CN114591953B - Construction method of CD163 gene pig-derived mouse model - Google Patents

Abstract

The invention discloses sgRNA, the expression cassette, the recombinant vector or the cell, and also discloses application of the sgRNA, the expression cassette, the recombinant vector or the cell and 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 swine-derived mouse model and a method for identifying the CD163 gene swine-derived mouse. According to the invention, a CRISPRP/Cas 9 gene modification method is adopted to replace a mouse Cd163 gene with a pig CD163 gene, so that a CD163 swine-derived mouse model is prepared, the mouse model has a pig functional gene, has very important significance for researches such as infection, invasion and immunity of swine-origin related viruses, and can become an ideal animal model for screening and evaluating related medicines and diagnostic products such as African swine fever and blue-ear disease, and new ideas and strategies are brought for controlling epidemic development.

Description

Construction method of CD163 gene pig-derived 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 pig-derived mouse model.

Background

The epidemic situation of African swine fever spreading in recent years brings serious hit to the pig industry in China, and is the first killer in the pig industry in China at present. African swine fever (African swine fever, ASF) is a swine-virulent infectious disease caused by African swine fever virus (African swine fever virus, ASFV) that presents significant difficulties and challenges to the global pig industry. The mortality rate of hyperacute and acute infection caused by virulent strains is almost as high as 100%. In 2018, 8 months, ASF is transmitted into China, and 32 provinces in China have epidemic outbreaks at present.

African Swine Fever Virus (ASFV) is a unique double-stranded DNA virus, which enters cells in a receptor-mediated "endocytosis" mode mainly through entering tonsils from oral and nasal mucosa, and rapidly spreads to the whole body through lymph fluid and blood after replication. Immunization is the first barrier of pigs against ASFV. Monocytes and macrophages are the most dominant infected cells for ASFV, and highly mature, cells express high levels of specifically labeled macrophages 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 receptor CD163, surface antigen 4E9 (porcine CD107a or lysosomal associated membrane protein I) are susceptible to ASFV infection. Studies have also shown that CD163 receptor is a necessary but inadequate condition for ASFV (e.g., highly pathogenic Georgia 2007/1 strain) infection, suggesting that infection with ASFV requires additional receptor assistance. In addition, a plurality of cases of gene knockout pigs constructed aiming at the CD163 gene at home and abroad have been reported, and the pig with the gene deletion is proved to resist the highly Pathogenic porcine reproductive and respiratory syndrome virus infection (PRRSV), which is the most important receptor for mediating PRRSV infection. CD163 gene modification significantly inhibited PRRSV replication both in vitro and in vivo, and these findings suggest that modification of CD163 may provide a potential strategy for anti-PRRSV treatment.

One of the best ways to prevent the spread of epidemic disease is to vaccinate against it, but so far no effective vaccine has been available worldwide. Vaccine development has progressed slowly because african swine fever virus itself is complex in structure, has multiple immune escape mechanisms, and does not induce the production of neutralizing antibodies, and the specific mechanism by which scientists stimulate protective immune responses with african swine fever strains is not known. Vaccines are indispensable for controlling african swine fever, attenuated vaccines are manufactured using live viruses, and there is a risk that microorganisms may persist throughout the swine herd and cause unacceptable adverse reactions.

Vaccine drug research and screening experiments need to be evaluated on animal models, rodents are the most widely used experimental animal models, and are one of the best experimental animal models due to small size, short growth and breeding period and easy operation, so that no mouse model aiming at swine CD163 gene preparation exists at present.

