CN114591953A - Construction method of CD163 gene swine mouse model - Google Patents

Abstract

The invention discloses sgRNA, the expression cassette, the recombinant vector or the cell, and also discloses the sgRNA, the expression cassette, the recombinant vector or the cell, and application of the primer pair in CD163 gene editing. The invention also discloses a targeting vector and a preparation method and application thereof. The invention finally discloses a construction method of the CD163 gene humanized mouse model and a method for identifying the CD163 gene humanized mouse. According to the invention, by the CRISRP/Cas9 gene modification method, CD163 gene of a pig is replaced by Cd163 gene of a mouse, a CD163 swine-derived mouse model is prepared, the CD163 swine-derived mouse model has functional genes of the pig, has very important significance for research on infection, invasion, immunity and the like of swine-derived related viruses, can possibly become an ideal animal model for screening and evaluating related medicines and diagnostic products of African swine fever, blue-ear disease and the like, and brings new ideas and strategies for controlling the development of epidemic situations.

Description

Construction method of CD163 gene swine mouse model

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method of a CD163 gene humanized mouse model.

Background

The epidemic situation of African swine fever spread in recent years brings serious striking to the pig industry in China, and is the number one killer of the pig industry in China at present. African Swine Fever (ASF) is a swine virulent infectious disease caused by African Swine Fever Virus (ASFV), which presents great difficulties and challenges to the swine industry worldwide. The death rate of hyperacute and acute infection caused by virulent strain is nearly as high as 100%. In 8 months in 2018, ASF is introduced into China, and epidemic outbreaks occur in 32 provinces in China at present.

The African Swine Fever Virus (ASFV) is a unique double-stranded DNA virus, mainly enters tonsil through oral and nasal mucosa, enters cells in a receptor-mediated endocytosis mode, and rapidly spreads to the whole body through lymph fluid and blood after replication. Immunization is the first barrier against ASFV in pigs. Monocytes and macrophages are the most prevalent infected cells of ASFV, and macrophages that are highly mature, with cells expressing high levels of specific markers, are most susceptible to ASFV.

The CD163 scavenger receptor is a receptor on the surface of porcine macrophages and monocytes, and studies have suggested that macrophages expressing the CD163 receptor, surface antigen 4E9 (porcine CD107a or lysosome-associated membrane protein I) are susceptible to ASFV infection. It has also been shown that the CD163 receptor is a necessary but insufficient condition for infection with ASFV (e.g.highly pathogenic Georgia 2007/1strain), suggesting that additional receptor assistance is required for infection with ASFV. In addition, many reports of gene knockout pigs constructed by aiming at the CD163 gene are reported at home and abroad, and the fact that the gene-deleted pigs can resist highly Pathogenic and highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection, which is the most important receptor for mediating PRRSV infection, is proved. The CD163 gene modification significantly inhibited PRRSV replication both in vitro and in vivo, and these findings suggest that modifying CD163 may provide a potential strategy for anti-PRRSV therapy.

One of the best ways to prevent the spread of epidemic diseases is to vaccinate with an effective vaccine, but no effective vaccine against it is available worldwide to date. The research and development of the vaccine are slow because the African swine fever virus has a complex structure, has various immune escape mechanisms and does not induce the generation of neutralizing antibodies, and scientists do not know about the specific mechanism for stimulating protective immune response by the African swine fever virus. Vaccines are indispensable for controlling african swine fever, and the use of live viruses to make attenuated vaccines carries the risk that microorganisms may persist throughout the herd and cause unacceptable adverse reactions.

The research and development and screening experiments of vaccine drugs need to be evaluated on animal models, and rodents are used as the most widely applied experimental animal models, are one of the best experimental animal models because of small body size, short growth and breeding period and easy operation, and no mouse model prepared aiming at the porcine CD163 gene exists up to now.

The gene editing function of the CRISPR/Cas9 system is mainly applied to genome targeting of multiple species, modification (such as knockout, mutation and knock-in) operation is carried out on alleles, various modification models can be established quickly and efficiently to research gene functions or establish disease models, compared with a traditional gene editing mouse model manufactured based on an embryonic stem cell targeting technology, the gene editing mouse model is simple in design and convenient to operate, a complex in-vitro splicing process of targeting vectors is omitted, and work such as screening and culturing of embryonic stem cells is carried out, so that the gene editing mouse model has a great application prospect regardless of the manufacturing cost or period of the model. Although the CRISPR/Cas9 gene editing system has high cleavage efficiency and is generally applied to the establishment of a gene knockout model, the gene knockout model is faced with more complex gene modification requirements, for example, when the gene of a longer fragment is targeted, the targeting efficiency is obviously reduced along with the increase of the length of a knock-in fragment. In addition, when the CRISPR/Cas9 system containing the donor recombinant fragment is directly micromanipulated on fertilized eggs, the donor fragment can be recombined at a target site, and also can be randomly integrated into the genome of a mouse with high probability, the integrated site is random, high-copy knock-in can occur, and the mouse with random integration can have unexpected phenotypes such as exogenous gene ectopic expression, non-target site endogenous gene silencing or activation and the like under the influence of the integrated site and copy number, so that the experimental result is interfered. Therefore, the recombination efficiency of the CRISPR/Cas9 system is yet to be further researched and optimized, and at the same time, how to reduce or avoid the random integration probability of donor during targeting and improve the detection rate of random integration undoubtedly brings a series of challenges to gene editing workers.

Disclosure of Invention

The purpose of the invention is as follows: according to the invention, by the CRISRP/Cas9 gene modification method, CD163 gene of a pig is replaced by Cd163 gene of a mouse, a CD163 swine-derived mouse model is prepared, the CD163 swine-derived mouse model has functional genes of the pig, has very important significance for research on infection, invasion, immunity and the like of swine-derived related viruses, can possibly become an ideal animal model for screening and evaluating related medicines and diagnostic products of African swine fever, blue-ear disease and the like, and brings new ideas and strategies for controlling the development of epidemic situations.

The invention aims to solve the technical problem of providing the sgRNA.

The technical problem to be solved by the invention is to provide an expression cassette, a recombinant vector or a cell containing the sgRNA.

The technical problem to be solved by the present invention is to provide a primer pair for amplifying or identifying the sgRNA, the expression cassette, the recombinant vector or the cell.

The invention also aims to solve the technical problem of providing the sgRNA, the expression cassette, the recombinant vector or the cell and the application of the primer pair in CD163 gene editing.

The invention also aims to solve the technical problem of providing a method for constructing a CD163 gene humanized mouse model.

The technical problem to be solved finally by the invention is to provide a method for identifying CD163 gene humanized mice.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the present invention provides sgrnas having:

(i) as shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; or

(ii) As shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; or

(iii) (iii) a sequence which encodes the same protein as the nucleotide sequence of (i) or (ii) but which differs from the nucleotide sequence of (i) or (ii) by virtue of the degeneracy of the genetic code; or

(iv) A sequence having at least 70% homology to the sequence of (i) or (ii) or (iii).

The present disclosure also includes expression cassettes, recombinant vectors, or cells containing the sgrnas.

Wherein the recombinant vector is pUC57 kan-T7-delG-sgRNA.

The invention also comprises a primer pair for amplifying or identifying the sgRNA, the expression cassette, the recombinant vector or the cell.

Wherein the sequence of the primer pair is shown as SEQ ID NO:3 and SEQ ID NO: 4 is shown in the specification; or/and SEQ ID NO: 5 and SEQ ID NO: and 6, respectively.

The invention also comprises the sgRNA, the expression cassette, the recombinant vector or the cell and the application of the primer pair in CD163 gene editing.

The present disclosure also includes a targeting vector comprising the following elements: an upstream homology arm, a porcine-derived CD163 gene, a Stop element and a downstream homology arm, wherein the sequence of the upstream homology arm is shown as SEQ ID NO:34, and the porcine CD163 gene sequence is shown as SEQ ID NO:35, the Stop element sequence is shown as SEQ ID NO:37, and the sequence of the downstream homology arm is shown as SEQ ID NO: shown at 38.

Wherein the sequence of the targeting vector is shown as SEQ ID NO: shown at 39.

