CN109097393A - People's tctp gene whole body strikes the animal model, preparation method and application subtracted - Google Patents
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Abstract
Application itself discloses the tctp gene conditional gene based on recombinase identifying system and strikes the non-human mammal model's building kit subtracted, the construction method of targeting vector and its targeting vector, the whole body clpp gene of non-human mammal tctp gene subtracts animal model, its sperm, egg cell, fertilized eggs, embryo, filial generation, tissue or cell, and the said goods and method are in immunological investigation, immunotoxicology, the research of immunological rejection and its mechanism, study cancer cell apoptosis mechanism and cancer target, the research of targeting cancer therapy, safety evaluatio and the purposes in terms of pharmacodynamic evaluation, make cancer pathology model, application in the medicine preparation for the treatment of disease.The tctp gene whole body knock-out mice for stablizing heredity is constructed using homologous recombination technique and embryonic stem cell technologies, whole body is avoided and knocks out embryonic death caused by the gene, is of great significance for the cancer occurrence and development and treatment of cancer of studying tctp gene.
Description
Technical field
The present invention relates to production whole body clpp genes to subtract the field of animal model, specifically, is related to a kind of preparing tctp gene
Whole body strikes the method and application of the non-human mammal subtracted.
Background technique
Cancer is to influence one of the major chronic disease of China's residents ' health, first of the column city cause of the death.In China, every year
New cancer cases are up to 4,290,000, that is to say, that about 10,000 people make a definite diagnosis cancer daily in the whole nation, and about 7 people make a definite diagnosis and suffer from cancer within every 1 minute.In if
State is 85 years old the average life expectancy of the people, then everyone accumulative cancer risk of suffering from is up to 36%.Worldwide, about 22%
Newly-increased cases of cancer and 27% cancer mortality occur in China.Lung cancer be China so that the most common malignant tumour in the whole world with
And the first cancer mortality reason.With air pollution caused by China's industrialization, the further speeding up of urbanization process with
And smoking rate is high, the harm of lung cancer will continue to aggravate.Liver cancer is one of highest malignant tumour of case fatality rate.China is every
Year, there are about 38.3 ten thousand people to die of liver cancer, accounts for the 51% of global PLC mortality case load.It is expected that within coming few decades, China's cancer
Morbidity and mortality are in rising trend by whole continuation.Severe situation brings heavy bear to the society in China and medical treatment
Load.Heavy cancer burden needs to solve using the control strategy of synthesization.Regulate and control and contain cancer from molecular biological mechanism
The technique study of disease progress becomes the important directions for the treatment of of cancer research.
Clonorchiasis Sinensis (TCTP, also known as P21 mouse TCTP), is that one kind is widely present in animal, plant and ferment
Protein family that is highly conserved in sequence and having very high homology in mother, encoding gene are TPT1 tumor.TCTP is also known as
For Histamine-releasing factor (HRF).The albumen of mankind's TPT1 gene coding claims fortilin, is a new class of
Anti-apoptotic proteins matter.In recent years research prompt TCTP may have very important biological function, including adjust the cell cycle
Process and pernicious transfer, calbindin, extracellular histamine release albumen, anti-apoptotic and anti-malarial effect, anti-inflammatory response
With protect cells from a variety of irritability damages, cancer eliminating etc..
The structure of 1.TCTP gene and TCTP mRNA
RupecRA et al. has isolated TCTP/TPT1 gene in 1998 from No. 13 chromosomes, and 1999,
The gene motif is further positioned between 13q12-q14 by MacDonaldSM et al..ThieleH etc. analyzes TCTP/
TPT1 gene is made of 5 exons and 6 intrones, 3819 nucleotide of overall length, and all introne/exon regions are equal
Meet GT/AG rule.Promoter region is located at the TATA box and Binding site for transcription factor at the place -30bp, in Sp1 mammal
Deposit NF1, AP1, CP2 and MZF1 etc..Tctp gene has many very important biological functions, including calcium binding, encloses
It sees and combines, bores release, adjusting the cell cycle, anti-apoptotic and stem cell differentiation etc..Many important physiology courses are given birth in participation into the cell,
Including cell growth, the vicious transformation of the process of cell cycle, tumour and adjusting Apoptosis.In view of above-mentioned mechanism, TCTP egg
It is played in the white regulation development in kinds cancers such as breast cancer, prostate cancer, lung cancer, glioma, lymthomas important
The important biomolecule marker of role and liver cancer cells reversal procedures.The cancer occurrence and development for studying tctp gene are sent out in the process
The effect waved, is of great significance to treatment of cancer.Tctp gene to the double action of tumor cell proliferation and apoptosis and
Strong downward in the tumour cell of reverse makes it be expected to become the ideal targets treated in tumour, has and potentially answer
With value.
Currently, how to evaluate the anti-tumour cell proliferative of tctp gene and apoptosis-promoting effect still has bottleneck.Due to open country
Raw type lower animal all expresses tctp gene, therefore the relevant tumour of evaluation tctp gene can not be gone using wild type lower animal
Apoptosis.The tumour progression of tctp gene animals showing positive baboon can only be used as receptor is investigated, and this animal rareness is very
Hardly possible is universal to be used.Therefore, how evaluating tctp gene, there is bottlenecks in the relationship developed with animal tumor.
It is lethal that the whole body knockout of tctp gene will lead to mice embryonic, domestic at present without carrying out gene for this gene
The genetic engineering mouse of transformation exists, and is unable to get relevant animal pattern at home.It is mentioned in view of the deficiencies of the prior art with defect, spy
It is of the invention out.
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide the tctp gene conditionity base based on recombinase identifying system
Subtract non-human mammal model's building kit, targeting vector and construction method, animal model, filial generation, embryo or cell because striking
And preparation method thereof and the said goods and method in immunological investigation, immunotoxicology, immunological rejection and its mechanism
Research, the application in safety evaluatio, in terms of pharmacodynamic evaluation purposes, production cancer pathology model purposes and
Purposes in the medicine preparation for the treatment of disease.