The gene editing function of the CRISPR/Cas9 system is mainly applied to genome targeting of a plurality of species, alleles are subjected to modification (such as knockout, mutation and knock-in) operation, various modification models can be quickly and efficiently built to study gene functions or build disease models, and compared with a traditional gene editing mouse model manufactured based on an embryonic stem cell targeting technology, the gene editing function is simple in design and convenient to operate, the complex in-vitro splicing process of targeting vectors is omitted, and the screening and culturing of embryonic stem cells and other works are omitted, so that the method has great application prospects in terms of manufacturing cost and period of the model. Although CRISPR/Cas9 gene editing systems have high cleavage efficiency and have been commonly used in the creation of gene knockout models, targeting efficiency is significantly reduced with increasing length of the knockin fragments when more complex genetic modification requirements, such as longer fragment gene targeting, are faced. In addition, when the CRISPR/Cas9 system containing the donor recombination fragment is directly subjected to micromanipulation on fertilized eggs, the donor fragment can be recombined at a target site, and also has a high probability of being randomly integrated into the genome of a mouse, the integrated site is random, high copy number knockin can occur, and the randomly integrated mouse can have unexpected phenotypes such as ectopic expression of an exogenous gene, silencing or activation of an endogenous gene at a non-target site and the like under the influence of the integrated site and the copy number, so that experimental results are interfered. Therefore, the recombination efficiency of the CRISPR/Cas9 system remains to be further explored and optimized, and at the same time, how to reduce or avoid the random integration probability of the donor during targeting, and to increase the detection rate of random integration, undoubtedly brings a series of challenges to the gene editing workers.

Disclosure of Invention

The invention aims to: according to the invention, a CRISPRP/Cas 9 gene modification method is adopted to replace a mouse Cd163 gene with a pig CD163 gene, so that a CD163 swine-derived mouse model is prepared, the mouse model has a pig functional gene, has very important significance for researches such as infection, invasion and immunity of swine-origin related viruses, and can become an ideal animal model for screening and evaluating related medicines and diagnostic products such as African swine fever and blue-ear disease, and new ideas and strategies are brought for controlling epidemic development.

The technical problem to be solved by the invention is to provide the sgRNA.

The invention also solves the technical problem of providing an expression cassette, a recombinant vector or a cell containing the sgRNA.

The invention also solves the technical problem of providing primer pairs for amplifying or identifying the sgrnas, the expression cassettes, the recombinant vectors or the cells.

The invention also solves 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 solves the technical problem of providing a construction method of a CD163 gene pig-derived mouse model.

The final technical problem to be solved by the invention is to provide a method for identifying a CD163 gene pig-derived mouse.

In order to solve the technical problems, the invention adopts the following technical scheme: the present invention provides sgrnas having:

(i) The sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, a nucleotide sequence shown in seq id no; or (b)

(ii) The sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, and a nucleotide sequence complementary to the nucleotide sequence shown in (a); or (b)

(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 (b)

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

The invention also includes an expression cassette, recombinant vector or cell containing said sgRNA.

Wherein the recombinant vector is pUC57kan-T7-delG-sgRNA.

The invention also includes primer pairs for amplifying or identifying the sgrnas, the expression cassettes, recombinant vectors or cells.

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

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 invention also provides a targeting vector, which comprises the following elements: an upstream homology arm, a swine CD163 gene, a Stop element, and a downstream homology arm, wherein the upstream homology arm sequence is as set forth in SEQ ID NO:34, the swine CD163 gene sequence is shown in SEQ ID NO:35, the Stop element sequence is shown as SEQ ID NO:37, said downstream homology arm sequence is as set forth in SEQ ID NO: shown at 38.

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

The invention also discloses 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 by adopting a fusion PCR (polymerase chain reaction) mode, and performing slec connection on the fused product, the stop element and the pMD18T vector.

The invention also discloses application of the targeting vector in preparation of a CD163 gene pig-derived animal model.

The invention also discloses a construction method of the CD163 gene pig-derived mouse model, which comprises the following steps:

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

2) Performing 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 mixed solution obtained after targeting vector, injecting the mixed solution into fertilized eggs of mice, transplanting the fertilized eggs into pseudopregnant female mice, and after birth of the mice, identifying and screening out medium-target mice, namely positive mice by gene;

4) And (3) breeding the obtained positive mice and wild mice, and carrying out gene identification on the born mice to obtain a stably inherited CD163 swine-derived mouse animal model.