The invention also comprises a construction method of the targeting vector, which comprises the following steps: and fusing the upstream homology arm, the downstream homology arm and the synthesized pCD163 gene fragment in a fusion PCR mode, and performing slic connection on a fused product, a stop element and a pMD18T vector to obtain the recombinant plasmid.

The invention also comprises the application of the targeting vector in the preparation of the CD163 gene swine animal model.

The invention also discloses a construction method of the CD163 gene swine humanized mouse model, which comprises the following steps:

1) cloning the DNA sequence of the sgRNA into a vector to construct a recombinant plasmid;

2) carrying out PCR amplification on the recombinant plasmid obtained in the step 1) as a template to obtain a template, and transcribing the template to obtain sgRNA;

3) incubating sgRNA and Cas9 protein, adding a targeting vector to obtain a mixed solution, injecting the mixed solution into mouse fertilized eggs, transplanting the fertilized eggs into a pseudopregnant female mouse, and after the mouse is born, identifying genes of a screened targeted mouse, namely a positive mouse;

4) and breeding the obtained positive mouse and a wild mouse, and carrying out gene identification on the born mouse to obtain the stably inherited CD163 humanized mouse animal model.

The gene identification of the step 3) and the gene identification of the step 4) comprise the PCR identification of 5 'end and 3' end of mouse tail genome DNA, if expected bands are detected by the PCR identification, the target fragments are recombined at the target sites, and the PCR products are sequenced at the same time, and the positive mouse is obtained if the sequencing is correct.

Wherein, the PCR primer pair identified by the PCR of the 5 'end and the 3' end is SEQ ID NO: 11 and SEQ ID NO: 12 or/and SEQ ID NO: 13 and SEQ ID NO: as shown at 14.

Wherein, the primer pairs for PCR sequencing of the 5 'end and the 3' end are respectively SEQ ID NO: 15 and SEQ ID NO: 16 or/and SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

Wherein, the gene identification of the step 4) also comprises PCR identification and sequencing verification of internal sequences.

Wherein, the PCR identification primer pairs of the internal sequences are respectively SEQ ID NO: 19 and SEQ ID NO: 20 is shown in the figure; SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

Wherein, the sequencing verification primer of the internal sequence in the step 4) comprises SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28 or/and SEQ ID NO: 29.

the gene identification of the step 4) further comprises qPCR detection, wherein a qPCR detection primer pair is designed according to a 5' end homology arm, a wild mouse is used as a control, the copy number of the inserted exogenous targeting fragment in a genome is detected, and if the copy number of the mouse is between 0.75 and 1.25, no random insertion is indicated.

Wherein the qPCR detection primer pairs in the step are respectively SEQ ID NO: 30 and SEQ ID NO: 31, shown in the figure; or/and SEQ ID NO: 32 and SEQ ID NO: shown at 33.

The present disclosure also includes a method of identifying a CD163 gene suinized mouse comprising the steps of: by extracting

The obtained positive mouse tail genome DNA adopts SEQ ID NO:3 and SEQ ID NO: 4 is shown in the specification; or/and SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification; or/and SEQ ID NO: 11 and SEQ ID NO: 12 or/and SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure; or/and SEQ ID NO: 15 and SEQ ID NO: 16 or/and SEQ ID NO: 17 and SEQ ID NO: 18 is shown in the figure; or/and SEQ ID NO: 19 and SEQ ID NO: 20 is shown in the figure; SEQ ID NO: 21 and SEQ ID NO: 22; or/and SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28 or/and SEQ ID NO: 29; or/and SEQ ID NO: 30 and SEQ ID NO: 31, shown in the figure; or/and SEQ ID NO: 32 and SEQ ID NO: shown at 33.

The invention also comprises the application of the model mouse constructed by the method in screening or preparing drugs and diagnostic products for swine fever or blue ear disease.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. the CD163 gene swine mouse constructed by the invention has a functional gene of a pig, the knocked-in gene is from a swine-origin CD163 gene and knocked into a Cd163 gene locus of the mouse, and the regulation and control element of the mouse is reserved, so that the expression of the swine-origin CD163 is controlled by using the regulation and control mechanism of the mouse, and the Cd163 gene of the mouse is silenced, so that the drug cross reaction caused by the homology relation of species amino acids can be avoided. The model has functional genes of pigs, and has important guiding significance for researches such as infection, invasion, immunity, drug development and the like of porcine-derived related viruses.

2. The length of the knocked-in pig source gene sequence is 3330bp (SEQ ID NO:35), in order to regulate the transcription of the pig source gene, a transcript termination element Stop (SEQ ID NO:37) with the length of 830bp is knocked in after the CDS of the pig source gene, the element contains 3 copies of a PolyA structure so as to enhance the transcription regulation function of the gene, and the length of a model insertion element reaches 4160 bp. Mouse offspring obtained after microinjection and transplantation are successfully screened by PCR and sequencing detection means from 119F 0 mice to obtain 2 positive mice.

3. The invention verifies the correct integration of the target site gene by a PCR and sequencing method, simultaneously carries out QPCR detection on the mouse with positive targets, detects the possibility of other site integration in the mouse genome, further verifies that the positive mouse is the correct target, and has no random insertion of other non-target sites of the positive mouse.

Drawings

FIG. 1, a schematic diagram of pCD163 porcine-derived mouse model;

FIG. 2, 1-44 # F0 mouse primary screening and identification PCR results; representing a Positive control, adding a donor plasmid before the target of the project into a PCR system as a Positive control template control; WT: WT is C57BL/6J wild type, and is a negative control; n: n is negative blank control; /: the lane is without sample; 1-44: 1-44 # F0 mouse; m: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp;

FIG. 3, 45-119 # F0 mouse primary screening and identification PCR results; representing a Positive control, adding a donor plasmid before the target of the project into a PCR system as a Positive control template control; WT: WT is C57BL/6J wild type, and is a negative control; n: n is negative blank control; /: the lane is without sample; 45-119: 45-119 # F0 mouse; m: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp;

FIG. 4, F0 generation mouse 5' end PCR identification electrophoretogram; p: representing a Positive control, adding a donor plasmid before the target of the project into a PCR system as a Positive control template control; WT: WT is C57BL/6J wild type, and is a negative control; n: n is negative blank control; 5 'and 3' end PCR results: 14#, 23#, 26#, 31#, 40#, recombination occurs.

FIG. 5, 3' end PCR identification electrophoretogram of F0 mouse; p: representing the Positive control, and adding a donor plasmid before the project is targeted into a PCR system to serve as a template control of the Positive control; WT: WT is C57BL/6J wild type, and is a negative control; n: n is negative blank control;

FIG. 6, F1 generation mouse 5' end PCR identification electrophoretogram;

FIG. 7, 3' end PCR identification electrophoretogram of F1 mouse;

FIG. 8, internal PCR electrophoretogram of 58# and 59# mouse generation F1; m: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100 bp.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 pCD163 porcine-derived mouse model establishment

We used homologous recombination and CRISPR/Cas9 technology to replace the porcine CD163 gene with the murine CD163 gene on the zygote of B6 background mice to construct a mouse model that can express porcine CD163, and the targeting strategy is shown in figure 1.

1. Determining pig origin fragment replacement region and inserted pig origin sequence

According to the structure and the function of the porcine CD163, a CDS (coding sequence) and a transcript termination element PolyA (PolyA) of the porcine CD163 are selected to be inserted into a translation initiation codon site of the mouse Cd163, and the transcription and the translation of the mouse Cd163 gene are terminated while the CDS and the transcript termination element PolyA are inserted. The amino acid sequence of the selected porcine CD163 gene is shown as SEQ ID NO:37, and the strategy diagram is shown in FIG. 1.

2. Positive mice obtained by vector injection transplantation

1) Selecting two sides of a mouse Cd163 gene translation initiation codon as targeting sites, and designing a homologous DNA donor containing a swine-origin CD163 gene and an identification scheme;

2) preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; and designing sgRNA aiming at the murine sequence in the porcine-derived sequence insertion region. Designing, synthesizing and recognizing a target site, and constructing an sgRNA expression vector. The recognition site of the sgRNA is positioned on exon where the translation initiation codon of the mouse Cd163 gene is positioned, and the sequence of the target site of the sgRNA on the Cd163 is shown in Table 1. The DNA sequence of sgRNA was cloned into a pUC57kan-T7-delG vector (the resistance of PUC57 was modified from Amp + to Kana +, and the vector was named pUC57kan-T7-delG, in which PUC57 was from GenScript under the accession number SD1176) to construct a pUC57-sgRNA plasmid.

