CN110938651A

CN110938651A - Targeting vector, method for constructing BAC clone by targeting and integrating exogenous gene to mouse F4/80 exon 22 site and application

Info

Publication number: CN110938651A
Application number: CN201911076716.9A
Authority: CN
Inventors: 盛剑鹏; 汤江辉; 白雪莉; 梁廷波
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-03-31

Abstract

The invention belongs to the technical field of biology, and discloses a targeting vector, a method for constructing BAC clone by targeting and integrating an exogenous gene to a mouse F4/80 exon 22 site and application thereof, wherein the sequence of the targeting vector is shown as SEQ ID NO.14 and is named as a vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803 HA. The invention provides a new site sequence capable of realizing targeted integration, a targeting vector for targeted insertion of an exogenous gene can be constructed by utilizing the site, the fact that the exogenous gene can be efficiently integrated to a mouse F4/80BAC clone F4/80 exon 22 site by utilizing the targeting vector is verified, and the subsequently obtained F4/80BAC for targeted insertion of the exogenous gene can be used for constructing a mouse embryonic stem cell for targeted insertion of the exogenous gene, so that a foundation is established for constructing a transgenic cell line and a mouse.

Description

Targeting vector, method for constructing BAC clone by targeting and integrating exogenous gene to mouse F4/80 exon 22 site and application

Technical Field

The invention belongs to the technical field of biology, and mainly relates to a targeting vector, a method for constructing BAC clone by targeting integration of an exogenous gene to a mouse F4/80 exon 22 site, and application thereof.

Background

The exogenous gene is integrated to a certain site on the target cell genome by homologous recombination to achieve the aim of modifying a certain gene on a chromosome by site-directed modification, and the technology is called gene targeting technology. The targeted integration of exogenous genes to specific chromosomal loci by using gene targeting technology has important application values in gene therapy, cell engineering, genetic animal models, gene function research, drug development and the like, and all the applications depend on the reliability and predictability of functions of transferred genes on one hand and require that the transferred genes do not influence the functions of endogenous genes and/or other regulatory factors on the other hand. F4/80, also known as epidermal growth factor-containing mucin-like hormone receptor-like 1(EMR1), is widely expressed in many cells of myeloid lineage, including monocytes, macrophages, but not neutrophils. And the knockout of F4/80 did not affect macrophage survival and function. Therefore, F4/80 can be used as a gene knock-in site, so that the exogenous gene is specifically expressed in the mononuclear macrophage and is not expressed in other cells, and the function of the cells is not influenced.

Bacterial Artificial Chromosome (BAC) refers to a Bacterial chromosome cloning vector constructed based on an F plasmid. In the gene targeting technique, the homology arms of long fragments are required to ensure the efficiency of homologous recombination, and it is difficult to directly clone the homology arms of long fragments. As a vector for gene preservation, BAC can clone about 150kb DNA and has the characteristic of simple genetic background. Foreign genes needing targeted insertion can be firstly introduced into corresponding BAC clone by using a short homology arm, and then BAC is extracted to be further used as a gene targeting vector so as to improve the gene targeting efficiency.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a targeting vector, a method for constructing BAC clone by targeting and integrating an exogenous gene to a mouse F4/80 exon 22 site and application thereof.

The object of the present invention is achieved by the following technical means. A targeting vector HAs a sequence shown in SEQ ID NO.14 and is named as a vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803 HA. The vector can integrate the exogenous gene IRES-DTR and the resistance gene Neomycin in a targeted manner. After targeted integration IRES-DTR is transcribed under the control of F4/80 promoter, foreign gene DTR is translated under the control of IRES, and resistance gene Neomycin is expressed under the control of PGK (mammalian) and EM7 (E.coli). If it is desired to express other genes, the foreign gene DTR may be replaced with the gene of interest.

The invention discloses a preparation method of a targeting vector, which comprises the following steps:

(1) using RP23-212F14 BAC DNA as a template, and carrying out PCR by using a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO.2 to amplify a gene fragment F4/803' side homologous arm (F4/803HA) with a sequence shown as SEQ ID NO. 3;

(2) the target gene fragment F4/803HA is cloned to the vector PGK-EM7-Neo (counter-Selection BAC Modification Kit, Genebridges) with a positive Selection marker by utilizing the enzyme cutting sites NotI and BglII to obtain the intermediate vector PGK-EM7-Neo-F4/803 HA.

(3) Carrying out PCR by taking pLVX-EF1 α -IRES-mCherry plasmid (addge) as a template and utilizing a forward primer with a sequence shown in SEQ ID NO.4 and a reverse primer with a sequence shown in SEQ ID NO.5 to amplify a gene fragment IRES with a sequence shown in SEQ ID NO.6, carrying out PCR by utilizing a forward primer with a sequence shown in SEQ ID NO.7 and a reverse primer with a sequence shown in SEQ ID NO.8 by taking pIRES-proHB EGF WT plasmid (addge) as a template to amplify a gene fragment DTR with a sequence shown in SEQ ID NO.9, and obtaining a fusion gene fragment IRES-DTR with a sequence shown in SEQ ID NO.10 by OVerlap PCR;

(4) using RP23-212F14 BAC DNA as a template, and carrying out PCR by using a forward primer with a sequence shown as SEQ ID NO.11 and a reverse primer with a sequence shown as SEQ ID NO.12 to amplify a homologous arm (F4/805HA) at the F4/805' side of a gene fragment with a sequence shown as SEQ ID NO. 13;

(5) the target gene fragments F4/805HA and IRES-DTR are cloned to an intermediate vector PGK-EM7-Neo-F4/803HA by utilizing enzyme cutting sites BamHI, SalI and MluI, and a targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA with the sequence shown in SEQ ID NO.14 is obtained.