The gene identification in the step 3) and the step 4) comprises the step of carrying out PCR identification of the 5 'end and the 3' end of mouse rat tail genome DNA, and if the PCR identification detects the expected band, the target fragment is indicated to be recombined at the target site, and meanwhile, the PCR product is sequenced, and the positive mouse is obtained after 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: 14.

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

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

Wherein, the PCR identification primer pair of the internal sequence is SEQ ID NO:19 and SEQ ID NO: shown at 20; SEQ ID NO:21 and SEQ ID NO: shown at 22.

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 in the step 4) further comprises qPCR detection, a qPCR detection primer pair is designed according to a 5' -end homology arm, a wild type mouse is used as a control, the copy number of the inserted exogenous targeting fragment in the genome is detected, and if the copy number of the mouse is between 0.75 and 1.25, random insertion is not shown.

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

The invention also includes a method for identifying a CD163 gene pig-derived mouse, comprising the steps of: by extraction of

The obtained positive mouse tail genome DNA adopts SEQ ID NO:3 and SEQ ID NO:4 is shown in the figure; or/and SEQ ID NO:5 and SEQ ID NO:6 is shown in the figure; or/and SEQ ID NO:11 and SEQ ID NO:12 or/and SEQ ID NO:13 and SEQ ID NO: 14; or/and SEQ ID NO:15 and SEQ ID NO:16 or/and SEQ ID NO:17 and SEQ ID NO: shown at 18; or/and SEQ ID NO:19 and SEQ ID NO: shown at 20; SEQ ID NO:21 and SEQ ID NO: shown at 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; or/and SEQ ID NO:32 and SEQ ID NO: shown at 33.

The invention also discloses application of the model mouse constructed by the method in screening or preparing medicines for treating swine fever or blue-ear diseases and diagnostic products.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

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

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

3. The invention verifies the correct integration of target site genes by PCR and sequencing methods, and simultaneously carries out QPCR detection on mice with positive target, detects the possibility of other site integration in the genome of the mice, and further verifies that positive mice are correct targets, and other non-target sites of the positive mice are inserted randomly.

Drawings

FIG. 1, schematic diagrams of a pCD163 swine-derived mouse model;

FIG. 2, 1-44#F0 mice primary screening assay PCR results; representing the Positive control, the PCR system was added with the donor plasmid before targeting of the project as a Positive control template control; WT: WT is C57BL/6J wild type, negative control; n: n is a negative blank control; /: the lane is free of sample; 1 to 44: 1-44#F0 mice; m:8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

FIG. 3, 45-119#F0 mice primary screening assay PCR results; representing the Positive control, the PCR system was added with the donor plasmid before targeting of the project as a Positive control template control; WT: WT is C57BL/6J wild type, negative control; n: n is a negative blank control; /: the lane is free of sample; 45-119: 45-119#F0 mice; m:8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

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

FIG. 5, 3' end PCR identification electrophoretogram of F0 generation mice; p: representing the Positive control, the PCR system was added with the donor plasmid before targeting of the project as a Positive control template control; WT: WT is C57BL/6J wild type, negative control; n: n is a negative blank control;

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

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

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

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 construction of pCD163 swine-derived mouse model

The homologous recombination and CRISPR/Cas9 technology are used for replacing the pig CD163 gene with the mouse CD163 gene on fertilized eggs of a B6 background mouse, so that a mouse model capable of expressing pig-derived CD163 is constructed, and a targeting strategy is shown in figure 1.

1. Determination of porcine fragment substitution region and inserted porcine sequence

According to the structure and function of swine CD163, CDS of swine CD163 and a transcript termination element PolyA are selected to be inserted into a translation initiation codon locus of mouse Cd163, and the transcription and translation of mouse Cd163 genes are stopped at the same time of insertion. The amino acid sequence of the selected swine CD163 gene is shown as SEQ ID NO. 37, and the strategy diagram is shown as figure 1.