In a first aspect, the present invention provides a kind of tctp gene conditionity based on recombinase identifying system strike subtract it is inhuman
Mammalian animal model constructs kit, wherein it includes targeting vector element gram that the non-human mammal model, which constructs kit,
Grand primer, the targeting vector element cloning primer include:
LRJ-072-A-F (Seq ID.No.5): cgatGAATTCGTATACAAGATCCGGGAGATCGC,
LRJ-072-A-R (Seq ID.No.6): cgatAGCGCTAGTACTAAGTTGCTGTGGCCTCTCTC,
LRJ-072-B1-F (Seq ID.No.7): cgatGCGATCGCGGTTCCGCTTGATGAGAGTGAC,
LRJ-072-B1-R (Seq ID.No.:8): cgatGTCGACTAGCCATGTTGGCACACACC,
LRJ-072-B2-F (Seq ID.No.:9): cgatACGCGTGATATCGCATATGTGTTATTTTTGAGAACTA
GG,
LRJ-072-B2-R (Seq ID.No.:10): cgatCCGCGGTATACTTACACACATGACCCTGAG；
LRJ-072-C-F (Seq ID.No.:11): cgatCTCGAGTACGTACCACCATGCCCAGTTTAGA,
LRJ-072-C-R (in) (Seq ID.No.:12): CATGGAGAGACTGCTTGAAAGATATCTAGCAGTTACT
CAGTCTCAC,
LRJ-072-C-F(in)(Seq ID.No.:13):GTGAGACTGAGTAACTGCTAGATATCTTTCAAGCAGT
CTCTCCATG
LRJ-072-C-R(Seq ID.No.:14):cgatGCGGCCGCCTTGGTGTTCATAGTTATGCCG。
In some embodiments, it further includes tctp gene conditionity that the non-human mammal model, which constructs in kit,
Clpp gene subtracts the verifying primer of targeting vector composition element intermediate segment, detection probe prepares primer, sequencing primer, targeting vector
PCR screen primer, genotyping primer.Wherein
The intermediate segment verifies primer
LRJ-072-Atest-F (Seq ID.No.:15): GTCACCATGATCATCTACCGG,
LRJ-072-Atest-R (Seq ID.No.:16): acgggttcttctgttagtcc,
LRJ-072-Btest-F (Seq ID.No.:17): atgctatacgaagttatacgcg,
LRJ-072-Btest-R:(Seq ID.No.:18): acactgaaacatcctattgcatg,
LRJ-072-C1test-F(Seq ID.No.:19:gctttccgattgagcccagg,
LRJ-072-C1test-R (Seq ID.No.:20): tctctagagtcacagaacttatgg,
LRJ-072-C2test-F (Seq ID.No.:21): gctttccgattgagcccagg,
LRJ-072-C2test-R (Seq ID.No.:22): agctagcttggctggacgta,
The sequencing primer includes:
LRJ-072-B1-616F (Seq ID.No.:23): gctagaagacattacctattggagc and
LRJ-072-B1-713R (Seq ID.No.:24): ggtttctgctcttcaagtttgc；
The probe prepares primer
LRJ-072-5'Probe-F (Seq ID.No.25) gctatctatggggaaagaatat) and
LRJ-072-5'Probe-R ((Seq ID.No.26) gagcacttcatctgtcagca),
LRJ-072-3'Probe-F (Seq ID.No.27) agctagggagcaggttagaag) and
LRJ-072-3'Probe-R((Seq ID.No.28)ctctggtgagctcatctctg)；
The genotyping primer is to including:
LRJ-072-B1 Loxp-F (Seq ID.No.29): tcattgagactgttatctgtagaccagact,
LRJ-072-B1 Loxp-R (Seq ID.No.30): agcaaccataccatctggattcatgt；'
The PCR primer group includes 5 ' end screening primer pairs and screens primer pairs, 5 ' the end screening primer pair to 3 ' ends
Including
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat
It screens primer pair and includes in the 3 ' end
LRJ-072-PCR-F2 (Seq ID.No.:3): GCTCGACTAGAGCTTGCGGA,
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
Second aspect, the present invention provides it is a kind of based on recombinase identification building tctp gene condition strike subtract it is inhuman
The targeting vector of mammal, wherein
The whole body of the tctp gene strike subtract conditionity be based on exon 2 frameshift mutation caused by homologous recombination generate
Exon 3 and 4 product of exon are truncated protein；
The targeting vector pDTA-LRJ-072 that the tctp gene conditionity strikes the non-human mammal subtracted is ' suitable to 3 ' from 5
Sequence include sequentially connected B1 segment, B2 segment, B3 segment and C1 and C2 segment it is warm at the B2-B1- that is formed by connecting of C segment
A-C1-C2 sequence attachment；The B2 fragment sequence is as shown in SEQ ID NO:33, the B1 fragment sequence such as SEQ ID NO:
Shown in 32, the A fragment sequence is as shown in SEQ ID NO:31, the C1 fragment sequence such as SEQ ID NO:34 institute, the C2
Segment is as shown in SEQ ID NO:35；, the tctp gene whole body conditionity knockout targeting vector pDTA-LRJ-072 sequence is such as
Shown in SEQ ID NO:36.
The B1 segment includes positive screening element, wherein the positive screening element includes that sequentially connected 5 ' recombinase is known
The positive riddled basins box -3 ' of other sequence 1- reporter gene box-recombinates enzyme recognition sequence 1, in some embodiments, the report
It accuses box gene and shears receptor with a composing type, the pressure of the reporter gene mRNA can be mediated to shear, make the report
Gene can be expressed under the driving of target practice gene promoter to the expression of tctp gene described in tracer；The reporter gene box
In can terminate in advance the transcription of this target practice gene comprising polyadenylic acid (Poly A), to knock out tctp gene；The report
' and 3 ' be respectively that 5 ' recombination enzyme recognition sequence 1 and 3 ' recombinase is known for the 5 of box gene and the antibiotics resistance gene box dyad
It is recombination enzyme recognition sequence 2 among other sequence 1, the reporter gene box and the antibiotics resistance gene box, it is described heavy
Group enzyme recognition sequence 1 can be identified by recombinase, to realize the reporter gene box and the antibiotics resistance gene box
It deletes.
In some embodiments, the B1 segment includes from 5 ' to 5 ' recombination enzyme recognition sequence 1, positive screenings of 3 ' connections
Element, ' recombination enzyme recognition sequence 2, resistance screening box gene, 3 recombination enzyme recognition sequences 1,5- ' recombination enzyme recognition sequence 2, T are non-
People's mammal tctp gene exon 3-4, the B1 segment 5 ' and 3 ' be ScaI and Salt, EcoV restriction enzyme site respectively；
The B2 segment includes from 5 ' to 3- ' recombination enzyme recognition sequence 2 (3 '-lox P) non-human mammal of 3 ' connections
Tctp gene exon 5-6,3 ' homology arms；' and 3 ' be Salt, ScaI restriction enzyme site respectively for the 5 of the B12 segment；
The A segment includes from 5 ' to 3 ' connections, 5 ' homology arms, non-human mammal tctp gene exons 1-2, institute
State A segment 5 ' and 3 ' be EcoI and ScaI and restriction enzyme site respectively,
' and 3 ' be SnaBI and EcoV restriction enzyme site respectively, includes negative selection marker gene for the 5 of the C1 segment；
' and 3 ' be EcoV and EcoI restriction enzyme site respectively, and C1 and C2 fusion segment include negative selection mark for the 5 of the C2 segment
Remember gene.
In some embodiments, the positive riddled basins include encoding neomycin phosphotransferase (Neo), tide it is mould
The gene of plain B phosphotransferase, hypoxanthine phosphoribosyltransferase or puromycin acetyltransferase；The negative selection label
Gene includes the gene of encoding diphtheria toxin (DAT) or thymus gland deoxidation nucleosides.
In some embodiments, the recombination enzyme gene includes the gene for encoding Cre enzyme or Flp enzyme.
In a specific exemplary embodiment, in the positive screening element, the recombination enzyme recognition sequence 1 is FRT
Point, the reporter gene are EGFR-PA, and the reporter gene box is En2 SA-IRES-eGFP-PA, the antibiotic resistance
Gene is Neo, and the recombination enzyme recognition sequence 2 is the site LoxP, the nucleotide sequence of the nucleotide sequence of the targeting vector
Respectively as shown in SEQ ID NO:36.