The invention also discloses an application of the targeting vector in targeted integration of the exogenous gene to the F4/80 exon 22 site and construction of the escherichia coli BAC Clone in targeted integration of the exogenous gene to the F4/80 exon 22 site. The targeting vector can be used for constructing a mouse F4/80BAC clone by targeting and integrating a foreign gene to an F4/80 exon 22 site.

The invention also provides a method for constructing a mouse F4/80-DTR BAC clone inserted with the targeted foreign gene by utilizing the targeting vector and carrying out targeted integration on the foreign gene to the F4/80 exon 22 site. The method comprises the following specific steps:

(1) transforming the Red/ET expression plasmid pRED/ET into an F4/80BAC clone by an electroporation method, screening on an LB plate with resistance to chloramphenicol and tetracycline, selecting a single colony for amplification, and inducing the expression of the Red/ET by using L-arabinose;

(2) the targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA was linearized by digestion with BamHI, and the linearized vector was transformed into the above-described F4/80BAC clone expressing Red/ET by electroporation, so that IRES-DTR-PGK-EM7-Neo was inserted into F4/80BAC in a targeted manner;

(3) f4/80BAC clone successfully inserted into IRES-DTR-PGK-EM7-Neo expresses neomycin resistance, and LB plate with neomycin and chloramphenicol is used for screening to obtain BAC clone F4/80-IRES-DTR-EM 7-PGK-Neo;

(4) selecting a single clone, and successfully inserting an IRES-DTR-PGK-EM7-Neo F4/80BAC clone to obtain a 1209bp band by using a forward primer with a sequence shown as SEQ ID NO.15 and a reverse primer with a sequence shown as SEQ ID NO.16 as colony PCR. SpeI enzyme digestion identification positive BAC clone can see that one band is deleted between 3 kb and 3.5 kb.

Therefore, the invention provides a preparation and detection method for constructing a mouse F4/80BAC clone with targeted insertion of an exogenous gene by targeting a targeting vector and targeted integration of the exogenous gene to an F4/80 exon 22 site.

The invention has the beneficial effects that:

the invention provides a method for efficiently and safely integrating an exogenous gene to a mouse F4/80BAC clone F4/80 exon 22 site in a targeted manner and an application example.

The invention provides DNA sequence information of a left homologous arm and a right homologous arm of a targeting vector targeting a mouse F4/80 exon 22 site and a construction method thereof.

The invention provides a method for screening and identifying positive clones and an application example.

As mentioned in the background technology, F4/80 can be used as a site for knocking in a gene, so that an exogenous gene is specifically expressed in mononuclear macrophages and the cell function is not influenced, the invention provides a novel site sequence capable of realizing targeted integration, a targeting vector for targeted insertion of the exogenous gene can be constructed by utilizing the site, the targeting vector can be used for efficiently integrating the exogenous gene to a mouse F4/80BAC clone F4/80 exon 22 site, and the subsequent obtained F4/80BAC for targeted insertion of the exogenous gene can be used for constructing mouse embryonic stem cells for targeted insertion of the exogenous gene, thereby establishing a foundation for constructing a transgenic cell line and a mouse.

Drawings

FIG. 1: constructing a targeting plasmid flow diagram of a mouse F4/80 exon 22 site;

FIG. 2: the plasmid structure schematic diagram of an intermediate vector PGK-EM7-Neo-F4/803HA carrying resistance genes Neomycin and homologous arms at the side of F4/803';

FIG. 3: a plasmid structure schematic diagram of a targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA carrying exogenous genes IRES-DTR, resistance genes Neomycin and homology arms at two sides of F4/80;

FIG. 4: schematic diagram of primers (black arrows) for PCR screening and identification after targeting vector targeting principle and homologous recombination insert F4/80 BAC;

FIG. 5: PCR screening and identification of F4/80BAC monoclonal targeting F4/80 exon 22 site, wherein the correctly targeted monoclonal has about 1.2kb band;

FIG. 6: enzyme digestion identification successfully targets F4/80 exon 22 site F4/80BAC monoclonal.

Detailed Description

The invention will be described in detail below with reference to the following drawings:

example 1: constructing a targeting vector for targeted integration of exogenous genes to mouse F4/80 exon 22 site

Mouse F4/80 genome sequence is obtained by searching and downloading from genome, ucsc, edu website, and the position and sequence of each exon and intron are determined by combining mouse F4/80(Genbank No: NM-010130.4) sequence. BAC Clone containing the entire F4/80 Gene RP23-212F14 was purchased from Chieldren's Hospotal Oakland Research Institute and BAC DNA was utilized

HiPure Plasmid Filter Maxiprep Kit (Invitrogen) was prepared for use.

PCR amplification to obtain the homology arm at the F4/803' side of the target gene fragment (F4/803 HA): RP23-212F14 BACDNA is taken as a template, and forward primers F4/80-3HA-F (BglII) -ACCAC are utilizedAGATCTAACAATGTCTGAAGATTGTAAGTGC (SEQ ID NO.1), reverse primer F4/80-3HA-R (NotI) -AACAACGCGGCCGCTGTACCGTTGAAATAGGACGTG (SEQ ID NO.2) was used for PCR amplification of the 3' side homology arm F4/803HA (SEQ ID NO. 3). Underlined sequences are the restriction sites.

The PCR reaction system is as follows: (Table 1)

The PCR amplification conditions were as follows: (Table 2)

PCR products of a target gene fragment F4/803HA are cloned to a carrier PGK-EM7-Neo with a positive Selection marker gene, target bands are cut out respectively and recovered by using AxyPrep DNA Gel Extraction Kit (Axygen). the carrier PGK-EM7-Neo (counter-Selection BACmodification Kit, GeneBridgges) with the positive Selection marker of the Neomycin and the recovered F4/803HA are cut by BglII and NotI (NEB) double digestion, the digested PCR products are purified and recovered by using AxyPrep PCR Clean-Up Kit (Axygen), the digested carrier is separated by agarose Gel electrophoresis, the target bands are cut out and recovered by using the AxyPrep DNA Gel Extraction Kit (Axygen). the carrier and the target fragment are sequenced by using T4DNA Gel (NEB), the target fragment is transformed to Escherichia coli, a single strain of DNA Gel is extracted and cultured, and the result of the PCR products is inserted into a carrier PGK-EM7-Neo, the PCR products are analyzed by using T4DNA Gel electrophoresis, and the PCR products are inserted into a PCR product of Escherichia coli strain, and the PCR products are analyzed by using a PCR clone (SEQ ID strain), and the PCR products of the Escherichia coli, the PCR products are analyzed by using a PCR products of the Escherichia coli strain, the.