2. The positive mice are obtained by carrier injection transplantation

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

2) Preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; sgrnas for murine sequences were designed in the porcine sequence insertion region. Designing and synthesizing a recognition target site, and constructing an sgRNA expression vector. The sgRNA recognition site was located on exon where the translation initiation codon of the mouse Cd163 gene was located, and the target site sequence of the sgRNA on Cd163 is shown in table 1. The DNA sequence of the sgRNA was cloned into pUC57kan-T7-delG vector (the resistance of pUC57 was modified from Amp+ to Kana+, the vector was named pUC57kan-T7-delG, where PUC57 was from GenScript, cat# SD 1176) and the pUC57-sgRNA plasmid was constructed.

TABLE 1 DNA sequence of sgRNA

The preparation method of sgRNA transcription comprises the following steps: and (3) performing PCR (polymerase Star or PrimerStar Max) by taking a sgRNA-F, sgRNA-R as a primer and taking a puc57-sgRNA plasmid with correct sequencing as a template, and purifying a PCR product to prepare the sgRNA transcription template. Transcription was performed using the T7-ShortScript in vitro transcription kit (AM 1354) to give sgRNA.

TABLE 2 sgRNA primer sequences

sgRNA screening: after incubating the sgrnas with Cas9 protein, the mixture was injected into fertilized eggs for 0.5 day, and after culturing to blastula stage, identification of ko positive rate of mouse Cd163 gene was performed, thereby screening sgrnas with high cleavage activity.

sgRNA cleavage identification method: the collected blasts were subjected to PCR amplification (PCR protocol Table 3 below), the amplified bands were subjected to second generation sequencing (sequencing primer: cd163-intF 1), and compared with the wild type (wt) bands, and the probability of mutation was counted (the identification results are shown in Table 3 below).

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 are targeted respectively) and targeting vector are injected into fertilized eggs of mice for 0.5 day, transplanted into pseudopregnant female mice, and after the mice are born, the medium-target mice screened by 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 are fused by adopting a fusion PCR mode, and the fused product is subjected to slec connection with stop elements and a pMD18T vector. After in vitro connection and transformation to escherichia coli DH5 alpha, selecting a monoclonal to carry out PCR and enzyme digestion identification, and the successfully connected vector is named dsDNA-Cd163-S1/S2; bstz171 enzyme cuts dsDNA-Cd163-S1/S2, two bands of 7098bp and 3702bp are generated, and the 7092bp fragment is recovered to be the targeting vector.

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

A. the tail DNA of the F0 mice which were born were extracted, and first subjected to PCR preliminary screening, the PCR primers are shown in Table 5, the primers pCD163-KI-tF1/pCD163-KI-5tR1 are located on the upstream homology arm and the swine CD163 CDS, respectively, and the primers 3Stop-KI-3tF1/pCD163-KI-tR2 are located on the exogenous Stop element and the downstream homology arm, respectively. If PCR produces a band of the expected size, it indicates that the mouse genome has the target fragment inserted. The PCR primary screening identification results are shown in FIG. 2 and FIG. 3.

TABLE 5 F0 Generation mice Primary screening identification PCR protocol

Remarks: KI is the mid-target genotype; WT is wild-type.

As can be seen from fig. 2 and 3, the primary PCR results: no. 14, no. 23, no. 26, no. 31, no. 40, as positive.

B. For primary screening positive mice, recombination at the 5 'and 3' ends was further identified and sequencing confirmed if targets were present. PCR identification after mid-targeting was performed with 5 'and 3' PCR primers (see Table 6), the primers pCD163-KI-5tF2/pCD163-KI-5tR2 were located outside the upstream homology arm and within the porcine fragment of the targeting vector, respectively, and 3Stop-KI-3tF1/pCD163-KI-3tR1 were located outside the exogenous Stop element and downstream homology arm, respectively, and PCR amplification at the 5 'and 3' ends produced PCR products of the expected size, and sequencing was consistent with the theoretical sequence, indicating recombination of the target fragment at the target site.