In a specific exemplary embodiment, the non-human mammal is mouse, the end the 5` homology arm sequence point
Homology arm sequence with the end 3` is respectively that 5.6kb and 5.0kb is derived from tctp gene the 2nd on No. 14 chromosome of the mouse
The end introne 3 ` of exon to the end introne 5` of the 5th exon, 5 ' homology arms of the non-human mammal tctp gene
With the nucleotide sequences of 3 ' homology arms respectively as shown in SEQ ID NO:37 and SEQ ID NO:38.
The present invention is in the gene targeting carrier of building, using positive-negative selection strategy, therefore can be largely
The cell of random integration is excluded, target practice cell enrichment efficiency is improved.Sequence is identified by introducing recombinase in positive screening-gene two sides
Column, realize the deletion of the reporter gene box and the antibiotics resistance gene box.
In some embodiments, the positive riddled basins include encoding neomycin phosphotransferase (Neo), tide it is mould
The gene of plain B phosphotransferase, hypoxanthine phosphoribosyltransferase or puromycin acetyltransferase；In some embodiments
In, the negative selection marker gene includes the gene of encoding diphtheria toxin (DAT) or thymus gland deoxidation nucleosides.In some embodiments
Described in recombination enzyme gene be encode Cre enzyme gene, or in some embodiments the recombination enzyme gene be encode Flp enzyme
Gene.
In a specific illustrative embodiment, in the positive screening element, the recombination enzyme recognition sequence 1 is the site FRT,
The reporter gene is EGFR-PA, and the reporter gene box is En2SA-IRES-eGFP-PA, the antibiotics resistance gene
For Neo, the recombination enzyme recognition sequence 2 is the site LoxP, and the nucleotide sequence of the nucleotide sequence of the targeting vector is distinguished
As shown in SEQ ID NO:36.
Negative selection gene of the present invention is encoding diphtheria toxin (DTA) or thymus gland in a specific illustrative embodiment
Deoxygenate the gene of nucleosides etc.；Preferably, the negative selection gene is DTA.
In a specific illustrative embodiment, the non-human mammal is mouse, the end the 5` homology arm sequence point and
The homology arm sequence at the end 3` is respectively that 5.6kb and 5.0kb is derived from No. 14 chromosome of the mouse outside tctp gene the 2nd
Show son the end introne 3 ` to the end introne 5` of the 5th exon, 5 ' homology arms of the non-human mammal tctp gene and
The nucleotide sequence of 3 ' homology arms is respectively as shown in SEQ ID NO:37 and SEQ ID NO:38.
In a specific embodiment, the recombinase identifying system is Cre-LoxP system.The tctp gene is based on
The conditional gene of recombinase identifying system strikes the sequence for reducing the targeting vector of mouse model building as shown in SEQ ID NO.36,
The tctp gene conditionity strikes the targeting vector subtracted and is named as pDTA-LRJ-072 targeting vector.
In one embodiment, 1 acting sequences of recombinase are Frt, and 2 acting sequences 2 of recombinase are LoxP
Site, the reporter gene box is En2SA-IRES-eGFP-PA, and the antibiotics resistance gene is Neo.
Preferably, the recombination enzyme gene is the Cre gene with tACE specificity promoter.
The third aspect, the present invention provides the tctp gene whole body conditionitys based on recombinase identification described in second aspect
Clpp gene subtracts the construction method of targeting vector, comprising the following steps:
(1) from being template with the genomic DNA of target non-human mammal tissue extraction, described is expanded with PCR method
Section A, the segment B1, the segment B2 and segment C1 and C2；
(2) it connects, constructs intermediate vector: by digestion identification, correct B1 or B2 segment is sequenced and A segment is consecutively connected to
Forward direction screening starting vector 1 obtains intermediate vector-B1-B2-A；Meanwhile correct C1, C2 piece will be sequenced by Overlap PCR
Section is connected to the acquisition of negative selection gene starting vector 2 negative selection gene intermediate vector 2-TCTP-C after being fused to C segment；
(3) correct intermediate vector 1-TCTP-B1-B2-A will be connected, cut out with restriction enzyme and recycles recycling mesh
Segment A-NeoR-B, conversion the BAC bacterium containing pBCTG recombinated to obtain TCTP-NeoR BAC；Bacterium colony PCR detects TCTP-
NeoR BAC, identification amplified band are correct；
(4) linearisation of negative selection gene starting vector 2-TCTP-C and conversion AB recombinate BAC
I.e. digestion handles negative selection gene starting vector 2-TCTP-C, recycles purpose band negative selection gene-TCTP-C, electricity
Turn to have recombinated the BAC bacterium of NeoR；Negative selection gene-TCTP-C saves TCTP-NeoR BAC, obtains the B containing TCTP, A
The recombinant bacterium converted with the targeting vector of C segment；The recombinant bacterium of the targeting vector-TCTP-BAC conversion obtained after rescue is dropped into
Row PCR Testing and appraisal, correct recombinant clone is expanded, purified rear digestion verification.
In some embodiments, the positive riddled basins include encoding neomycin phosphotransferase (Neo), tide it is mould
The gene of plain B phosphotransferase, hypoxanthine phosphoribosyltransferase or puromycin acetyltransferase；The negative selection label
Gene includes the gene of encoding diphtheria toxin (DAT) or thymus gland deoxidation nucleosides.
The recombination enzyme gene is the gene for encoding Cre enzyme, or institute in some embodiments in some embodiments
Stating recombination enzyme gene is the gene for encoding Flp enzyme.
In a specific illustrative embodiment, in the positive screening element, the recombination enzyme recognition sequence 1 is the site FRT,
The reporter gene is EGFR-PA, and the reporter gene box is En2SA-IRES-eGFP-PA, the antibiotics resistance gene
For Neo, the recombination enzyme recognition sequence 2 is the site LoxP, and the nucleotide sequence of the nucleotide sequence of the targeting vector is distinguished
As shown in SEQ ID NO:36.
In one exemplary embodiment, the positive screening starting vector 1 is pKFCR-EGF, the intermediate vector 1-
TCTP-B1-B2-A is pKFCR-EGFP-TCTP-B1-B2-A, and the negative selection gene starting vector 2 is pDTA-Down carrier,
The negative selection marker gene is the gene dat, the negative selection gene starting vector 2-TCTP-C of encoding diphtheria toxin (DAT)
For pDTA-Down-C, the targeting vector of B, A and the C segment containing TCTP is pDTA-LRJ-072.
In one exemplary embodiment, the tctp gene conditionity based on recombinase identification strikes the structure for subtracting targeting vector
Construction method includes the following steps
(1) it is template from normal mouse separation genomic DNA, expands the segment A, the segment B1, institute with PCR method
State segment B2 and segment C；
(2) pKFCR-EGFP will be consecutively connected to by digestion identification, the correct B1 or B2 segment of sequence verification and A segment
Carrier obtains pKFCR-B2-B1-A intermediate vector, meanwhile, it is C piece that correct C1, C2 segment composition, which will be sequenced, by over-lap PCR
It is connected to pDTA-Down carrier after section and obtains pDTA-Down-TCTP-C；
(3) correct pKFCR-EGFP-TCTP-B1-B2-A will be connected, cuts out pKFCR-EGFP- with restriction enzyme
B1-B2-A band, recycles target fragment A-NeoR-B, and BAC bacterium of the conversion containing pBCTG is recombinated；Bacterium colony PCR detects A-
The BAC of NeoR-B segment recombination, identification amplified band are correct；
(4) linearization process of pDTA-down-TCTP-C, i.e. digestion handle pDTA-Down-TCTP-C, recycle purpose item
Band, electricity turn to have recombinated the BAC bacterium of NeoR；TCTP-NeoR BAC is saved with pDTA-TCTP-C；To the recombination after rescue
Bacterium colony carries targeting vector pDTA-LRJ-072.PCR Testing and appraisal is carried out, correct recombinant clone is expanded, it is pure through Stbl3
Digestion verification after change,.