PCR amplification to obtain target gene fragment IRES and DTR, using pLVX-EF1 α -IRES-mCherry plasmid (addrene) as template and utilizing forward primer IRES-F (SalI) -ACCACGTCGACGCCCCTCTCCCTCCCCCC (SEQ ID NO.4), and reverse primer IRES-R-CATGGTTGTGGCAAGCTTATCATCGTG (SEQ ID NO.5) as PCR amplified gene fragment IRES (SEQ ID NO. 6); pIRES-proHB EGF WT plasmid (adddge) is taken as a template, and a forward primer DTR-F-CACGATGATAAGCTTGCCACAACCATGAAGCTGCTGCCGTCG (SEQ ID NO.7) and a reverse primer DTR-R (MluI) -ACCAC are utilizedACGCGTTTAGTGGGAATTAGTCATGCCC (SEQ ID NO.8) was used for PCR amplification of DTR (SEQ ID NO. 9). Underlined sequences are the restriction sites. The PCR reaction system is as shown in Table 1, and the PCR amplification conditions are as shown in Table 2.

After the PCR products were separated by agarose gel electrophoresis, the desired bands were excised and recovered using the AxyPrep DNA Gelextraction Kit (Axygen). IRES and DTR are subjected to Overlap PCR to synthesize a fusion gene fragment IRES-DTR (SEQ ID NO.10), which comprises the following specific steps:

the Overlap PCR reaction system is as follows:

the conditions for the Overlap PCR amplification were:

PCR amplification of the homology arm at the F4/805' side of the target gene fragment (F4/805 HA): RP23-212F14 BAC DNA as template and forward primer F4/80-5HA-F (BamHI) -ACCACGGATCCGAAAAAGGATCAATAAGAATAGAAAGAATTG (SEQ ID NO.11), reverse primer F4/80-5HA-R (SalI) -ACCACGTCGACCCATAATATATTTAAAAGCAAGAAAGGAT G (SEQ ID NO.12) was used for PCR amplification of the 5' side homology arm F4/805HA (SEQ ID NO. 13). Underlined sequences are the restriction sites. The PCR reaction system is as shown in Table 1, and the PCR amplification conditions are as shown in Table 2.

Cloning the target gene fragment F4/805HA and IRES-DTR into intermediate vector PGK-EM7-Neo-F4/803HA after agarose Gel electrophoresis separation of PCR product, cutting out target band and recovering by AxyPrep DNA GelExtraction Kit (Axygen). after purification, F4/805HA is cut by BamHI and SalI (NEB) and IRES-DTR is cut by SalI and MluI (NEB). intermediate vector PGK-EM7-Neo-F4/803HA is cut by BamHI and MluI (NEB). after the cut PCR product is purified by AxyPrep PCR Clean-Up Kit (Axygen), the cut plasmid vector is cut by agarose Gel electrophoresis separation, cutting out target band and recovering by sequencing T4DNA extension Kit (Axygen). after agarose Gel electrophoresis separation of target band and inserting into target vector DNA, inserting into target vector DNA of mouse DNA, plasmid, etc. 7-5-15-5-T-Pro-5-T-103-T, DNA, genome, DNA, cloning.

Example 2: targeted integration of foreign genes into mouse F4/80BAC using targeting vectors

Electroporation pRed/ET was transformed into E.coli BAC Clone RP23-212F14 containing the entire F4/80 gene: coli BAC Clone RP23-212F14 containing the entire F4/80 gene was inoculated on LB plate containing chloramphenicol (15. mu.g/mL) and cultured at 37 ℃ for 16 h. 1 clone was picked into a 2mL centrifuge tube (one hole punched in the lid to allow aeration) with 1mL LB medium (containing 15. mu.g/mL chloramphenicol) and incubated overnight at 37 ℃ at 200 rpm. 30 μ L of E.coli cultured overnight was cloned into 2mL centrifuge tube containing 1.4mL LB medium (containing 15 μ g/mL chloramphenicol), and cultured at 37 ℃ for 2-3h to amplify E.coli in log phase. The amplified E.coli was centrifuged at 11000rpm at 2 ℃ for 30s, and the supernatant was discarded by aspiration. The Escherichia coli precipitate was precooled on 1mL iceThe resulting solution was washed 3 times with ddH20, and about 30. mu.L of ddH2O was left to resuspend E.coli, and 1. mu.L of pRed/ET (20 ng/. mu.L, counter-Selection BAC Modification Kit (Genebridges, USA)) was added and the mixture was gently mixed and allowed to stand on ice. Transfer of E.coli suspension to ice-precooled 1mm electroporation cuvette using

Electroporation was performed with Electroporator 2510, 1350V 10. mu.F 600Ohms, transferring pRed/ET into BAC Clone RP23-212F 14. The E.coli was resuspended in 1mL of antibiotic-free LB medium and transferred to a 2mL centrifuge tube and incubated at 30 ℃ 1000rpm for 70min to allow expression of the resistance gene.