Table 1 DNA sequences of sgrnas

The sgRNA transcription preparation method comprises the following steps: PCR is carried out by taking PrimerStar or PrimerStar Max system and sgRNA-F, sgRNA-R as primers and puc57-sgRNA plasmid with correct sequencing as a template, and a PCR product is purified to prepare an sgRNA transcription template. The sgRNA was transcribed using T7-ShortScript in vitro transcription kit (AM 1354).

Table 2 sgRNA primer sequences

sgRNA screening: after the sgRNAs and the Cas9 protein are respectively incubated, the mixed solution is injected into fertilized eggs of 0.5 day, and after the fertilized eggs are cultured to a blastocyst stage, the ko positive rate of the mouse Cd163 gene is identified, so that the sgRNAs with high cutting activity are screened.

The sgRNA cleavage identification method comprises the following steps: PCR amplification is carried out on the collected blastocysts (the PCR scheme is shown in the following table 3), second-generation sequencing is carried out on the amplified bands (sequencing primer: Cd163-intF1), the amplified bands are compared with wild type (wt) bands, and the probability of mutation occurrence is counted (the identification result is shown in the following table 3).

Table 3 sgRNA cleavage PCR identification protocol

Table 4 sgRNA cleavage activity

sgRNA name	Cutting efficiency
		sgRNA1(Cd163-S1)	87.0％
sgRNA2(Cd163-S2)	66.7％

3) Cas9/sgRNA system (S1 and S2 target respectively) and a targeting vector are injected into fertilized eggs of mice of 0.5 day, the fertilized eggs are transplanted into pseudopregnant female mice, and after the mice come to birth, the targeted mice screened out through gene identification are positive F0 generation mice.

The construction method of the targeting vector comprises the following steps: the upstream homology arm (SEQ ID NO:34), the downstream homology arm (SEQ ID NO:38) and the synthesized pCD163 gene fragment were fused by fusion PCR, and the fused product was slic-linked to the stop element and the pMD18T vector. After in vitro connection and transformation to escherichia coli DH5 alpha, selecting a single clone for PCR and enzyme digestion identification, and naming the successfully connected vector as dsDNA-Cd 163-S1/S2; bstz171 enzyme-cleaves the dsDNA-Cd163-S1/S2 to generate two bands of 7098bp and 3702bp respectively, and recovering 7092bp fragments to obtain the targeting vector.

Specifically, the screening steps of the positive F0 mouse are as follows:

A. the tail DNA of born F0 mice is extracted, and the initial PCR screening identification is firstly carried out, wherein the PCR primers are shown in Table 5, the primers pCD163-KI-tF1/pCD163-KI-5tR1 are respectively positioned on an upstream homology arm and a swine source CD163 CDS, and the primers 3Stop-KI-3tF1/pCD163-KI-tR2 are respectively positioned on an exogenous Stop element and a downstream homology arm. If PCR produces a band of the expected size, it indicates that the mouse genome has the target fragment inserted. The results of PCR preliminary screening are shown in FIGS. 2 and 3.

TABLE 5F 0 Primary Screen identification PCR protocol for mouse

Remarking: KI is an on-target genotype; WT was wild type.

As can be seen from fig. 2 and 3, the primary screening PCR results: and (5) primarily screening the 14#, 23#, 26#, 31#, and 40# to be positive.

B. And (5) aiming at the mice positive for primary screening, further identifying the recombination of the 5 'end and the 3' end, and sequencing to confirm whether the target is hit. After-target PCR identification was performed with 5 'and 3' PCR primers (see Table 6), primers pCD163-KI-5tF2/pCD163-KI-5tR2 were located outside the upstream homology arm and inside the pig-derived segment of the targeting vector, respectively, and 3Stop-KI-3tF1/pCD163-KI-3tR1 were located outside the exogenous Stop element and downstream homology arm, respectively, as PCR amplification at 5 'and 3' ends produced PCR products of the expected size, and sequencing was consistent with the theoretical sequence, it was suggested that the target fragment was recombined at the target site.

TABLE 6 genotype identification primers for 5-and 3-termini of CD163 suinized mice

Remarking: KI is an on-target genotype; WT was wild type.

The results of 5 'end and 3' end PCR identification are shown in FIG. 4 and FIG. 5, and the results of 5 'end and 3' end PCR: 14#, 23#, 26#, 31#, 40#, recombination occurs.

C. Sequencing protocol: the sequencing method comprises the steps of recovering 5-end and 3-end PCR products, sequencing the recovered PCR products according to the sequencing scheme shown in the table 7, and determining the mouse with the correct sequencing to be a positive mouse.

Sequencing primer: pCD163-KI-5tF1, detecting the 5-terminal homology arm and genome junction, sequencing primer: pCD163-KI-5tR1, detecting the junction of the pig CDS and the homology arm, sequencing primer: pCD163-KI-3seqF1, detecting the 3-terminal homology arm and genome joint; sequencing primer: pCD163-KI-3seqR1, detecting Stop and 3-terminal homology arm joints;

table 7 CD163 humanized mouse 5-and 3-terminal genotype sequencing protocols

The sequencing results were: 23#, 31 #: the 5 'end and the 3' end are sequenced correctly, namely 23#, 31# are positive F0 generation mice.

4) The 31# positive F0-generation mouse is selected to be bred with a wild type C57BL/6J mouse to generate a litter of mice-F1-generation mouse, the total number of the F1-generation mouse is 4, the number of the F1-generation mouse is 57-60 #, the born mice are subjected to genotype identification to obtain 2 positive mice, the number of the positive mice is 58#, the number of the positive mice is 59#, and the positive mice are CD163 humanized mice capable of stably inheriting an animal model.

Example 2 genotyping of F1 Generation mice

CD163 porcine-derived mouse 5 'end and 3' end genotype identification, two-end PCR identification (Table 6) is carried out on mouse tail genomic DNA of F1 generation mouse obtained in step 4) of example 1 after targeting by using two pairs of primers respectively, the identification scheme and the identification primers are the same as those in example 1, the primers pCD163-KI-5tF2/pCD163-KI-5tR2 are respectively positioned outside the 5 'end homology arm and in the porcine fragment of the targeting vector, and if the pair of primers is amplified, a PCR product is generated, which indicates that the target vector is effectively recombined at the 5' end of the mouse genome; 3stop-KI-3tF1/pCD163-KI-3tR1 is respectively positioned in the exogenous fragment and outside the 3 'homologous arm of the targeting vector, and if the pair of primers is amplified to generate a PCR product, the target vector is effectively recombined at the 3' end of the mouse genome. As can be seen in FIGS. 6 and 7, the pCD163 rat tail DNA was used to identify the electrophoretogram, and the 5 'and 3' identification bands of the 58 th and 59 th mice in the electrophoretogram were positive, indicating that the mice were positive mice correctly undergoing gene recombination, and the 5 'and 3' PCR products of the 58 th and 59 th mice were recovered and further confirmed by sequencing (see Table 11 for sequencing primers), and the mice correctly sequenced were prescreened positive mice.

Example 3 identification of internal sequences in F1 mouse generations

The 58 th and 59 th positive mice identified by the PCR preliminary screening at the two ends of the example 2 are further subjected to PCR amplification of internal sequences (figure 8 and table 10) and sequencing verification (table 11), PCR primers are designed aiming at inserted exogenous sequences, the homology arm of the target site, the genome joint and knocked-in exogenous gene sequences are determined to be all correct, and the mice with correct sequencing are identified as positive medium-target mice. The PCR reaction conditions and system are referred to in tables 8 and 9. The sequencing result shows that: the CD163 and Stop elements inserted into mice 58# (58#) and 59# (59#), and the linker sequences of the insertion site and the homology arm were correct, and the mice were positive.

TABLE 8 PCR reaction System

Reagent (Vazyme P112-03)	Volume (μ l)	Specification of
			2×Taq Master Mix，Dye Plus	12.5	\
ddH2O	9.5	\
			Primer A(10pmol/μl)	1	10pmol/μl
Primer B(10pmol/μl)	1	10pmol/μl
			Template(≈100ng/μl)	1	≈100ng/μl

TABLE 9 PCR reaction conditions

TABLE 10 internal identification primers for CD163 Swine-derived mice

Note: KI is an on-target genotype; WT was wild type.