The fourth aspect of the present invention provides a kind of non-human mammal tctp gene whole body item based on recombinase identification
Part strikes the animal model subtracted, its sperm, egg cell, fertilized eggs, embryo, filial generation, tissue or cell.
The fifth aspect of the present invention provides the non-human mammal tctp gene based on recombinase identification of fourth aspect
Whole body conditionity strikes the preparation method for subtracting animal model, filial generation, embryo or cell comprising following steps:
(1) tctp gene whole body conditional gene described in second aspect is struck and subtracts targeting vector and is transferred to non-human mammal embryo
Tire stem cell；
(2) it has recombinated tctp gene whole body conditional gene and has struck the screening and identification for subtracting the embryonic stem cell of targeting vector；
(3) chimeric non-human mammal is prepared；Recombination embryonic cell microinjection is entered in foster animal embryo, is transplanted to
In pseudo pregnant animal body, mate with intact animal；
(4) genotype identification and tctp gene whole body conditionity strike the screening for subtracting positive chimeric animal: embedding to what is obtained
Fit animal carries out genotype verifying and protein expression level verifying, screen positive tctp gene whole body conditionity strike subtract it is embedding
Fit animal；
(5) the chimeric non-human mammal for rejecting reporter gene and positive selection markers is obtained；
(6) breeding tctp gene whole body conditionity strikes the F1 generation heterozygote non-human mammal model subtracted.
In a preferred embodiment, the step (1) is to construct targeting vector pDTA-LRJ-072 using BAC carrier.
It is dynamic that the building process of targeting vector including the use of the BAC carrier containing non-human mammal TCTP extracts to obtain the inhuman lactation
The homologous recombination arm of object TCTP, the including 5 ' first recombination enzyme recognition site insertion exon 2s by way of homologous recombination
Sub- downstream is non-conservative and 3 ' introne non-conservative area in upstream is inserted into two the first reversed recombination enzyme recognition sites, in a forward direction
The second recombination enzyme recognition site, 3 ' non-conservative differentiations are swum in the non-conservative area of upstream introne and 5 ' introne upstreams of exon 5
It Cha Ru not 5 ' second recombination enzyme recognition sites and 3 ' second recombination enzyme recognition sites.Artificial synthesized is contained into reporter gene and antibiosis
The target practice segment of plain screening-gene is inserted into starting vector by homologous recombination mode, to obtain tctp gene in exon 2 and 3
Between by 5 the ' the 1st recombination enzyme recognition sites-reporter gene box-the second recombinate enzyme recognition site-antibiotics resistance gene box -3 ' the
One recombination enzyme recognition site insertion interrupts；The TCTP interrupted is inserted by 3 ' second recombination enzyme recognition sites between exon 4 and 5
Gene whole body, which strikes, subtracts targeting vector.It is identified with PCR and I digestion of Pst.
In a preferred embodiment, the step (2) is to be turned targeting vector pDTA-LRJ-072 using electroporation
It practices shooting in non-human mammalian embryo stem cell (ES cell), brings it about homologous recombination, pass through PCR and Southern
Blot filters out middle target clone.Wherein, the PCR reaction primer pair of 5 ' end screening is
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat,
It screens primer pair and includes in the 3 ' end
LRJ-072-PCR-F2 (Seq ID.No.:3): GCTCGACTAGAGCTTGCGGA
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
Preferably, 5 ' end screening PCR reaction condition be 95 DEG C 10 seconds, 68 DEG C 6 minutes, totally 35 circulation；3 ' end screenings
PCR reaction condition be 95 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 40 seconds, totally 35 circulation.
Preferably, 3 ' end screening PCR reaction condition be 95 DEG C 10 seconds, 68 DEG C 6 minutes, totally 40 circulation；3 ' end screenings
PCR reaction condition be 95 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 40 seconds, totally 35 circulation.
Step described in some embodiments (3) is that injection blastaea uses C57BL/6N inbred mouse (purchased from Beijing
Tie up experimental animal Technology Co., Ltd. of tonneau China), by superfecundation, natural conception is developed to blastocyst stage in embryoid body, uses
In injection；Pseudopregnant mouse receptor uterus is implanted into after injection, pseudopregnant recipients are the first-filial generation of C57BL/6N and CBA, are given birth to out
Chimeric male mouse of the chimeric rate greater than 50%.
Step described in some embodiments (4) be the high chimeric male mouse of the chimeric rate that will obtain successively with Flper mouse
And the mating of wild type C57BL/6N mouse, the inhuman lactation of chimera that reporter gene and Neo resistant gene are left out in acquisition completely are dynamic
Object.
Step described in some embodiments (5) is the chimera for leaving out reporter gene and antibiotics resistance gene completely again
Struck with whole body subtract cre Strains of Mouse mate obtain people's tctp gene whole body strike the mouse Chimera subtracted, people's tctp gene is complete
Body strikes the mouse Chimera subtracted and the extracted coda gene group DNA progress PCR of wild type C57BL/6N background female mice backcrossing is anti-
It should identify and carry out genotype, wild type, heterozygote or homozygote are judged according to the electrophoresis result of PCR product, it is complete to obtain tctp gene
Body strikes the F1 generation heterozygote subtracted.
In one embodiment, it is respectively as follows: 1. for the PCR of the F1 generation hybrid mice genotype identification primer reacted
Detect 5 ' loxP:LRJ-072-B1 Loxp-F (5 '-GGCTGGACGTAAACTCCTC-3 ' (SEQ ID NO.29).
2. detecting 3 ' loxP:LRJ-072-B2 Loxp-R (AGCAACCATACCATCTGGATTCATGT) (SEQ ID
NO.30)
Reaction condition are as follows: 95 DEG C 30 seconds；58 DEG C 30 seconds；72 DEG C 30 seconds；35 circulations.
6th aspect, the present invention, which provides a kind of non-human mammal tctp gene whole body and strikes, subtracts neural progenitor cell line.It is to use
The tctp gene whole body of the application second aspect strikes the targeting vector transfection non-human mammalian embryo stem cell subtracted, in acquisition
Target positive cell clone, as non-human mammal tctp gene whole body strike the neural progenitor cell line subtracted.Wherein, described for identifying
The Specific PCR primers of middle target positive cell clone include short PCR screening primer and long PCR screening primer, wherein short PCR is screened
Primer includes
LRJ-072-B1 Loxp-F (Seq ID.No.29): tcattgagactgttatctgtagaccagact
LRJ-072-B1 Loxp-R (Seq ID.No.30): agcaaccataccatctggattcatgt.；
The long PCR screening primer sets include 5 ' end screening primer pairs and hold screening primer pairs to 3 ',
It screens primer pair and includes in the 5 ' end
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat
It screens primer pair and includes in the 3 ' end
LRJ-072-PCR-F2 (Seq ID.No.:3): GCTCGACTAGAGCTTGCGGA
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
7th aspect, the present invention provide kit described in a kind of first aspect present invention, target practice described in second aspect
Carrier, the tctp gene conditional gene based on recombinase identification described in the third aspect strike the construction method for subtracting targeting vector,
Non-human mammal sperm, egg cell, fertilized eggs, embryo, filial generation, tissue or neural progenitor cell line described in fourth aspect, the 5th
Described in aspect based on recombinase identification non-human mammal tctp gene conditional gene strike subtract animal model, filial generation,
The preparation method of embryo or cell is pacified in the research of immunological investigation, immunotoxicology, immunological rejection and its mechanism, drug
Application in full property evaluation, pharmacodynamic evaluation.
