms being capable of homologous recombination with a target site, the expression gene comprising a first functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor 1 and a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor 1, a second functional region comprising a nucleic acid fragment encoding an extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding a transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding an intracellular domain of mouse type I interferon receptor2 and a transcription terminator, and a junction region for connecting the first functional region to the second functional region, the junction region comprising a nucleic acid fragment encoding a protein with self-cleaving function; and

gRNA, designed from the target site;

the nucleotide sequence of the target site is shown as SEQ ID NO. 1; the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2; the nucleotide sequence of the homology arm of the homologous recombination vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.