TABLE 11 CD163 porcine-derived mouse internal sequencing protocol

Example 4QPCR assay identification

QPCR detection: for the screened 58# and 59# positive targeted mice, mouse tail DNA was extracted, qPCR detection was further performed using the primers of table 12 (reaction system and reaction program at table 13 and table 14), pCD163-5q-tF1/pCD163-5q-tR1 was designed at the 5' end homology arm, wild type mice were used as control to detect the copy number of the inserted exogenously targeted fragment in the genome, with the oIMR3580 and oIMR1544 primer products as internal controls, qPCR data were corrected, and if the mouse copy number was between 0.75-1.25, no random insertion was indicated.

TABLE 12 QPCR protocol for CD163 porcine-derived mice

TABLE 13 QPCR reaction System Table 14QPCR reaction procedure

The QPCR assay results are shown in tables 15 and 16, and B6-1 and B6-2 are two C57BL/6J wild-type controls. QPCR results: the 58# and 59# copy numbers were consistent with wild type and no random insertions were detected.

Watch 15

Calibration data	Sample number	Random insertion copy number
			0.81	58#	Is consistent with the wild type
0.87	59#
			1.00	B6-1	Wild type
0.98	B6-2	Wild type

。

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<212> DNA

<213> XM004136-pCD163-KI-tF1(Artificial Sequence)

<400> 19

ggactctccc tttaagattg gctcc 25

<210> 20

<211> 22

<212> DNA

<213> XM004136-pCD163-long-tR1(Artificial Sequence)

<400> 20

caaaatgagc agaaccagtg gc 22

<210> 21

<211> 24

<212> DNA

<213> XM004136-pCD163-long-tF2(Artificial Sequence)

<400> 21

agaaagtggt gttgcctgca tagg 24

<210> 22

<211> 22

<212> DNA

<213> XM004136-pCD163-KI-tR2(Artificial Sequence)

<400> 22

ccatgtttca cacccaggac ag 22

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggactctccc tttaagattg gctcc 25

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agatgaggct ggtgaatgga gg 22

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atcttgagca gactacgccg ac 22

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

caaaatgagc agaaccagtg gc 22

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

agaaagtggt gttgcctgca tagg 24

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

tgtgtgtgac gactcctggg a 21

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cactttcata ggacccagat gcc 23

<210> 30

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ggactctccc tttaagattg gctcc 25

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tagaaagtgg atgaccacgg agg 23

<210> 32

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tcaccagtca tttctgcctt tg 22

<210> 33

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

cacgtgggct ccagcatt 18

<210> 34

<211> 1520

<212> DNA

<213> upstream homology arm (Artificial Sequence)

<400> 34

gcgccacgaa gaagacaaga cccaaagccg gctctcccat actcacaccc tcaggaccac 60

aacccccacg tctacgggca gttctactgt gctgtggaga caaggtgcag gacctgctca 120

cccgagtgct gtcagagaca gctccccagc tctcatgacc agctctccca tctgccgtgg 180

gtgatgagag gttcttccct cgctgccacc accacacagc agacaagagg tagggctgaa 240

tcacgcatgc tttttaccct tggggttgat ttagctgcaa tccccacatc caagaccagc 300

cctactgtga ggcccaggca aggtgcacgg cctgctttcc tgagtactac ccctaggtaa 360

gggtaggatc tgctctcaca gccccagggc cagctctttc aggatgccca ggtaaagggt 420

ggggccagtt ctacccagcc ctcagacatc agcatggccc cagaaagcag cccaaaccag 480

ggatgtctgt ctgcctagcc tttgatagta acagacccct gctgttttag ggtcacagac 540

ccagacatgg ccccaggtga tagctcaggc cagtacccca gctacttgcc ctctagtctt 600

cagttgtacc tctcttcatt gtgcccacat ccttctgttt ctctttctct tccatttctc 660

caccacttac ttgcttcttt tagtggtaaa cagagtctct gagtgtgtgg gttcatttta 720

ggagtagtct cgggagtgct atgtactacc catacattac gacactggtc aggggtcatc 780

tcaggcattg tctgccccta ggcctgtgct gcaattgact agtgctcatc tcaagctaga 840

tccgtgttca ggctccatgg agctggactg gtgatcacaa tgcccaggcc ttcctgagtg 900

gccctatgta agggtcagct gtcttgggct cactccttcc ccaaccccgc tcccacccct 960

gaacccgtgg tccgaggtag ggttcttatg gtctctggct tactccctac tctgggagct 1020

catccaggcc tccaaggcac cagactcatg gttgtctcgg actttctttt ttccaggtgt 1080

gctaggctac taatcattca tgagttcaca ggtcagaaca ctgagcatag tcacagcatc 1140

tctcctctct gccaccaact aacgcaggtg tgacacagaa gccacacttg caacatctct 1200

aagagcagat taatcaatca attaattaaa ctattcaatc aagagcaaca tatgaataaa 1260

tacactccct agtttttgga aatctatgaa aactgttaaa ctttcctttg gttgttctgt 1320

gttccaaagt gggaggagaa agaggataaa agaaataagg actctccctt taagattggc 1380

tcctccctcc ttctagagcc tttccagtct taagagtgag atgattctct ccctccgtgg 1440

tcatccactt tctacagaga acacgtctat gaaatagtat caggagacac acggagccat 1500

caaaatcatc aagctttgga 1520

<210> 35

<211> 3333

<212> DNA

<213> porcine-derived CD163 CDS Sequence (Artificial Sequence)