The eighth aspect present invention provides kit described in a kind of first aspect present invention, and target practice described in second aspect carries
Body, the tctp gene conditional gene based on recombinase identification described in the third aspect strike the construction method for subtracting targeting vector, the
Four aspect described in non-human mammalian embryo, filial generation, tissue or neural progenitor cell line, sperm, egg cell, fertilized eggs, the 5th side
Described in face based on recombinase identification tctp gene conditionity strike the non-human mammal animal model subtracted, filial generation, embryo or
The preparation method of cell, non-human mammal tctp gene knock out application of the precursor in treatment disease, uncomfortable drug.
Preferably, the disease is tumor disease.
In one embodiment, the disease is liver tumour disease.
Tctp gene whole body conditionity of the invention, which is struck, to be subtracted targeting vector and deletes outside the functional areas the 3rd and 4 of tctp gene
Aobvious son, and generate the truncated protein of a 73aa (34aa comes from N-terminal, and 39aa comes from C-terminal).
The present invention takes first whole body to knock out, and the design scheme that postcondition knocks out, this layout strategy is in normal condition
Property knock out on the basis of, be inserted into the reporter gene box Reporter that two sides have Frt sequence in the 5 ' upstreams loxP
Cassette and neomycin resistance gene box (Neo cassette), Reporter cassette is with a composing type shearing
Receptor (En2SA) can mediate the pressure of mRNA to shear, and enable Reporter in the driving following table of target practice gene promoter
It reaches, therefore can be used to the expression of tracer target practice gene.Simultaneously because in Reporter cassette polyA presence, mention
Before terminate the transcription of this target practice gene, thus achieved the purpose that knock out this gene.Additionally, due to Reporter cassette
Two sides with Neo cassette are the site Frt, can be identified by Flp recombinase, and Reporter cassette and Neo are removed
Conventional conditionity can be transformed into after cassette to knock out.To sum up, this design scheme can get reserve and knock out potential
Strike mouse model, i.e. KO first, conditional ready entirely.
Detailed description of the invention
Fig. 1 .TCTP gene whole body conditional gene, which strikes, subtracts animal model preparation flow figure
Fig. 2 .TCTP gene whole body conditional gene, which strikes, subtracts targeting vector construction strategy figure
Fig. 3 .TCTP gene whole body conditional gene, which strikes, subtracts targeting vector LRJ-072 targeting vector element connection schematic diagram
Fig. 4 .TCTP gene whole body conditional gene, which strikes, subtracts targeting vector digestion verification strategy schematic diagram
Fig. 5 .TCTP gene whole body conditional gene strikes A, B1, B2, C1, C2 and the C segment for subtracting targeting vector building process
Agarose gel electrophoresis 5-A proof diagram after PCR product digestion
5-A, 5-B, 5-C, 5-D, 5-E, 5-F, respectively A, B1, B2, C1, C2 and C2 and C fragmentary views
5-G, 5-H are respectively 3 ' probe and 5 ' probes
Fig. 6 restriction endonuclease analysis: agarose gel electrophoresis after pDTA-down-C carrier digestion with restriction enzyme
PDTA-down-C carrier r
Restriction enzyme: XbaI, it is contemplated that product: 1859bp+4492bp
Restriction enzyme: EcoRV+SalI, it is contemplated that product 1415bp+4936bp 3952bp
The result of Fig. 7 pKFCR-B2 carrier digestion verification
With PstI digestion, swimming lane 1-4 primer size is 261bp+525bp+1418bp+5728bp
It is 922bp+3058bp+3952bp with SacI digestion swimming lane 1-4 with SacI digestion products size
Swimming lane M:DNA marker
With the DNA detection Southern BLOT probe from mouse ES cell, (A ScaI enzymic digestion probe, B are used Fig. 8
EcoRV digests probe)
The restriction endonuclease analysis of Fig. 9 A-Neo-B vector
Restriction enzyme: ScaI expects product s:1478bp+7905bp
Restriction enzyme: BstZ17I expects product s:2895bp+6488bp
The PCR of Figure 10 LRJ-072BAC AB recombinant vector is verified
(primer: Atest-F/R；Btest-F/RExpected, product length: 713bp；580bpB)
Agarose gel electrophoresis figure after the restriction enzyme digestion of Figure 11 pDTA-LRJ-072- targeting vector
Figure 12 pDTA-LRJ-072- targeting vector largely prepares Ago-Gel electricity after the cutting of product restriction enzyme
Swimming
1. restriction enzyme: SnaBI, desired product 21822bp
2. restriction enzyme: HindIII, desired product: 471bp+5197bp+6342bp+9812bp
3. restriction enzyme: EcoRI+EcoRV, desired product: 1251bp+2646bp+4078bp+4939bp+
8908bp
4 restriction enzymes: SalI+ScaI, desired product: 1495bp+2717bp+4582bp+5626bp+7402bp
The PCR of Figure 13 LRJ-072 targeting vector is screened,
Template: the BAC of transformation, wild type gene group DNA
PCR-F1/PCR-R1
1.200ng BAC, 2.100ng BAC, 3.50ng BAC, 4.10ng BAC, 5.1ng BAC,
6.500pg BAC；7.WT；8.p-DAT ABC,
PCR-1.200ng BAC, 2.100ng BAC, 3.50ng BAC, 4.10ng BAC, 5.1ng BAC, 6.500pg
BAC；7.WT；8.p-DAT ABC F2/PCR-R2
The LR-PCR screening of Figure 14 positive ES clone
Figure 15 SR-PCR screens positive ES clone
The Neo of Figure 16 5 ', 3 ' and 3 ' probe SOUTHERN blot verifies positive ES clone
The genotyping primer design of Figure 17 ES clone
The Genotyping of Figure 18 F1 heterozygote
Figure 19 carries out the detection of TCTP protein expression with Westhern blot
Specific embodiment
Technical solution of the present invention is made further explanation below.
The present invention relates to a kind of method for making Gene Knock-Out Animal Model model and the animal models in cancer cell-apoptosis,
Purposes in the research of its mechanism for the treatment of of cancer, preclinical safety evaluation (such as immunological evaluation, implantation experiment)；And by
Cell, tissue, organ and the embryo that the animal pattern is derived are in above-mentioned field.