L-arabinose induces Red/ET expression: mu.L of E.coli transferred to pRed/ET and induced to express the resistance gene was spread on an LB plate containing tetracycline (3. mu.g/mL) and chloramphenicol (15. mu.g/mL) uniformly and cultured at 30 ℃ for 16 hours in the absence of light. 1 single clone was picked up in a centrifuge tube with a 2mL lid-pierced hole containing 1mL LB medium (containing 3. mu.g/mL tetracycline, 15. mu.g/mL chloramphenicol), and incubated overnight at 30 ℃ at 200 rpm. mu.L of E.coli was placed in 1 centrifuge tube with 1mL LB medium (containing 3. mu.g/mL tetracycline, 15. mu.g/mL chloramphenicol) 2mL lid-pierced holes and incubated at 1100rpm for 2h at 30 ℃ until the OD600 was approximately 0.3. Adding 50 mu L L-arabinose to the final concentration of 0.3-0.4%, culturing at 37 ℃ and 200rpm for 45-60min, and inducing Red/ET expression.

Electroporation the linearized targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA was transformed into a BAC Clone RP23-212F14 expressing Red/ET: coli expressing Red/ET was centrifuged at 2 ℃ and 11000rpm for 30s and the supernatant was discarded. The E.coli pellet was washed 3 times with 1mL of ddH20 pre-cooled on ice, leaving about 30. mu.L of ddH2O to resuspend the E.coli, 1-2. mu.l (100 and 200ng) of the linearized targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA was digested with BamHI (NEB), gently mixed, and allowed to stand on ice. Transfer of E.coli suspension to ice-precooled 1mm electroporation cuvette using

Electroporation was performed with Electroporator 2510 at 1350V 10. mu.F 600Ohms and the linearized targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA was transferredBACclone RP23-212F14 was incorporated. The E.coli was resuspended in 1mL of antibiotic-free LB medium and transferred to a 2mL centrifuge tube, incubated at 30 ℃ 1000rpm for 70min to insert IRES-DTR-PGK-EM7-Neo homologous recombination into F4/80BAC and express the neomycin resistance gene. mu.L of E.coli was applied to LB plates containing tetracycline (3. mu.g/mL), chloramphenicol (15. mu.g/mL) and neomycin (15. mu.g/mL), spread evenly and cultured at 30 ℃ for more than 20 hours to obtain an E.coli clone inserted into IRES-DTR-PGK-EM7-Neo, which was named BACCloneF4/80-IRES-DTR-PGK-EM 7-Neo.

Example 3: screening and identification of BAC clone with exogenous gene targeted integrated to mouse F4/80 exon 22 site

Colony PCR screening of E.coli BAC Clone targeted for insertion of IRES-DTR-PGK-EM 7-Neo: 12 single clones were picked up and blown into 1.5mL centrifuge tubes containing 10. mu.l of ddH2O and mixed well. Mu.l of the bacterial suspension was used as a template, and colony PCR was performed using forward primer F4/80-189503-F-TGAAATAACCCAGACACAGAAGTTT (SEQ ID NO.15) and reverse primer IRES screen-R-CGGCAATATGGTGGAAAATAACA (SEQ ID NO. 16). The remaining bacterial solution was added to a 15mL centrifuge tube containing 5mL LB medium (containing 15. mu.g/mL chloramphenicol and 15. mu.g/mL neomycin) and incubated at 37 ℃ and 220rpm for 16h, and pRed/ET was lost at 37 ℃.

The colony PCR reaction system is as follows:

colony PCR reaction conditions were as follows:

the PCR product was identified by 1% agarose gel electrophoresis, and a band of about 1.2kb was observed in the BAC-positive clone of E.coli inserted targeted to the site of exon 22 of IRES-DTR-PGK-EM7-Neo to F4/80, as shown in FIG. 5.

Enzyme digestion identification of positive BAC clones: 4mL of the overnight amplified positive BAC clone was collected and BAC was extracted using Counter-selection (advanced) BAC Modification Kit protocol version 3.0(Gene Bridges, USA). The positive clone BAC is cut by SpeI (NEB) and identified by electrophoresis on 1% agarose gel, and a band is deleted in 3-3.5kb, as shown in figure 6. And centrifuging residual bacteria liquid of the positive BAC clone with the correct target insertion foreign gene in PCR and enzyme digestion identification, removing supernatant, and resuspending the supernatant by using LB culture medium containing 20% of glycerol and storing the supernatant at the temperature of-80 ℃.

It should be understood that equivalent substitutions and changes to the technical solution and the inventive concept of the present invention should be made by those skilled in the art to the protection scope of the appended claims.

SEQUENCE LISTING

<110> Zhejiang university medical college affiliated to the first hospital

<120> targeting vector, method for constructing BAC clone by targeting and integrating exogenous gene to mouse F4/80 exon 22 site and application

<160>

<170>PatentIn version 3.3

<210>1

<211>37

<212>DNA

<213> primer F4/80-3HA-F (BglII)

<400>1

ACCACCAGAT CTAACAATGT CTGAAGATTG TAAGTGC

<210>2

<211>36

<212>DNA

<213> primer F4/80-3HA-R (NotI)

<400>2

AACAACGCGG CCGCTGTACC GTTGAAATAG GACGTG

<210>3

<211>281

<212>DNA

<213> Gene fragment F4/80-3HA

<400>3

ACCACCAGAT CTAACAATGT CTGAAGATTG TAAGTGCTTT AAGGATCATA CTTTTAATAA 60

CTATCTAGCT TATGGAAATT TAGGGTATCA TGAGTTGATG GCAGTTTTGT TTTTAGCTCA 120

AAACATGGAT AGATGGAACC AAACTCCAGG AGTTTCATGC ACCAAGGAGA AACACAGGAT 180

ATCTACCCAA ATAAGTTTGT CTTTTGTGCC TTACAACTAT GAAGCTCCAC ATGTTTTGAA 240

AAGAGCACGT CCTATTTCAA CGGTACAGCG GCCGCTTGTT G

<210>4

<211>30

<212>DNA

<213> primer IRES-F (SalI)