<400> 35

atggtgctac ttgaagactc tggatctgca gactttagaa gatgttctgc ccatttaagt 60

tccttcactt ttgctgtagt cgctgttctc agtgcctgct tggtcactag ttctcttgga 120

ggaaaagaca aggagctgag gctaacgggt ggtgaaaaca agtgctctgg aagagtggag 180

gtgaaagtgc aggaggagtg gggaactgtg tgtaataatg gctgggacat ggatgtggtc 240

tctgttgttt gtaggcagct gggatgtcca actgctatca aagccactgg atgggctaat 300

tttagtgcag gttctggacg catttggatg gatcatgttt cttgtcgagg gaatgagtca 360

gctctctggg actgcaaaca tgatggatgg ggaaagcata actgtactca ccaacaggat 420

gctggagtaa cctgctcaga tggatctgat ttagagatga ggctggtgaa tggaggaaac 480

cggtgcttag gaagaataga agtcaaattt caagagcggt ggggaacagt gtgtgatgat 540

aacttcaaca taaatcatgc ttctgtggtt tgtaaacaac ttgaatgtgg aagtgctgtc 600

agtttctctg gttcagctaa ttttggagaa ggttctggac caatctggtt tgatgatctt 660

gtatgcaatg gaaatgagtc agctctctgg aactgcaaac atgaaggatg gggaaagcac 720

aattgcgatc atgctgagga tgctggagtg atttgcttaa atggagcaga cctgaaactg 780

agagtggtag atggactcac tgaatgttca ggaagattgg aagtgaaatt ccaaggagaa 840

tggggaacaa tctgtgatga tggctgggat agtgatgatg ccgctgtggc atgtaagcaa 900

ctgggatgtc caactgctgt cactgccatt ggtcgagtta acgccagtga gggaactgga 960

cacatttggc ttgacagtgt ttcttgccat ggacacgagt ctgctctctg gcagtgtaga 1020

caccatgaat ggggaaagca ttattgcaat cataatgaag atgctggtgt gacatgttct 1080

gatggatcag atctggaact gagacttaaa ggtggaggca gccactgtgc tgggacagtg 1140

gaggtggaaa ttcagaaact ggtaggaaaa gtgtgtgata gaagctgggg actgaaagaa 1200

gctgatgtgg tttgcaggca gctgggatgt ggatctgcac tcaaaacatc atatcaagtt 1260

tattccaaaa ccaaggcaac aaacacatgg ctgtttgtaa gcagctgtaa tggaaatgaa 1320

acttctcttt gggactgcaa gaattggcag tggggtggac ttagttgtga tcactatgac 1380

gaagccaaaa ttacctgctc agcccacagg aaacccaggc tggttggagg ggacattccc 1440

tgctctggtc gtgttgaagt acaacatgga gacacgtggg gcaccgtctg tgattctgac 1500

ttctctctgg aggcggccag cgtgctgtgc agggaactac agtgcggcac tgtggtttcc 1560

ctcctggggg gagctcactt tggagaagga agtggacaga tctgggctga agaattccag 1620

tgtgaggggc acgagtccca cctttcactc tgcccagtag caccccgccc tgacgggaca 1680

tgtagccaca gcagggacgt cggcgtagtc tgctcaagat acacacaaat ccgcttggtg 1740

aatggcaaga ccccatgtga aggaagagtg gagctcaaca ttcttgggtc ctgggggtcc 1800

ctctgcaact ctcactggga catggaagat gcccatgttt tatgccagca gcttaaatgt 1860

ggagttgccc tttctatccc gggaggagca ccttttggga aaggaagtga gcaggtctgg 1920

aggcacatgt ttcactgcac tgggactgag aagcacatgg gagattgttc cgtcactgct 1980

ctgggcgcat cactctgttc ttcagggcaa gtggcctctg taatctgctc agggaaccag 2040

agtcagacac tatccccgtg caattcatca tcctcggacc catcaagctc tattatttca 2100

gaagaaagtg gtgttgcctg catagggagt ggtcaacttc gcctggtcga tggaggtggt 2160

cgttgtgctg ggagagtaga ggtctatcct ggggcatcct ggggcaccat ctgtgatgac 2220

agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc tgagctgtgg atgggccatt 2280

aatgccactg gttctgctca ttttggggaa ggaacagggc ccatttggct ggatgagata 2340

aactgtaatg gaaaagaatc tcatatttgg caatgccact cacatggttg ggggcggcac 2400

aattgcaggc ataaggagga tgcaggagtc atctgctcag agttcatgtc tctgagactg 2460

atcagtgaaa acagcagaga gacctgtgca gggcgcctgg aagtttttta caacggagct 2520

tggggcagcg ttggcaggaa tagcatgtct ccagccacag tgggggtggt atgcaggcag 2580

ctgggctgtg cagacagagg ggacatcagc cctgcatctt cagacaagac agtgtccagg 2640

cacatgtggg tggacaatgt tcagtgtcct aaaggacctg acacactatg gcagtgcccc 2700

tcatctccat ggaagaagag actggccagc ccctcagagg agacatggat cacatgtgcc 2760

aacaaaataa gacttcaaga aggaaacact aattgttctg gacgtgtgga gatctggtac 2820

ggaggttcct ggggcactgt gtgtgacgac tcctgggacc ttgaagatgc tcaggtggtg 2880

tgccgacagc tgggctgtgg ctcagctttg gaggcaggaa aagagcccgc atttggccag 2940

gggactgggc ccatatggct caatgaagtg aagtgcaagg ggaatgaacc ctccttgtgg 3000

gattgtcctg ccagatcctg gggccacagt gactgtggac acaaggagga tgctgctgtg 3060

acgtgctcag aaattgcaaa gagccgagaa tccctacatg ccacaggtcg ctcatctttt 3120

gttgcacttg caatctttgg ggtcattctg ttggcctgtc tcatcgcatt cctcatttgg 3180

actcagaagc gaagacagag gcagcggctc tcagttttct caggaggaga gaattctgtc 3240

catcaaattc aataccggga gatgaattct tgcctgaaag cagatgaaac ggatatgcta 3300

aatccctcag gagaccactc tgaagtacaa tga 3333

<210> 36

<211> 1110

<212> PRT

<213> porcine-derived CD163 amino acid Sequence (Artificial Sequence)