Non-human mammal model of the invention has artificially been picked out functional areas the 3rd and 4 exons of tctp gene.Benefit
With bacterial artificial chromosome homologous recombination technique, with antibiotic resistance genes and it is attached to the end 5` (upstream) and the end 3` (downstream)
Homology arm constitute targeting vector, replacementTCTPThe functional areas the 3rd of gene and 4 exons.The approach for implementing the invention mainly wraps
Include targeting vector building, recombination embryonic cell clone and blastaea microinjection.Divide first from the genomic DNA of C57BL mouse
FromTCTPGene, through long-chain PCR method expand obtain homology arm, then with the compound building targeting vector of antibiotic resistance genes；It beats
Targeting vector construction of strategy is as shown in Figure 2.Embryonic cell is separated from the embryo of C57BL mouse, for beating after short-term expansion culture
The electroporation of targeting vector, screening obtain positive embryos cell clone；Embryonic cell, which will be recombinated, through micro-injection method injects mouse embryo
In tire, then it is transplanted in false pregnancy mouse body；Obtain blackspot shape allophenic mice；Further with wild typeTCTPMouse mating, is obtained
Obtain F1 generation heterozygote；Genotype verifying, screening-gene are carried out to obtained F1 generation hybrid mice using molecular biology method
The hybrid mice that the positive gene of successful knockout knocks out；Post-coitum obtains two chromosomes and is picked out between F1 generation heterozygote
Homozygote mouse；After Southern Blot method carries out genotype identification, building is to obtain Gene Knock-Out Animal Model population.Herein
On the basis of, it can further obtain the cell, organ and embryo of the animal model.
It is rejected from mouse genome specifically, the invention proposes one kindTThe functional areas the 3rd of CTP gene and 4 exons
Non-human mammal especially rodent Gene Knock-Out Animal Model production method (Fig. 2).Tctp gene used in the present invention is complete
It is about 4.08kb, wild-type template gene order is selected from the 14th DNA sequence DNA of C57BL/6J mouse.TCTPGene contains
6 exons, functional areas are located at 3-4 exon.Tctp gene knockout targeting vector constructed by the present invention is as shown in Figure 3
Length is 21.822kb, has 5 ' sequentially to contain 5` (upstream) homology arm (5.6kb), exons 1 and exon 2, (Frt sequence to 3 '
Column-the positive riddled basins box-Frt sequence of reporter gene box-loxP-, 5 ' loxP, exon 3-4 and 3 ' loxP, outer aobvious
Son 5 and exon 6 and 3` homology arm (5.0kb) and negative selection mark DAT.Wherein the reporter gene tape is formed by one
Type shears receptor (En2SA), and the pressure of mRNA can be mediated to shear, enable reporter gene in the driving of target practice gene promoter
Lower expression, the FRT sequence can be identified by Flp recombinase；Include in the reporter gene box polyadenylic acid (Poly A).With
NeoR-PA replaces the 5th exon.
Intermediate vector pKFCR-EGFR, 5` (upstream) homology arm that 5 ' ScaI endonuclease bamhi of tctp gene is cloned
(5.6kb) includes tctp gene exons 1 and 2；3 ' EcoV endonuclease bamhi be cloned into carrier, 3 ' homology arms include tctp gene
Exon 3,4,5 and 6.NEO is placed on NEO gene two in the introne between the 2nd and the 3rd exon into as positive selection marker
Respectively there is a LoxP sequence in side；Negative riddled basins are placed on 5, and ' on the outside of homology arm, carrier can use PstI linearization for enzyme restriction.
It utilizesTwo sides with Frt sequence-the positive riddled basins box of reporter gene box-loxP- -- Frt reject TCTP base
3rd and 4 exon 2s of cause are topmost wild type gene site (WT locus), contain 6 exons (Exon1-6)；It inserts
The antibiotic resistance genes (neo, PGK-gb2-neomycin selection cassette) entered are instead of the portion of exon 2
Divide 3 ' terminal sequences；Targeting vector includes containing 2~4 exon 2-4, insert Frt sequence-reporter gene box-loxP- just screens
Marker gene box-Frt sequence, 5 ' loxP, exon exon 3-4 and 3 ' loxP upstream homology arm and insert negative selection base
The downstream homology arm of cause；Neo sequence is taken off after the completion of recombination, schematic diagram is as shown in Figure 2.
By the process of microinjection producer gene knock-out animal the following steps are included: the male mouse of preparation infertility and false pregnancy are female
Mouse；Super ovulation；Harvest fertilized eggs；Recombinate the preparation of embryonic cell；The positive embryonic cell of recombination is imported into fertilized eggs；Fertilized eggs
Transplanting；The acquisition of allophenic mice, further mates with wild-type mice, obtains hybrid mice (head builds mouse)；Head builds mouse
(Founder) detection of recombinant DNA integration in；Mouse germline is picked out by crossbreed gene.
The tctp gene that the present invention obtains, which knocks out allophenic mice, has new phenotype.General vital signs are normal, growth
Development is normal.Since the report that tctp gene rejects animal model is few, phenotypic characteristic needs to be further looked at.
It is built using the mating of following scheme and is: then mated, select with the male mouse of expression recombinase for female chimeras mouse
Week old at 10 weeks there is mating experience, healthy and strong C57BL mouse to mate therewith.Start to check feelings of becoming pregnant after living together 1 week
Condition, if it find that pregnancy individually raises female mice, until production.If it is Male chimeras body mouse, select 8 weeks or more,
Healthy and strong female mice mates therewith.Offspring's separate marking of each homozygote mouse, is to treat as one.Detection discovery gene
Heredity can be stablized in offspring by picking out, and litter size amount is no different with normal mouse, generally can successfully feed, rare to eat the mothers such as son
Property bad phenomenon.It is to lay the foundation that these advantageous features are built for next step large-scale breeding.
The gene level phenotypic evaluation of TCTP uses the RT-PCR identification method of TCTP mRNA.The protein level table of TCTP
Type identification is identified using TCTP western blot.The missing that TCTP whole body strikes reduction mouse tctp gene expression will will lead to entirely
Body respectively organizes TCTP expression to reduce.Tctp gene rejecting strikes the life cycle for subtracting animal and wild animal without significant difference, not
See the different report of life cycle.In the present invention, the life cycle of allophenic mice has no shortening phenomenon, and general state is just
Often.
The present invention relates to the methods of production Gene Knock-Out Animal Model model and the animal model in a variety of biogenic materials
(animal derived, be derived from human body), tissue or the immunological investigation of organ, the research of immunological rejection and its mechanism, clinic
Purposes in preceding safety evaluatio (such as immunological evaluation, implantation experiment)；And be derived by the animal pattern cell,
Tissue, organ and embryo and its purposes in above-mentioned field.
The present invention is in the gene targeting carrier of building, using positive-negative selection strategy, therefore can be largely
The cell of random integration is excluded, target practice cell enrichment efficiency is improved.Sequence is identified by introducing recombinase in positive screening-gene two sides
Column, to facilitate the deletion of subsequent marker gene.
The application obtains functional areas the 3rd and the 4 exon animal models for artificially having picked out TCTP.Currently, not yet people grinds
Study carefully a kind of tool and method for being used as research cancer cell apoptosis mechanism and cancer target；The present invention utilizes homologous recombination skill
The tctp gene whole body knock-out mice of heredity is stablized in art and embryonic stem cell technologies building, for studying cancer cell apoptosis machine
The research of system and cancer target；The research of targeting cancer therapy avoids whole body and knocks out embryonic death caused by the gene, for
The effect played during the cancer occurrence and development of tctp gene is studied, is of great significance to treatment of cancer.