<400>4

ACCACCGTCG ACGCCCCTCT CCCTCCCCCC

<210>5

<211>27

<212>DNA

<213> primer IRES-R

<400>5

CATGGTTGTG GCAAGCTTAT CATCGTG

<210>6

<211>602

<212>DNA

<213> Gene fragment IRES

<400>6

ACCACCGTCG AGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA 60

ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA 120

TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC 180

TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC 240

TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG 300

CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA 360

ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCTCCTCAAG 420

CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT 480

GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC 540

CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAAGCTTG CCACAACCAT 600

GA

<210>7

<211>42

<212>DNA

<213> primer DTR-F

<400>7

CACGATGATA AGCTTGCCAC AACCATGAAG CTGCTGCCGT CG

<210>8

<211>34

<212>DNA

<213> primer DTR-R (MluI)

<400>8

ACCACCACGC GTTTAGTGGG AATTAGTCAT GCCC

<210>9

<211>663

<212>DNA

<213> Gene fragment DTR

<400>9

CACGATGATA AGCTTGCCAC AACCATGAAG CTGCTGCCGT CGGTGGTGCT GAAGCTCTTT 60

CTGGCTGCAG TTCTCTCGGC ACTGGTGACT GGCGAGAGCC TGGAGCGGCT TCGGAGAGGG 120

CTAGCTGCTG GAACCAGCAA CCCGGACCCT CCCACTGTAT CCACGGACCA GCTGCTACCC 180

CTAGGAGGCG GCCGGGACCG GAAAGTCCGT GACTTGCAAG AGGCAGATCT GGACCTTTTG 240

AGAGTCACTT TATCCTCCAA GCCACAAGCA CTGGCCACAC CAAACAAGGA GGAGCACGGG 300

AAAAGAAAGA AGAAAGGCAA GGGGCTAGGG AAGAAGAGGG ACCCATGTCT TCGGAAATAC 360

AAGGACTTCT GCATCCATGG AGAATGCAAA TATGTGAAGG AGCTCCGGGC TCCCTCCTGC 420

ATCTGCCACC CGGGTTACCA TGGAGAGAGG TGTCATGGGC TGAGCCTCCC AGTGGAAAAT 480

CGCTTATATA CCTATGACCA CACAACCATC CTGGCCGTGG TGGCTGTGGT GCTGTCATCT 540

GTCTGTCTGC TGGTCATCGT GGGGCTTCTC ATGTTTAGGT ACCATAGGAG AGGAGGTTAT 600

GATGTGGAAA ATGAAGAGAA AGTGAAGTTG GGCATGACTA ATTCCCACTA AACGCGTGGT 660

GGT

<210>10

<211>1238

<212>DNA

<213> Gene fragment IRES-DTR

<400>10

ACCACCGTCG ACGCCCCTCT CCCTCCCCCC CCCCTAACGT TACTGGCCGA AGCCGCTTGG 60

AATAAGGCCG GTGTGCGTTT GTCTATATGT TATTTTCCAC CATATTGCCG TCTTTTGGCA 120

ATGTGAGGGC CCGGAAACCT GGCCCTGTCT TCTTGACGAG CATTCCTAGG GGTCTTTCCC 180

CTCTCGCCAA AGGAATGCAA GGTCTGTTGA ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG 240

CTTCTTGAAG ACAAACAACG TCTGTAGCGA CCCTTTGCAG GCAGCGGAAC CCCCCACCTG 300

GCGACAGGTG CCTCTGCGGC CAAAAGCCAC GTGTATAAGA TACACCTGCA AAGGCGGCAC 360

AACCCCAGTG CCACGTTGTG AGTTGGATAG TTGTGGAAAG AGTCAAATGG CTCTCCTCAA 420

GCGTATTCAA CAAGGGGCTG AAGGATGCCC AGAAGGTACC CCATTGTATG GGATCTGATC 480

TGGGGCCTCG GTGCACATGC TTTACATGTG TTTAGTCGAG GTTAAAAAAC GTCTAGGCCC 540

CCCGAACCAC GGGGACGTGGTTTTCCTTTG AAAAACACGA TGATAAGCTT GCCACAACCA 600

TGAAGCTGCT GCCGTCGGTG GTGCTGAAGC TCTTTCTGGC TGCAGTTCTC TCGGCACTGG 660

TGACTGGCGA GAGCCTGGAG CGGCTTCGGA GAGGGCTAGC TGCTGGAACC AGCAACCCGG 720

ACCCTCCCAC TGTATCCACG GACCAGCTGC TACCCCTAGG AGGCGGCCGG GACCGGAAAG 780

TCCGTGACTT GCAAGAGGCA GATCTGGACC TTTTGAGAGT CACTTTATCC TCCAAGCCAC 840

AAGCACTGGC CACACCAAAC AAGGAGGAGC ACGGGAAAAG AAAGAAGAAA GGCAAGGGGC 900

TAGGGAAGAA GAGGGACCCA TGTCTTCGGA AATACAAGGA CTTCTGCATC CATGGAGAAT 960

GCAAATATGT GAAGGAGCTC CGGGCTCCCT CCTGCATCTG CCACCCGGGT TACCATGGAG 1020

AGAGGTGTCA TGGGCTGAGC CTCCCAGTGG AAAATCGCTT ATATACCTAT GACCACACAA 1080

CCATCCTGGC CGTGGTGGCT GTGGTGCTGT CATCTGTCTG TCTGCTGGTC ATCGTGGGGC 1140

TTCTCATGTT TAGGTACCAT AGGAGAGGAG GTTATGATGT GGAAAATGAA GAGAAAGTGA 1200

AGTTGGGCAT GACTAATTCC CACTAAACGC GTGGTGGT

<210>11

<211>43

<212>DNA

<213> primer F4/80-5HA-F (BamHI)

<400>11

ACCACCGGAT CCGAAAAAGG ATCAATAAGA ATAGAAAGAA TTG

<210>12

<211>42

<212>DNA

<213> primer F4/80-5HA-R (SalI)