<400> 36

Met Val Leu Leu Glu Asp Ser Gly Ser Ala Asp Phe Arg Arg Cys Ser

1 5 10 15

Ala His Leu Ser Ser Phe Thr Phe Ala Val Val Ala Val Leu Ser Ala

20 25 30

Cys Leu Val Thr Ser Ser Leu Gly Gly Lys Asp Lys Glu Leu Arg Leu

35 40 45

Thr Gly Gly Glu Asn Lys Cys Ser Gly Arg Val Glu Val Lys Val Gln

50 55 60

Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp Asp Met Asp Val Val

65 70 75 80

Ser Val Val Cys Arg Gln Leu Gly Cys Pro Thr Ala Ile Lys Ala Thr

85 90 95

Gly Trp Ala Asn Phe Ser Ala Gly Ser Gly Arg Ile Trp Met Asp His

100 105 110

Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp Asp Cys Lys His Asp

115 120 125

Gly Trp Gly Lys His Asn Cys Thr His Gln Gln Asp Ala Gly Val Thr

130 135 140

Cys Ser Asp Gly Ser Asp Leu Glu Met Arg Leu Val Asn Gly Gly Asn

145 150 155 160

Arg Cys Leu Gly Arg Ile Glu Val Lys Phe Gln Glu Arg Trp Gly Thr

165 170 175

Val Cys Asp Asp Asn Phe Asn Ile Asn His Ala Ser Val Val Cys Lys

180 185 190

Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser Gly Ser Ala Asn Phe

195 200 205

Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp Leu Val Cys Asn Gly

210 215 220

Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu Gly Trp Gly Lys His

225 230 235 240

Asn Cys Asp His Ala Glu Asp Ala Gly Val Ile Cys Leu Asn Gly Ala

245 250 255

Asp Leu Lys Leu Arg Val Val Asp Gly Leu Thr Glu Cys Ser Gly Arg

260 265 270

Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr Ile Cys Asp Asp Gly

275 280 285

Trp Asp Ser Asp Asp Ala Ala Val Ala Cys Lys Gln Leu Gly Cys Pro

290 295 300

Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala Ser Glu Gly Thr Gly

305 310 315 320

His Ile Trp Leu Asp Ser Val Ser Cys His Gly His Glu Ser Ala Leu

325 330 335

Trp Gln Cys Arg His His Glu Trp Gly Lys His Tyr Cys Asn His Asn

340 345 350

Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Leu Arg

355 360 365

Leu Lys Gly Gly Gly Ser His Cys Ala Gly Thr Val Glu Val Glu Ile

370 375 380

Gln Lys Leu Val Gly Lys Val Cys Asp Arg Ser Trp Gly Leu Lys Glu

385 390 395 400

Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Lys Thr

405 410 415

Ser Tyr Gln Val Tyr Ser Lys Thr Lys Ala Thr Asn Thr Trp Leu Phe

420 425 430

Val Ser Ser Cys Asn Gly Asn Glu Thr Ser Leu Trp Asp Cys Lys Asn

435 440 445

Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr Asp Glu Ala Lys Ile

450 455 460

Thr Cys Ser Ala His Arg Lys Pro Arg Leu Val Gly Gly Asp Ile Pro

465 470 475 480

Cys Ser Gly Arg Val Glu Val Gln His Gly Asp Thr Trp Gly Thr Val

485 490 495

Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser Val Leu Cys Arg Glu

500 505 510

Leu Gln Cys Gly Thr Val Val Ser Leu Leu Gly Gly Ala His Phe Gly

515 520 525

Glu Gly Ser Gly Gln Ile Trp Ala Glu Glu Phe Gln Cys Glu Gly His

530 535 540

Glu Ser His Leu Ser Leu Cys Pro Val Ala Pro Arg Pro Asp Gly Thr

545 550 555 560

Cys Ser His Ser Arg Asp Val Gly Val Val Cys Ser Arg Tyr Thr Gln

565 570 575

Ile Arg Leu Val Asn Gly Lys Thr Pro Cys Glu Gly Arg Val Glu Leu

580 585 590

Asn Ile Leu Gly Ser Trp Gly Ser Leu Cys Asn Ser His Trp Asp Met

595 600 605

Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys Cys Gly Val Ala Leu

610 615 620

Ser Ile Pro Gly Gly Ala Pro Phe Gly Lys Gly Ser Glu Gln Val Trp

625 630 635 640

Arg His Met Phe His Cys Thr Gly Thr Glu Lys His Met Gly Asp Cys

645 650 655

Ser Val Thr Ala Leu Gly Ala Ser Leu Cys Ser Ser Gly Gln Val Ala

660 665 670

Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr Leu Ser Pro Cys Asn

675 680 685

Ser Ser Ser Ser Asp Pro Ser Ser Ser Ile Ile Ser Glu Glu Ser Gly

690 695 700

Val Ala Cys Ile Gly Ser Gly Gln Leu Arg Leu Val Asp Gly Gly Gly

705 710 715 720

Arg Cys Ala Gly Arg Val Glu Val Tyr Pro Gly Ala Ser Trp Gly Thr

725 730 735

Ile Cys Asp Asp Ser Trp Asp Leu Asn Asp Ala His Val Val Cys Lys

740 745 750

Gln Leu Ser Cys Gly Trp Ala Ile Asn Ala Thr Gly Ser Ala His Phe

755 760 765

Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu Ile Asn Cys Asn Gly

770 775 780

Lys Glu Ser His Ile Trp Gln Cys His Ser His Gly Trp Gly Arg His

785 790 795 800

Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile Cys Ser Glu Phe Met

805 810 815

Ser Leu Arg Leu Ile Ser Glu Asn Ser Arg Glu Thr Cys Ala Gly Arg

820 825 830

Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser Val Gly Arg Asn Ser

835 840 845

Met Ser Pro Ala Thr Val Gly Val Val Cys Arg Gln Leu Gly Cys Ala

850 855 860

Asp Arg Gly Asp Ile Ser Pro Ala Ser Ser Asp Lys Thr Val Ser Arg

865 870 875 880

His Met Trp Val Asp Asn Val Gln Cys Pro Lys Gly Pro Asp Thr Leu

885 890 895

Trp Gln Cys Pro Ser Ser Pro Trp Lys Lys Arg Leu Ala Ser Pro Ser

900 905 910

Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile Arg Leu Gln Glu Gly

915 920 925

Asn Thr Asn Cys Ser Gly Arg Val Glu Ile Trp Tyr Gly Gly Ser Trp

930 935 940

Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu Asp Ala Gln Val Val

945 950 955 960

Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Glu Ala Gly Lys Glu Pro

965 970 975

Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu Asn Glu Val Lys Cys

980 985 990

Lys Gly Asn Glu Pro Ser Leu Trp Asp Cys Pro Ala Arg Ser Trp Gly

995 1000 1005

His Ser Asp Cys Gly His Lys Glu Asp Ala Ala Val Thr Cys Ser Glu

1010 1015 1020

Ile Ala Lys Ser Arg Glu Ser Leu His Ala Thr Gly Arg Ser Ser Phe

1025 1030 1035 1040

Val Ala Leu Ala Ile Phe Gly Val Ile Leu Leu Ala Cys Leu Ile Ala

1045 1050 1055

Phe Leu Ile Trp Thr Gln Lys Arg Arg Gln Arg Gln Arg Leu Ser Val

1060 1065 1070

Phe Ser Gly Gly Glu Asn Ser Val His Gln Ile Gln Tyr Arg Glu Met

1075 1080 1085

Asn Ser Cys Leu Lys Ala Asp Glu Thr Asp Met Leu Asn Pro Ser Gly

1090 1095 1100

Asp His Ser Glu Val Gln

1105 1110

<210> 37

<211> 830

<212> DNA

<213> Stop Sequence (Artificial Sequence)

<400> 37

cgcgatgaat aaatgaaagc ttgcagatct gcgactctag aggatctgcg actctagagg 60

atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 120

ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 180

gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 240

tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 300

tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa 360

aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 420

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 480

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 540

atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat ttgtagaggt 600

tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc 660

aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 720

cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 780

catcaatgta tcttatcatg tctggatccc catcaagctg atccggaacc 830

<210> 38

<211> 1409

<212> DNA

<213> downstream homology arm (Artificial Sequence)

<400> 38

ggtggacacc gcatggttct tcttggaggt gctggatctc ctggtaaaag tgtcttattg 60

atatctatcc tttaatttga gttgagagat gttctgacag tgaatggatg tgattggcat 120

aggagagcga ggaatagcaa ggggcatctg ggtcctatga aagtgctttt catcaaccat 180

agaccccaaa ctgaaatcgt aaagtaggaa gcaatgcttt tattcatttt ataaaattgt 240

tgaggtgaaa tcaagtcagg acctgaagta agcaaaactg tcctgggtgt gaaacatggg 300

ctgttaggaa agacaaagtg taggggtcaa tgctgattaa aagttgattt ccaataggaa 360

atgtggaaaa ccaactcagt gcaaaattct gcttcatgat gtttatgggc tggctaaact 420

cattcttcag tttcgcatga gtgctgagtt gtggtcactg tggagctcag agctatatgg 480

cactttttta tgctgtatgg gggatggtga taagattaac atttgggtgt ttgaggatgt 540

aaagtagact atttgtcaca caactgagaa tgcaggtcct gttggtggaa cagtcatgat 600

gtaagaaata ccagccattc tttcatgaag ttgttaggaa actttgtgga ccagatagac 660

atgcgggtgg gggtgtgttt aattgatgcc aagaaagtag agggacagag agagagctca 720

gaaagctgtc ctataatgta cagagaacat gctcggtcac tagtttggtt gatgtctctt 780

tcaagagctc aggcttaaga agctgccttt aaagtttgcc tccatgccca gcgttttcac 840

aaatcctcac tcctgggctg cacgtaaaca ataaacagtt gtatttctga agttgttcta 900

ccagccctgt cgttctcgat cggaatcttt cccacttgcc aaaggaaggg tctttctttg 960

tcttggttta gtctttgtca gatcccaaac tcaggacctt ctcttctcca aaatctcttg 1020

ttagtaaatt tcactcctgc cccttgtttc acaggttgta aaaggtttgt ccatctaggt 1080

ttctttgttg tggctgtgag ctcacttctc agtgcctctg ctgtcactaa cgctcctggt 1140

gagtattttg atcaacttac ttattgcctt ggcctgctct gatatgaatt gtcaaacaaa 1200

atcacaattc tcctgtatgg caaataacat ctaaaaatcc atcctctgca gagctgaaga 1260

actaaatatc agaactatac aattatattc taaccagacc caaaatgatg agcatggtct 1320

tccctcatat aaaacaacac catgttacat aaagactaaa agcagtacat agaagactaa 1380

aggaaaggag aagaaagctc agagttatc 1409

<210> 40

<211> 7092

<212> DNA

<213> targeting vector Sequence (Artificial Sequence)