A specific embodiment of the invention is only limitted to be explained further and illustrate the present invention, not to contents of the present invention structure
At limitation.
Example is carried out below by way of specific embodiment, wherein agents useful for same can be bought by market channel, Qi Take
With the laboratory manual with reference to this field corresponding PCR and gene sequencing
Carrier design is completed by the applicant in following embodiment, and sequencing is completed by Beijing Hua Da gene, Taq enzyme,
T4DNA ligase, restriction enzyme are purchased from Beijing NEB company, other reagents are import packing.Used in somatic cell clone
Reagent is purchased from Sigma company.
LIF (leukocyte inhibitory factor), DMEM soup-stock culture medium, L-Glutamine, nonessential amino acid, neomycin Rong
Youngster, mercaptoethanol, fetal calf serum (the small certification of ES), tearing mycin C, dimethyl sulfoxide (DMSO), PBS buffer solution (Ph7.2),
EDTA- trypsase, G418, gelatin, Brinster BMOC-3
The reagents such as culture solution are purchased from Sigma and HyClone company respectively.
TCTP targeting vector pDTA-LRJ-072: laboratory building；
ES cell strain TC1 comes from National Institutes of Health；The mouse embryonic fibroblasts of G418 resistance are military affairs
The present of Academy of Medical Sciences Bioengineering Research Institute.
Mouse C57BL/6J is purchased from Kunming Military Medical Science Institute experimental animal center.
The routine experiments operating procedure such as digestion, connection, recycling, conversion, PCR amplification is detailed in " molecular cloning (third edition) ".
The building of 1 targeting vector of embodiment
1. targeting vector element and strategy design of practicing shooting
The present embodiment mouse tctp gene strike subtract targeting vector using first strike entirely, then conditionity knock out design scheme, this
Kind layout strategy is to be inserted into the report that a two sides have Frt sequence in the 5 ' upstreams loxP on the basis of normal condition knocks out
Accuse box gene (Reporter cassette) and antibiotics resistance gene box (Neo box gene), reporter gene box Reporter
Cassette shears receptor (En2SA) with a composing type, and the pressure of mRNA can be mediated to shear, make reporter gene box
Reporter cassette can be expressed under the driving of target practice gene promoter, therefore can be used to the table of tracer target practice gene
Up to situation.Simultaneously because in reporter gene box Reporter cassette polyA presence, terminate this target practice gene in advance
Transcription, thus achieved the purpose that knock out this gene.Additionally, due to Reporter cassette loxP-Neo cassette
Two sides be the site Frt, can be identified by Flp recombinase, to remove (Reporter cassette) and antibiotic resistance base
It is knocked out because conventional conditionity can be transformed into after box (Neo cassette).To sum up, this design scheme can get reserve
Property knock out potential strike mouse model, i.e. KO first, conditional ready entirely.
Target practice site schematic diagram is as shown in Figure 2: people's tctp gene sequence is selected from the 4th DNA sequence of C57BL/6J mouse
DNA(GRCm38.p1C57BL/6J).Striking the target gene mouse tctp gene overall length subtracted is 4.08Kb, by the function deleted
Can area be located at mouse tctp gene the 3rd and the 4th exon, length is including the 3rd exon function is trivial and the 4th exon function
It is trivial to add the upstream 5`, generate the truncated protein of a 73aa (34aa comes from N-terminal, and 39aa comes from C-terminal).
Targeting vector length used in the present invention is 21.822kb (Fig. 1-A), is named as pDTA-LRJ-072- targeting vector,
Contain (upstream) homology arm (5.6kb): 5`UTR- exons 1 and 2,5 ' Frt-En2-SA-IRES-eGFP-PA-LoxP-Neo-3
' Frt, 5 ' LoxP '-exon 3-exon 4-3 ' LoxP, 3` (downstream) homology arm (5.0kb): exon 5- exon 6-3 '
UTR。
The wherein B2 segment (SEQ ID No.33) of 476bp: 5 ' the 3 of the aobvious son 2 of the site LoxP-people's tctp gene-outer 4 are ' interior
Containing son non-conservative area-' people's tctp gene exon 3-exon 4- exon 43 ' the non-conservative area of introne, the B1 of 1120bp
Segment (SEQ ID No.32): 5 ' reporter gene box-LoxP site-of the Frt sequence-with composing type shearing receptor (En2SA) is anti-
Raw -3 ' Frt sequence of element riddled basins box；
The A segment (SEQ ID No.31) of 391bp: 5` (upstream) homology arm (5.6kb)-tctp gene 1 and 2-5 ' Frt
Point-；
The C1 segment (SEQ ID No.34) of 467bp: ' non-conservative area's introne-is aobvious outside for the 3 of the 4th exon of tctp gene
Sub- 5- exon 6；
The C2 segment (SEQ ID No.35) of 462bp: 3 ' non-conservative area and DAT gene -3- of tctp gene exon 6
Catchment knocks out tctp gene prediction translation after exon 3 and exon 4 are as follows:
MIIYRDLISHDELFSDIYKIREIADGLCLEVEGKFFIGENMNPDGMVALLDYREDGVTPFMIFFKDGLE
MEKC
2. designing the primer of anamorphic zone restriction enzyme restriction enzyme site
Primer such as table 1 used in the sequence and the above-mentioned segment of amplification of above-mentioned segment
3. with normal C57BL/6J mouse (being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) separation genome
DNA is template, with the composition segment B1 of the targeting vector of the tctp gene of primer amplification step 1 design of step 2 design
(112bp), B2 (476bp), C1 (467bp), C2 (462bp) segment and B segment and 5 ' probe (350bp) and 3 ' probes
(452bp), clones A segment system and program is as shown in table 2
2 A segment PCR reaction system of table and program
Amplification system and the program for cloning B1 segment are as shown in table 3:
Table 3: the amplification system and program of clone's B1 segment
Amplification system and the program for cloning B2 segment are as shown in table 4:
Table 4: the amplification system and program of clone's B2 segment
Amplification system and the program for cloning C1 segment are as shown in table 5:
Table 5: the amplification system and program of clone's C1 segment
Amplification system and the program for cloning C2 segment are as shown in table 6:
Table 6: the amplification system and program of clone's C2 segment
Recycling and sequencing, digestion, the electrophoresis verifying of the pcr amplification product of the composition segment of 4.TCTP gene targeting carrier,
It is connected to pBlunt carrier, constructs intermediate vector.Reclaimer operation presses " molecular cloning (third edition) " related experiment generally in the art
Chapters and sections, electrophoresis verification result are as shown in Figure 5
It is recycled with PCR product QIAquick Gel Extraction Kit (Shanghai Jierui Biology Engineering Co., Ltd, article No.: GK2043-200), it will
The segment of recycling is connected respectively to pBlunt plasmid, and (pBlunt Cloning Kit is the limited public affairs of golden biotechnology entirely from Beijing
Department, coupled reaction system and condition press kit specification), after digestion, agarose gel electrophoresis, ultraviolet imagery result such as Fig. 5
A~I shown in.
PBlunt-A:1# and 2# are correct, and 1# is used for lower pacing sequence.