<400>12

ACCACCGTCG ACCCATAATA TATTTAAAAG CAAGAAAGGA TG

<210>13

<211>1025

<212>DNA

<213> Gene fragment F4/805HA

<400>13

ACCACCGGAT CCGAAAAAGG ATCAATAAGA ATAGAAAGAA TTGAATAGAG GATAACATGA 60

TAAATATTAT CAATGTACAT GATGTATGTG CATAAAACCA CTACAGAGAA ACACATCATT 120

TTGTTCAATA TACTAGTAAA TAAAAAACAA ATAGCATATA CATGTTTATA TATATTCTGT 180

GATTATTATA TATCACATAA TGTACATATA ATATTTATTT AATTTAGTCA ATGATTTTTG 240

TATCAAATTT TACTTTAACA TAACTTCATG CTGTGACCTC ACTATTCACT TAGTGCATAC 300

CAAATACTTG GTCAACTGGG TCTGTCTTAA TTTCAGTTCC TTTGACTATA CCTTGACAAA 360

AGCAACTTAG TAGAAAAAGA GTTTATTTTA TCTCAAAGTT ACAGAAAAAC AATTCATCAT 420

CAGGGAAGTC ACTACAGTAA GATATTGACA GAATTGCCAC ATTACATTCA CACTCAGCAG 480

CAGAGAGCAA CAAATTAGAG CATGTATGCA GTGCTCAGCA CATTGCCCAT TCTTACACAG 540

TTCAGAATCA TATTTCTAGG AATTGGTGCC ACCCATGGTG GCTGGGTATT TCTATATCAG 600

TCAGTATAAA AGATAAATCT ATTGGTCAGT CTGGTCTGAT AATCCCAAAC TGAGACTCTC 660

AGGTGATTCT AGATTGTGTC TAGTTGACAG TTACAATTAA TCATTACGTT TGTTATATTC 720

TTTGTTGCTG AACCCCTGAT AAATGGACCA TGATCCAGAA AATCACCTCG CCCAAAGATG 780

TTCACACAAG TAACTAGTTA AATTCAGTGA ATCACAAAGT AGGGCAATTG TGTTTAGCAT 840

TTCAATGTTT ATGCTTATAG CCTGATTCTA TATTGATATC CATTTACATG TGAGACAGTT 900

GTCCCCACAG ATAGGACTGG ATAATTTTTA GATCAATTTT GCTTCTAACT GAGGTCATTC 960

CCTTTTGTCT TTTTCAGGGT TAACATCCTT TCTTGCTTTT AAATATATTA TGGGTCGACG 1020

GTGGT

<210>14

<211>7266

<212>DNA

<213> targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA

<400>14

CTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 60

ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGACCGA 120

GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 180

CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CCACTACGTG AACCATCACC 240

CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG 300

CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG GCGAGAAAGG AAGGGAAGAA 360

AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG GTCACGCTGC GCGTAACCAC 420

CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCC CATTCGCCAT TCAGGCTGCG 480