<400> 40

gcgccacgaa gaagacaaga cccaaagccg gctctcccat actcacaccc tcaggaccac 60

aacccccacg tctacgggca gttctactgt gctgtggaga caaggtgcag gacctgctca 120

cccgagtgct gtcagagaca gctccccagc tctcatgacc agctctccca tctgccgtgg 180

gtgatgagag gttcttccct cgctgccacc accacacagc agacaagagg tagggctgaa 240

tcacgcatgc tttttaccct tggggttgat ttagctgcaa tccccacatc caagaccagc 300

cctactgtga ggcccaggca aggtgcacgg cctgctttcc tgagtactac ccctaggtaa 360

gggtaggatc tgctctcaca gccccagggc cagctctttc aggatgccca ggtaaagggt 420

ggggccagtt ctacccagcc ctcagacatc agcatggccc cagaaagcag cccaaaccag 480

ggatgtctgt ctgcctagcc tttgatagta acagacccct gctgttttag ggtcacagac 540

ccagacatgg ccccaggtga tagctcaggc cagtacccca gctacttgcc ctctagtctt 600

cagttgtacc tctcttcatt gtgcccacat ccttctgttt ctctttctct tccatttctc 660

caccacttac ttgcttcttt tagtggtaaa cagagtctct gagtgtgtgg gttcatttta 720

ggagtagtct cgggagtgct atgtactacc catacattac gacactggtc aggggtcatc 780

tcaggcattg tctgccccta ggcctgtgct gcaattgact agtgctcatc tcaagctaga 840

tccgtgttca ggctccatgg agctggactg gtgatcacaa tgcccaggcc ttcctgagtg 900

gccctatgta agggtcagct gtcttgggct cactccttcc ccaaccccgc tcccacccct 960

gaacccgtgg tccgaggtag ggttcttatg gtctctggct tactccctac tctgggagct 1020

catccaggcc tccaaggcac cagactcatg gttgtctcgg actttctttt ttccaggtgt 1080

gctaggctac taatcattca tgagttcaca ggtcagaaca ctgagcatag tcacagcatc 1140

tctcctctct gccaccaact aacgcaggtg tgacacagaa gccacacttg caacatctct 1200

aagagcagat taatcaatca attaattaaa ctattcaatc aagagcaaca tatgaataaa 1260

tacactccct agtttttgga aatctatgaa aactgttaaa ctttcctttg gttgttctgt 1320

gttccaaagt gggaggagaa agaggataaa agaaataagg actctccctt taagattggc 1380

tcctccctcc ttctagagcc tttccagtct taagagtgag atgattctct ccctccgtgg 1440

tcatccactt tctacagaga acacgtctat gaaatagtat caggagacac acggagccat 1500

caaaatcatc aagctttgga atggtgctac ttgaagactc tggatctgca gactttagaa 1560

gatgttctgc ccatttaagt tccttcactt ttgctgtagt cgctgttctc agtgcctgct 1620

tggtcactag ttctcttgga ggaaaagaca aggagctgag gctaacgggt ggtgaaaaca 1680

agtgctctgg aagagtggag gtgaaagtgc aggaggagtg gggaactgtg tgtaataatg 1740

gctgggacat ggatgtggtc tctgttgttt gtaggcagct gggatgtcca actgctatca 1800

aagccactgg atgggctaat tttagtgcag gttctggacg catttggatg gatcatgttt 1860

cttgtcgagg gaatgagtca gctctctggg actgcaaaca tgatggatgg ggaaagcata 1920

actgtactca ccaacaggat gctggagtaa cctgctcaga tggatctgat ttagagatga 1980

ggctggtgaa tggaggaaac cggtgcttag gaagaataga agtcaaattt caagagcggt 2040

ggggaacagt gtgtgatgat aacttcaaca taaatcatgc ttctgtggtt tgtaaacaac 2100

ttgaatgtgg aagtgctgtc agtttctctg gttcagctaa ttttggagaa ggttctggac 2160

caatctggtt tgatgatctt gtatgcaatg gaaatgagtc agctctctgg aactgcaaac 2220

atgaaggatg gggaaagcac aattgcgatc atgctgagga tgctggagtg atttgcttaa 2280

atggagcaga cctgaaactg agagtggtag atggactcac tgaatgttca ggaagattgg 2340

aagtgaaatt ccaaggagaa tggggaacaa tctgtgatga tggctgggat agtgatgatg 2400

ccgctgtggc atgtaagcaa ctgggatgtc caactgctgt cactgccatt ggtcgagtta 2460

acgccagtga gggaactgga cacatttggc ttgacagtgt ttcttgccat ggacacgagt 2520

ctgctctctg gcagtgtaga caccatgaat ggggaaagca ttattgcaat cataatgaag 2580

atgctggtgt gacatgttct gatggatcag atctggaact gagacttaaa ggtggaggca 2640

gccactgtgc tgggacagtg gaggtggaaa ttcagaaact ggtaggaaaa gtgtgtgata 2700

gaagctgggg actgaaagaa gctgatgtgg tttgcaggca gctgggatgt ggatctgcac 2760

tcaaaacatc atatcaagtt tattccaaaa ccaaggcaac aaacacatgg ctgtttgtaa 2820

gcagctgtaa tggaaatgaa acttctcttt gggactgcaa gaattggcag tggggtggac 2880

ttagttgtga tcactatgac gaagccaaaa ttacctgctc agcccacagg aaacccaggc 2940

tggttggagg ggacattccc tgctctggtc gtgttgaagt acaacatgga gacacgtggg 3000

gcaccgtctg tgattctgac ttctctctgg aggcggccag cgtgctgtgc agggaactac 3060

agtgcggcac tgtggtttcc ctcctggggg gagctcactt tggagaagga agtggacaga 3120

tctgggctga agaattccag tgtgaggggc acgagtccca cctttcactc tgcccagtag 3180

caccccgccc tgacgggaca tgtagccaca gcagggacgt cggcgtagtc tgctcaagat 3240

acacacaaat ccgcttggtg aatggcaaga ccccatgtga aggaagagtg gagctcaaca 3300

ttcttgggtc ctgggggtcc ctctgcaact ctcactggga catggaagat gcccatgttt 3360

tatgccagca gcttaaatgt ggagttgccc tttctatccc gggaggagca ccttttggga 3420

aaggaagtga gcaggtctgg aggcacatgt ttcactgcac tgggactgag aagcacatgg 3480

gagattgttc cgtcactgct ctgggcgcat cactctgttc ttcagggcaa gtggcctctg 3540

taatctgctc agggaaccag agtcagacac tatccccgtg caattcatca tcctcggacc 3600

catcaagctc tattatttca gaagaaagtg gtgttgcctg catagggagt ggtcaacttc 3660

gcctggtcga tggaggtggt cgttgtgctg ggagagtaga ggtctatcct ggggcatcct 3720

ggggcaccat ctgtgatgac agctgggacc tgaatgatgc ccatgtggtg tgcaaacagc 3780

tgagctgtgg atgggccatt aatgccactg gttctgctca ttttggggaa ggaacagggc 3840

ccatttggct ggatgagata aactgtaatg gaaaagaatc tcatatttgg caatgccact 3900

cacatggttg ggggcggcac aattgcaggc ataaggagga tgcaggagtc atctgctcag 3960

agttcatgtc tctgagactg atcagtgaaa acagcagaga gacctgtgca gggcgcctgg 4020

aagtttttta caacggagct tggggcagcg ttggcaggaa tagcatgtct ccagccacag 4080

tgggggtggt atgcaggcag ctgggctgtg cagacagagg ggacatcagc cctgcatctt 4140

cagacaagac agtgtccagg cacatgtggg tggacaatgt tcagtgtcct aaaggacctg 4200

acacactatg gcagtgcccc tcatctccat ggaagaagag actggccagc ccctcagagg 4260

agacatggat cacatgtgcc aacaaaataa gacttcaaga aggaaacact aattgttctg 4320

gacgtgtgga gatctggtac ggaggttcct ggggcactgt gtgtgacgac tcctgggacc 4380

ttgaagatgc tcaggtggtg tgccgacagc tgggctgtgg ctcagctttg gaggcaggaa 4440

aagagcccgc atttggccag gggactgggc ccatatggct caatgaagtg aagtgcaagg 4500

ggaatgaacc ctccttgtgg gattgtcctg ccagatcctg gggccacagt gactgtggac 4560

acaaggagga tgctgctgtg acgtgctcag aaattgcaaa gagccgagaa tccctacatg 4620

ccacaggtcg ctcatctttt gttgcacttg caatctttgg ggtcattctg ttggcctgtc 4680

tcatcgcatt cctcatttgg actcagaagc gaagacagag gcagcggctc tcagttttct 4740

caggaggaga gaattctgtc catcaaattc aataccggga gatgaattct tgcctgaaag 4800

cagatgaaac ggatatgcta aatccctcag gagaccactc tgaagtacaa tgacgcgatg 4860

aataaatgaa agcttgcaga tctgcgactc tagaggatct gcgactctag aggatcataa 4920

tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 4980

tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 5040

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 5100

attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgcgact 5160

ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct 5220

cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt 5280

tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 5340

atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 5400

ctggatctgc gactctagag gatcataatc agccatacca catttgtaga ggttttactt 5460

gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 5520

gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 5580

ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 5640

gtatcttatc atgtctggat ccccatcaag ctgatccgga accggtggac accgcatggt 5700

tcttcttgga ggtgctggat ctcctggtaa aagtgtctta ttgatatcta tcctttaatt 5760

tgagttgaga gatgttctga cagtgaatgg atgtgattgg cataggagag cgaggaatag 5820

caaggggcat ctgggtccta tgaaagtgct tttcatcaac catagacccc aaactgaaat 5880

cgtaaagtag gaagcaatgc ttttattcat tttataaaat tgttgaggtg aaatcaagtc 5940

aggacctgaa gtaagcaaaa ctgtcctggg tgtgaaacat gggctgttag gaaagacaaa 6000

gtgtaggggt caatgctgat taaaagttga tttccaatag gaaatgtgga aaaccaactc 6060

agtgcaaaat tctgcttcat gatgtttatg ggctggctaa actcattctt cagtttcgca 6120

tgagtgctga gttgtggtca ctgtggagct cagagctata tggcactttt ttatgctgta 6180

tgggggatgg tgataagatt aacatttggg tgtttgagga tgtaaagtag actatttgtc 6240

acacaactga gaatgcaggt cctgttggtg gaacagtcat gatgtaagaa ataccagcca 6300

ttctttcatg aagttgttag gaaactttgt ggaccagata gacatgcggg tgggggtgtg 6360

tttaattgat gccaagaaag tagagggaca gagagagagc tcagaaagct gtcctataat 6420