TABLE 6 5-and 3-terminal genotyping primers for CD163 swine-derived mice

Remarks: KI is the mid-target genotype; WT is wild-type.

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

C. Sequencing protocol: the sequencing method adopts the recovered 5-end and 3-end PCR products, the recovered PCR products are sequenced according to the sequencing scheme of Table 7, and the mice with correct sequencing are positive mice.

Sequencing primer: pCD163-KI-5tF1, detecting 5-terminal homology arm and genomic adaptor, sequencing primer: pCD163-KI-5tR1, detecting pig source CDS and homology arm joint, sequencing primer: pCD163-KI-3seqF1, detecting the 3-terminal homology arm and genome linker; sequencing primer: pCD163-KI-3seqR1, detecting the homologous arm joint of Stop and 3 end;

TABLE 7 5-and 3-terminal genotype sequencing protocol for CD163 swine-derived mice

The sequencing result is as follows: 23#,31#: the 5 'and 3' ends were sequenced correctly, i.e., 23# and 31# were positive F0 mice.

4) The 31# positive F0 mice and the wild C57BL/6J mice are selected to be bred, and then the bred mice are bred, namely F1 mice, which are 4 mice in total, the number of the mice is 57-60 #, the born mice are subjected to genotype identification, 2 positive mice are obtained through identification, the number of the positive mice is 58#, and the number of the positive mice is 59#, and the positive mice are CD163 swine-derived mice animal models which can be inherited stably.

Example 2 genotyping of F1 mice

Identification of 5 'and 3' end gene type of CD163 swine derived mice, two pairs of primers are respectively used for carrying out two-end PCR identification after targeting on rat tail genome DNA of F1 generation mice obtained in the step 4) of the example 1 (Table 6), the identification scheme and the identification primers are the same as those of the example 1, the primers pCD163-KI-5tF2/pCD163-KI-5tR2 are respectively positioned outside homologous arms of the 5 'end and in pig derived fragments of targeting vectors, and PCR products are generated by amplifying the primers, so that the target vectors are effectively recombined at the 5' end of the rat genome; the 3stop-KI-3tF1/pCD163-KI-3tR1 is respectively positioned in the exogenous fragment and outside the 3 'homology arm of the targeting vector, and if the pair of primers are amplified to generate PCR products, the target vector is proved to be effectively recombined at the 3' end of the mouse genome. As can be seen from FIGS. 6 and 7, the pCD163 rat tail DNA identification electropherograms, the 5 'and 3' identification of mice No. 58 and No. 59 in the electrophoresis bands were positive, indicating that the mice were positive mice for correct gene recombination, the PCR products at the 5 'and 3' ends of the mice No. 58 and No. 59 were recovered, further sequencing was confirmed (sequencing primers are shown in Table 11), and the mice with correct sequencing were initially screened positive mice.

EXAMPLE 3 F1 Generation mouse internal sequence identification

For the 58 th and 59 th positive mice identified by the PCR primary screening at both ends of example 2, further PCR amplification (FIG. 8 and Table 10) and sequencing verification (Table 11) of the internal sequences were performed, PCR primers were designed for the inserted exogenous sequences, and it was determined that all the homologous arms of the target sites were correct with the genomic linker and the sequence of the knocked-in exogenous gene, and the mice with correct sequencing were identified as positive mid-target mice. PCR reaction conditions and systems are referred to in tables 8 and 9. Sequencing results showed that: the CD163 and Stop elements inserted in the 58 th mouse (58#) and the 59 th mouse (59#) are correct, and the insertion site and the linker sequence of the homology arm are both correct, thus being positive mice.

TABLE 8 PCR reaction System

Reagent (Vazyme P112-03)	Volume (mul)	Specification of specification
			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 CD163 pig-derived mouse internal identification primers

Note that: KI is the mid-target genotype; WT is wild-type.