CAACTGTTGG GAAGGGCGAT CGGTGCGGGC CTCTTCGCTA TTACGCCAGC TGGCGAAAGG 540

GGGATGTGCT GCAAGGCGAT TAAGTTGGGT AACGCCAGGG TTTTCCCAGT CACGACGTTG 600

TAAAACGACG GCCAGTGAAT TGTAATACGA CTCACTATAG GGCGAATTGG GATCCGAAAA 660

AGGATCAATA AGAATAGAAA GAATTGAATA GAGGATAACA TGATAAATAT TATCAATGTA 720

CATGATGTAT GTGCATAAAA CCACTACAGA GAAACACATC ATTTTGTTCA ATATACTAGT 780

AAATAAAAAA CAAATAGCAT ATACATGTTT ATATATATTC TGTGATTATT ATATATCACA 840

TAATGTACAT ATAATATTTA TTTAATTTAG TCAATGATTT TTGTATCAAA TTTTACTTTA 900

ACATAACTTC ATGCTGTGAC CTCACTATTC ACTTAGTGCA TACCAAATAC TTGGTCAACT 960

GGGTCTGTCT TAATTTCAGT TCCTTTGACT ATACCTTGAC AAAAGCAACT TAGTAGAAAA 1020

AGAGTTTATT TTATCTCAAA GTTACAGAAA AACAATTCAT CATCAGGGAA GTCACTACAG 1080

TAAGATATTG ACAGAATTGC CACATTACAT TCACACTCAG CAGCAGAGAG CAACAAATTA 1140

GAGCATGTAT GCAGTGCTCA GCACATTGCC CATTCTTACA CAGTTCAGAA TCATATTTCT 1200

AGGAATTGGT GCCACCCATG GTGGCTGGGT ATTTCTATAT CAGTCAGTAT AAAAGATAAA 1260

TCTATTGGTC AGTCTGGTCT GATAATCCCA AACTGAGACT CTCAGGTGAT TCTAGATTGT 1320

GTCTAGTTGA CAGTTACAAT TAATCATTAC GTTTGTTATA TTCTTTGTTG CTGAACCCCT 1380

GATAAATGGA CCATGATCCA GAAAATCACC TCGCCCAAAG ATGTTCACAC AAGTAACTAG 1440

TTAAATTCAG TGAATCACAA AGTAGGGCAA TTGTGTTTAG CATTTCAATG TTTATGCTTA 1500

TAGCCTGATT CTATATTGAT ATCCATTTAC ATGTGAGACA GTTGTCCCCA CAGATAGGAC 1560

TGGATAATTT TTAGATCAAT TTTGCTTCTA ACTGAGGTCA TTCCCTTTTG TCTTTTTCAG 1620

GGTTAACATC CTTTCTTGCT TTTAAATATA TTATGGGTCG ACGCCCCTCT CCCTCCCCCC 1680

CCCCTAACGT TACTGGCCGA AGCCGCTTGG AATAAGGCCG GTGTGCGTTT GTCTATATGT 1740

TATTTTCCAC CATATTGCCG TCTTTTGGCA ATGTGAGGGC CCGGAAACCT GGCCCTGTCT 1800

TCTTGACGAG CATTCCTAGG GGTCTTTCCC CTCTCGCCAA AGGAATGCAA GGTCTGTTGA 1860

ATGTCGTGAA GGAAGCAGTT CCTCTGGAAG CTTCTTGAAG ACAAACAACG TCTGTAGCGA 1920

CCCTTTGCAG GCAGCGGAAC CCCCCACCTG GCGACAGGTG CCTCTGCGGC CAAAAGCCAC 1980

GTGTATAAGA TACACCTGCA AAGGCGGCAC AACCCCAGTG CCACGTTGTG AGTTGGATAG 2040

TTGTGGAAAG AGTCAAATGG CTCTCCTCAA GCGTATTCAA CAAGGGGCTG AAGGATGCCC 2000

AGAAGGTACC CCATTGTATG GGATCTGATC TGGGGCCTCG GTGCACATGC TTTACATGTG 2060

TTTAGTCGAG GTTAAAAAAC GTCTAGGCCC CCCGAACCAC GGGGACGTGG TTTTCCTTTG 2220

AAAAACACGA TGATAATATG GCCACAACCA TGAAGCTGCT GCCGTCGGTG GTGCTGAAGC 2280

TCTTTCTGGC TGCAGTTCTC TCGGCACTGG TGACTGGCGA GAGCCTGGAG CGGCTTCGGA 2340

GAGGGCTAGC TGCTGGAACC AGCAACCCGG ACCCTCCCAC TGTATCCACG GACCAGCTGC 2400

TACCCCTAGG AGGCGGCCGG GACCGGAAAG TCCGTGACTT GCAAGAGGCA GATCTGGACC 2460

TTTTGAGAGT CACTTTATCC TCCAAGCCAC AAGCACTGGC CACACCAAAC AAGGAGGAGC 2520

ACGGGAAAAG AAAGAAGAAA GGCAAGGGGC TAGGGAAGAA GAGGGACCCA TGTCTTCGGA 2580

AATACAAGGA CTTCTGCATC CATGGAGAAT GCAAATATGT GAAGGAGCTC CGGGCTCCCT 2640

CCTGCATCTG CCACCCGGGT TACCATGGAG AGAGGTGTCA TGGGCTGAGC CTCCCAGTGG 2700

AAAATCGCTT ATATACCTAT GACCACACAA CCATCCTGGC CGTGGTGGCT GTGGTGCTGT 2760

CATCTGTCTG TCTGCTGGTC ATCGTGGGGC TTCTCATGTT TAGGTACCAT AGGAGAGGAG 2820

GTTATGATGT GGAAAATGAA GAGAAAGTGA AGTTGGGCAT GACTAATTCC CACTAAACGC 2880

GTACCGGGCC CCCCCTCGAG GTCGACGGTA TCGATAAGCT TGATATCGAA TTCCGAAGTT 2940

CCTATTCTCT AGAAAGTATA GGAACTTCAG GTCTGAAGAG GAGTTTACGT CCAGCCAAGC 3000

TAGCTTGGCT GCAGGTCGTC GAAATTCTAC CGGGTAGGGG AGGCGCTTTT CCCAAGGCAG 3060

TCTGGAGCAT GCGCTTTAGC AGCCCCGCTG GGCACTTGGC GCTACACAAG TGGCCTCTGG 3020

CCTCGCACAC ATTCCACATC CACCGGTAGG CGCCAACCGG CTCCGTTCTT TGGTGGCCCC 3080

TTCGCGCCAC CTTCTACTCC TCCCCTAGTC AGGAAGTTCC CCCCCGCCCC GCAGCTCGCG 3240

TCGTGCAGGA CGTGACAAAT GGAAGTAGCA CGTCTCACTA GTCTCGTGCA GATGGACAGC 3300

ACCGCTGAGC AATGGAAGCG GGTAGGCCTT TGGGGCAGCG GCCAATAGCA GCTTTGCTCC 3360

TTCGCTTTCT GGGCTCAGAG GCTGGGAAGG GGTGGGTCCG GGGGCGGGCT CAGGGGCGGG 3420

CTCAGGGGCG GGGCGGGCGC CCGAAGGTCC TCCGGAGGCC CGGCATTCTG CACGCTTCAA 3480

AAGCGCACGT CTGCCGCGCT GTTCTCCTCT TCCTCATCTC CGGGCCTTTC GACCTGCAGC 3540

CTGTTGACAA TTAATCATCG GCATAGTATA TCGGCATAGT ATAATACGAC AAGGTGAGGA 3600

ACTAAACCAT GGGATCGGCC ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT 3660

GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG 3720

CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG 3780

GTGCCCTGAA TGAACTGCAG GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG 3840

TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG 3900

GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA 3960

TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC 4020

ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC 4080

AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA 4040

AGGCGCGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA 4200

ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG 4260

CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG 4320

AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG 4380

CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGGGGATCA ATTCTCTAGA GCTCGCTGAT 4440

CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT 4500

CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT 4560

CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG GACAGCAAGG 4620

GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT ATGGCTTCTG 4680

AGGCGGAAAG AACCAGCTGG GGCTCGACTA GAGCTTGCGG AACCCTTCGA AGTTCCTATT 4740

CTCTAGAAAG TATAGGAACT TCATCAGTCA GGTACATAAT AGATCTAACA ATGTCTGAAG 4800

ATTGTAAGTG CTTTAAGGAT CATACTTTTA ATAACTATCT AGCTTATGGA AATTTAGGGT 4860

ATCATGAGTT GATGGCAGTT TTGTTTTTAG CTCAAAACAT GGATAGATGG AACCAAACTC 4920

CAGGAGTTTC ATGCACCAAG GAGAAACACA GGATATCTAC CCAAATAAGT TTGTCTTTTG 4980

TGCCTTACAA CTATGAAGCT CCACATGTTT TGAAAAGAGC ACGTCCTATT TCAACGGTAC 5040

AGCGGCCGCC ACCGCGGTGG AGCTCCAGCT TTTGTTCCCT TTAGTGAGGG TTAATTTCGA 5000

GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CTCACAATTC 5060

CACACAACAT ACGAGCCGGA AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT 5220