gtacagagaa catgctcggt cactagtttg gttgatgtct ctttcaagag ctcaggctta 6480

agaagctgcc tttaaagttt gcctccatgc ccagcgtttt cacaaatcct cactcctggg 6540

ctgcacgtaa acaataaaca gttgtatttc tgaagttgtt ctaccagccc tgtcgttctc 6600

gatcggaatc tttcccactt gccaaaggaa gggtctttct ttgtcttggt ttagtctttg 6660

tcagatccca aactcaggac cttctcttct ccaaaatctc ttgttagtaa atttcactcc 6720

tgccccttgt ttcacaggtt gtaaaaggtt tgtccatcta ggtttctttg ttgtggctgt 6780

gagctcactt ctcagtgcct ctgctgtcac taacgctcct ggtgagtatt ttgatcaact 6840

tacttattgc cttggcctgc tctgatatga attgtcaaac aaaatcacaa ttctcctgta 6900

tggcaaataa catctaaaaa tccatcctct gcagagctga agaactaaat atcagaacta 6960

tacaattata ttctaaccag acccaaaatg atgagcatgg tcttccctca tataaaacaa 7020

caccatgtta cataaagact aaaagcagta catagaagac taaaggaaag gagaagaaag 7080

ctcagagtta tc 7092

Claims (21)

1. A sgRNA, characterized in that it has:

   (i) as shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; or

   (ii) As shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; or

   (iii) (iii) a sequence which encodes the same protein as the nucleotide sequence of (i) or (ii) but which differs from the nucleotide sequence of (i) or (ii) due to the degeneracy of the genetic code; or

   (iv) A sequence having at least 70% homology to the sequence of (i) or (ii) or (iii).
2. 2. An expression cassette, recombinant vector, or cell containing the sgRNA of claim 1.
3. 3. The recombinant expression vector of claim 2, wherein the recombinant vector is pUC57 kan-T7-delG-sgRNA.
4. 4. A primer pair for amplifying or identifying the sgRNA of claim 1, the expression cassette, the recombinant vector, or the cell of claim 2.
5. 5. The primer pair of claim 4, wherein the sequence of the primer pair is as shown in SEQ ID NO:3 and SEQ ID NO: 4 is shown in the specification; or/and SEQ ID NO: 5 and SEQ ID NO: and 6.
6. 6. Use of the sgRNA of claim 1, the expression cassette, recombinant vector or cell of claim 2, the primer pair of claim 4 or 5 for CD163 gene editing.
7. 7. A targeting vector, comprising the following elements: an upstream homology arm, a porcine-derived CD163 gene, a Stop element and a downstream homology arm, wherein the sequence of the upstream homology arm is shown as SEQ ID NO:34, and the sequence of the swine CD163 gene is shown as SEQ ID NO:35, the Stop element sequence is shown as SEQ ID NO:37, and the sequence of the downstream homology arm is shown as SEQ ID NO: shown at 38.
8. 8. The targeting vector of claim 7, wherein the sequence of said targeting vector is as set forth in SEQ ID NO: shown at 39.
9. 9. The method for constructing a targeting vector according to claim 7 or 8, comprising the steps of: and fusing the upstream homology arm, the downstream homology arm and the synthesized pCD163 gene fragment by adopting a fusion PCR (polymerase chain reaction) mode, and performing slic connection on a fused product, a stop element and a pMD18T vector to obtain the recombinant plasmid.
10. 10. Use of the targeting vector of claim 7 or 8 for the preparation of a CD163 gene swine ized animal model.
11. 11. A method for constructing a CD163 gene humanized mouse model is characterized by comprising the following steps:

    1) cloning the DNA sequence of the sgRNA of claim 1 into a vector to construct a recombinant plasmid;

    2) carrying out PCR amplification on the recombinant plasmid obtained in the step 1) as a template to obtain a template, and transcribing the template to obtain sgRNA;

    3) incubating sgRNA and Cas9 protein, adding the targeting vector of claim 7 or 8, injecting the obtained mixed solution into mouse fertilized eggs, transplanting the fertilized eggs into a pseudopregnant female mouse, and genetically identifying the screened targeted mouse, namely a positive mouse after the mouse is born;

    4) and breeding the obtained positive mouse and a wild mouse, and carrying out gene identification on the born mouse to obtain the stably inherited CD163 humanized mouse animal model.
12. 12. The method for constructing a CD163 gene-suinized mouse model, according to claim 11, wherein the gene identification in steps 3) and 4) comprises PCR identification of 5 'end and 3' end of mouse tail genomic DNA, if the PCR identification detects an expected band, it indicates that the target fragment is recombined at the target site, and the PCR product is sequenced at the same time, and the sequencing is correct, which is a positive mouse.
13. 13. The method for constructing a CD163 gene suinized mouse model according to claim 12, wherein the PCR primer pairs identified by PCR at the 5 'end and the 3' end are SEQ ID NO: 11 and SEQ ID NO: 12 or/and SEQ ID NO: 13 and SEQ ID NO: as shown at 14.
14. 14. The method for constructing a CD163 gene suinized mouse model according to claim 12, wherein the primer pairs for PCR sequencing of the 5 'end and the 3' end are SEQ ID NOs: 15 and SEQ ID NO: 16 or/and SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
15. 15. The method for constructing a humanized mouse model of a CD163 gene according to claim 12, wherein the gene identification of step 4) further comprises PCR identification and sequencing verification of internal sequences.
16. 16. The method for constructing a CD163 gene suinized mouse model according to claim 12, wherein the PCR identification primer pairs of the internal sequences are SEQ ID NOs: 19 and SEQ ID NO: 20 is shown; SEQ ID NO: 21 and SEQ ID NO: 22, respectively.
17. 17. The method for constructing a mouse model derived from a CD163 gene from swine according to claim 13, wherein the primers for verifying sequencing of the internal sequence in step 4) comprise SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28 or/and SEQ ID NO: 29.
18. 18. the method for constructing a CD163 gene suinized mouse model according to claim 11, wherein the gene identification in step 4) further comprises qPCR detection, wherein qPCR detection primer pairs are designed according to 5' end homology arms, wild-type mice are used as controls, and the copy number of the inserted exogenous targeting fragment in the genome is detected, and if the copy number of the mice is between 0.75 and 1.25, no random insertion is indicated.
19. 19. The method for constructing a CD163 gene porcine-derived mouse model, according to claim 18, wherein the qPCR detection primer pairs are SEQ ID NOs: 30 and SEQ ID NO: 31, shown in the figure; or/and SEQ ID NO: 32 and SEQ ID NO: shown at 33.
20. 20. A method for identifying CD163 gene suinized mice comprising the steps of: extracting positive mouse tail genome DNA obtained according to claims 9-17, respectively using the sequences of SEQ ID NO:3 and SEQ ID NO: 4 is shown in the specification; or/and SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification; or/and SEQ ID NO: 11 and SEQ ID NO: 12 or/and SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure; or/and SEQ ID NO: 15 and SEQ ID NO: 16 or/and SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or/and SEQ ID NO: 19 and SEQ ID NO: 20 is shown; the amino acid sequence of SEQ ID NO: 21 and SEQ ID NO: 22; or/and SEQ ID NO: 23. the amino acid sequence of SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 28 or/and SEQ ID NO: 29; or/and SEQ ID NO: 30 and SEQ ID NO: 31, shown in the figure; or/and SEQ ID NO: 32 and SEQ ID NO: shown at 33.
21. 21. The use of the model mouse constructed by the method of claims 11-19 in screening or preparing drugs and diagnostic products for swine fever or blue ear disease.

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