TABLE 11 CD163 pig-derived mouse internal sequencing protocol

Example 4QPCR assay identification

QPCR detection: for screened 58# and 59# positive mid-target mice, rat tail DNA was extracted and qPCR assays were further performed using the primers of table 12 (table 13 and 14 position reaction system and reaction program, respectively), pCD163-5q-tF1/pCD163-5q-tR1 was designed at the 5' end homology arm, using wild-type mice as controls for detecting the copy number of the inserted exogenous targeting fragment in the genome, with oIMR3580 and oIMR1544 primer products as internal controls, correcting qPCR data, if the mouse copy number was between 0.75 and 1.25, indicating no random insertion.

TABLE 12 CD163 pig-derived mouse QPCR protocol

TABLE 13 QPCR reaction System Table 14QPCR reaction procedure

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

TABLE 15

Calibration data	Sample number	Random insertion of copy number
			0.81	58#	Corresponding to wild type
0.87	59#
			1.00	B6-1	Wild type
0.98	B6-2	Wild type

。

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<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> swine 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> swine 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 (9)

1. The construction method of the CD163 gene pig-derived mouse model is characterized by comprising the following steps of:

1) The sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2, cloning the DNA sequence of the sgRNA of the nucleotide sequence shown in the formula 2 into a vector to construct a recombinant plasmid;

2) Performing 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 mixed solution obtained after targeting vector, injecting the mixed solution into fertilized eggs of mice, transplanting the fertilized eggs into pseudopregnant female mice, and after birth of the mice, identifying and screening out medium-target mice, namely positive mice by gene; the targeting vector comprises the following elements: an upstream homology arm, a swine CD163 gene, a Stop element, and a downstream homology arm, wherein the upstream homology arm sequence is as set forth in SEQ ID NO:34, the swine CD163 gene sequence is shown in SEQ ID NO:35, the Stop element sequence is shown as SEQ ID NO:37, said downstream homology arm sequence is as set forth in SEQ ID NO: shown at 38;

4) And (3) breeding the obtained positive mice and wild mice, and carrying out gene identification on the born mice to obtain a stably inherited CD163 swine-derived mouse animal model.

2. The method for constructing a swine-derived mouse model of a CD163 gene according to claim 1, wherein the gene identification in the step 3) and the step 4) comprises PCR identification of the 5 'end and the 3' end of mouse rat tail genome DNA, and if the PCR identification detects the 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 positive mouse is obtained after the correct sequencing.

3. The method for constructing a swine-derived mouse model of a CD163 gene according to claim 2, wherein the PCR primer pair identified by 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: 14.

4. The method for constructing a swine derived mouse model of a CD163 gene according to claim 2, wherein the primer pairs for PCR sequencing of the 5 'and 3' ends are SEQ ID NOs: 15 and SEQ ID NO:16 or/and SEQ ID NO:17 and SEQ ID NO: shown at 18.

5. The method for constructing a swine-derived mouse model of a CD163 gene according to claim 2, wherein the gene identification of step 4) further comprises PCR identification and sequencing verification of internal sequences.

6. The method for constructing a swine-derived mouse model of a CD163 gene according to claim 5, wherein the PCR identification primer pairs of the internal sequences are SEQ ID NOs: 19 and SEQ ID NO: shown at 20; SEQ ID NO:21 and SEQ ID NO: shown at 22.

7. The method of claim 5, wherein the primer for sequencing the internal sequence in step 4) is set as the primer set in 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.

8. the method according to claim 2, wherein the gene identification in step 4) further comprises qPCR detection, designing qPCR detection primer pairs according to 5' homology arms, and detecting the copy number of the inserted exogenous targeting fragment in the genome by using a wild-type mouse as a control, wherein no random insertion is indicated if the copy number of the mouse is between 0.75 and 1.25.

9. The method for constructing a swine-derived mouse model of a CD163 gene according to claim 8, wherein the qPCR detection primer pairs are SEQ ID NOs: 30 and SEQ ID NO: 31; or/and SEQ ID NO:32 and SEQ ID NO: shown at 33.