AACTCACATT AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC 5280

AGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG TTTGCGTATT GGGCGCTCTT 5340

CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 5400

CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 5460

TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT 5520

TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 5580

GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 5640

CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 5700

TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 5760

AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 5820

ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 5880

ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 5940

ACTACGGCTA CACTAGAAGA ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 6000

TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 6060

TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 6020

TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 6080

TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA TTAAAAATGA AGTTTTAAAT 6240

CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA CCAATGCTTA ATCAGTGAGG 6300

CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT TGCCTGACTC CCCGTCGTGT 6360

AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG TGCTGCAATG ATACCGCGAG 6420

ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA GCCAGCCGGA AGGGCCGAGC 6480

GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC TATTAATTGT TGCCGGGAAG 6540

CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT TGTTGCCATT GCTACAGGCA 6600

TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG CTCCGGTTCC CAACGATCAA 6660

GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA 6720

TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT GGTTATGGCA GCACTGCATA 6780

ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT GACTGGTGAG TACTCAACCA 6840

AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC TTGCCCGGCG TCAATACGGG 6900

ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT CATTGGAAAA CGTTCTTCGG 6960

GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG TTCGATGTAA CCCACTCGTG 7020

CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT TTCTGGGTGA GCAAAAACAG 7080

GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG GAAATGTTGA ATACTCATAC 7040

TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA 7200

TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG 7260

TGCCAC

<210>15

<211>25

<212>DNA

<213> primer F4/80-189503-F

<400>15

TGAAATAACC CAGACACAGA AGTTT

<210>16

<211>23

<212>DNA

<213> primer IRES screen-R

<400>16

CGGCAATATG GTGGAAAATA ACA

Claims

1. The targeting vector is characterized in that the sequence of the targeting vector is shown in SEQ ID NO.14 and is named as a vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803 HA.

2. A preparation method of a targeting vector is characterized by comprising the following steps: the method comprises the following steps:

(1) using RP23-212F14 BAC DNA as a template, and carrying out PCR by using a forward primer with a sequence shown as SEQ ID NO.1 and a reverse primer with a sequence shown as SEQ ID NO.2 to amplify a homologous arm at the side of a gene fragment F4/803' with a sequence shown as SEQ ID NO.3, namely F4/803 HA;

(2) cloning a target gene fragment F4/803HA to a vector PGK-EM7-Neo with a positive selection marker to obtain an intermediate vector PGK-EM7-Neo-F4/803 HA;

(3) carrying out PCR by taking pLVX-EF1 α -IRES-mCherry plasmid as a template and utilizing a forward primer with a sequence shown as SEQ ID NO.4 and a reverse primer with a sequence shown as SEQ ID NO.5 to amplify a gene fragment IRES with a sequence shown as SEQ ID NO.6, carrying out PCR by utilizing a forward primer with a sequence shown as SEQ ID NO.7 and a reverse primer with a sequence shown as SEQ ID NO.8 by taking pIRES-proHB EGF WT plasmid as a template to amplify a gene fragment DTR with a sequence shown as SEQ ID NO.9, and carrying out Overlap PCR to obtain a fusion gene fragment IRES-DTR with a sequence shown as SEQ ID NO. 10;

(4) using RP23-212F14 BAC DNA as a template, and carrying out PCR by using a forward primer with a sequence shown as SEQ ID NO.11 and a reverse primer with a sequence shown as SEQ ID NO.12 to amplify a homologous arm at the F4/805' side of a gene fragment with a sequence shown as SEQ ID NO.13, namely F4/805 HA;

(5) the target gene fragment F4/805HA and IRES-DTR are cloned to an intermediate vector PGK-EM7-Neo-F4/803HA, and a targeting vector F4/805HA-IRES-DTR-PGK-EM 7-Neo-F4/803HA with the sequence shown in SEQ ID NO.14 is obtained.

3. An application of a targeting vector in constructing an escherichia coli BAC Clone for targeting integration of an exogenous gene to a F4/80 exon 22 site by targeting integration of the exogenous gene to the F4/80 exon 22 site.

4. The use of the construction of an E.coli BACClone for targeted integration of an exogenous gene into the F4/80 exon 22 site according to claim 3, wherein the construction method of the E.coli BACClone for targeted integration of an exogenous gene comprises the following steps:

(1) electroporation the Red/ET expression plasmid pRED/ET was transformed into F4/80BAC clones and the expression of Red/ET was induced with L-arabinose;

(2) converting the enzyme-digested linearized targeting vector into the F4/80BAC clone expressing Red/ET by an electroporation method, and inserting IRES-DTR-PGK-EM7-Neo into F4/80BAC in a targeted manner;

(3) f4/80BAC clone successfully inserted into IRES-DTR-PGK-EM7-Neo expresses neomycin resistance, and LB plate with neomycin and chloramphenicol is used for screening to obtain BAC clone F4/80-IRES-DTR-EM7-PGK-Neo targeted to be inserted into the exon 22 sites of IRES-DTR-PGK-EM7-Neo to F4/80;

(4) a single clone is selected, a forward primer with a sequence shown as SEQ ID NO.15 and a reverse primer with a sequence shown as SEQ ID NO.16 are used as colony PCR, and an F4/80BAC clone successfully inserted into IRES-DTR-PGK-EM7-Neo can obtain a band with about 1.2 kb.