The correct 3# of pBlunt-B1:1# and 3# is used for lower pacing sequence.
PBlunt-B2:1# and correct 1# is used for lower pacing sequence.
PBlunt-C:1# is correct, and 1# is used for lower pacing sequence.
PBlunt-5 ' probe: 1# and 2# are correct, and 1# is used for lower pacing sequence.
PBlunt-3 ' probe: 1# and 2# are correct, and 1# is used for lower pacing sequence.
Segment B1 sequencing primer
| Primer | Sequence |
| LRJ-072-B1-616F | GCTAGAAGACATTACCTATTGGAGC |
| LRJ-072-B1-713R | GGTTTCTGCTCTTCAAGTTTGC |
5. constructing intermediate vector
(1) digestion
According to the forward and reverse primer in B1 add respectively AsisI and SalI restriction enzyme site or B2 segment forward direction and
Reverse primer is added with MluI/EcoRV and ScaII/BstZ restriction enzyme site respectively, and the forward and reverse primer of A segment is added with
The site EcoRI/BstZ171 and Eco47III/Sca restriction enzyme site, respectively with identical digestion with restriction enzyme pKFCR-
EGFP。
With PCR QIAquick Gel Extraction Kit (being purchased from Shanghai Jierui Biology Engineering Co., Ltd, article No.: GK2043-200)
Above-mentioned segment is recycled, agarose gel electrophoresis testing result is as shown in Figure 5.
(2) it connects: distinguishing digestion B1 .B2 and A segment by above-mentioned restriction enzyme site, segment B1 or B2 are successively connected to
PKFCR-EGFP reconnects A segment,
Can with AsisI and Eco47III/ScaI or MluI/EcoRV and Eco47III/ScaI digestion by B1-NEO-A or
B2-NEO-A is by B-Neo-A from connecting correct intermediate vector pKFCR-EGFP-B1-A or pKFCR-EGFP-B2-A digestion.
According to the 5 of C1 ' and 3 ' both ends add respectively XhoI/SnaBI and EcoRV and C2 segment 5 ' and 3 ' both ends add respectively
Add the site EcoRV and Not.By C1 and C2 segment composition be C segment by fusion DNA vaccine, 5 ' for sites SnaBI, 3 ' of C segment for
The site EcoRV '.The 5 of pDTA-down ' ' uses SnaBI, 3 ' using the site EcoRV ' effects.
Then using PCR method expand respectively the 5th exon 5 ' end probe A (391bp) and 3 ' hold probe B1 (112bp),
B2 (476bp), C1 (467bp), C2 (462bp) segment, amplified fragments carry out PCR verifying with verifying primer again, and verifying primer is such as
Shown in table 2, the agarose gel electrophoresis of the PCR product of intermediate vector (is purchased from the east Beijing Jun Yi electrophoresis equipment Co., Ltd, goods
Number: JY300HC) verification result is as shown in Figure 5.
The PCR of 7 intermediate vector of table verifies primer
Restriction enzyme: ScaI, product: 1478bp+7905bp
Restriction enzyme:: BstZ17I, product: 2895bp+6488bp.
5:TCTP systemic gene, which strikes, subtracts targeting vector building
(1) digestion is identified and correct B1 or B2 segment is sequenced and A segment is consecutively connected to pKFCR carrier, obtain
PKFCR-EGFP-TCTP-B1-A or pKFCR-TCTP-EGFP-B2-A, digestion and then sequencing.
According to the restriction enzyme site of band in amplimer, segment B1 or B2 are connected into pKFCR- by digestion connection method
Obtaining pKFCR-EGFP-B2 or pKFCR-EGFP-B1 in EGFP carrier (Biovector Co., LTD), (digestion verification result is such as
Shown in Fig. 7), all clone is correct, and No. 1 clone is used for lower step.Then segment A is connected, obtains intermediate vector pKFCR-EGFP-
B2-B1A (digestion verification result such as Fig. 9),
The pKFCR-EGFP carrier for having recombinated B is subjected to PCR verifying (table 8).
The reaction system and program of 8 pKFCR-EGFP-B1-B2-A carrier PCR of table verifying A segment
All clones are correct, and No. 1 clone is in next step.
The pKFCR-EGFP carrier for having recombinated AB is subjected to PCR verifying B segment (table 9).
The pKFCR-EGFP-B1-B2-A that table 9 has recombinated AB carries out the reaction system and program of PCR verifying B segment
The verification result of its PCR reaction product such as Figure 12.In addition to No. 2, the correct connection of other clones, No. 1 clone is used for
Lower step.
The agarose gel electrophoresis results of PCR reaction product are as shown in Figure 10.
PCR verification result 1 clone is correct.
(2) 6 will be sequenced correct C1 and C2 segment composition by over-lap PCR is connected to for C segment (883bp) clone
PDTA-down carrier (general such as spit of fland biotechnology (Beijing) Co., Ltd), obtains pDTA-down-TCTP-C, passes through PCR and enzyme
Verification result such as Fig. 6 is cut, wherein No. 1 clone is correct, is used for lower step.
(3) by the linearization process of pDTA-down-TCTP-C, i.e. XhoI/SnaBI and NotI digestion handles pDTA-
Down-TCTP-C, recycling purpose band are the pDTA-down-TCTP-C, digestion verification result such as Fig. 6 of linearisation.-
(4) the exon information of mouse tctp gene is searched for by webpage www.ensembl.org, we select to knock out and be somebody's turn to do
The 3 of gene, 4 exons (as shown in Figure 3).The 3 and 4 exon sequences knocked out include tctp gene critical function structure
Domain selects left and right homology arm according to site is knocked out.5 ' homology arms for the 5.5kb that we select have (long-armed), and the 3 ' of 5.0kb are homologous
Brachium (galianconism), respectively selects the sequence of 500bp in the outside of homology arm respectively, design amplification tctp gene 5 ' homology arm and 3 ' homologous
The primer (table 10) of arm.
Primer used in the homology arm of 10 PCR amplification tctp gene of table
5. transformed competence colibacillus cell
Target fragment A-NeoR-B is transferred to the BAC containing pBCTG by electroporation apparatus (U.S. BTX, model: ECM-630)
Strain (RP23-212C13 is purchased from invitrogen).
(1) C57BL/ the BAC inquiry and order of C57BL/6J mouse species: is searched for by webpage www.ensembl.org
6J mouse tctp gene is in No. 4 chromosomes, and choosing the BAC comprising people's tctp gene full gene group, (RP23-212C13 is purchased from
Invitrogen it) places an order.Exon information, we select to knock out the 3 of the gene and 4 exons (such as the tactful institute of Fig. 2 target practice
Show).
(2) extracting and purifying, the verifying of BAC
It is biochemical purchased from Tiangeng that the BAC clone for the C57BL/6J mouse species tctp gene ordered is loaded into bacterial strain TOP10
Scientific and technological (Beijing) Co., Ltd, article No.: CB104-02) come by carrier mailing of agar, we expand strain, to mention
Take BAC.
Reagent required for the extracting and purifying of table 11.BAC
(3) electricity turns competent bacteria Stbl3 (testing handbook by this field 9) and is transferred to target fragment A-NeoR-B electricity to contain
The BAC bacterium of pBCTG is recombinated,.
By Stbl3 competent cell (being purchased from Beijing Quanshijin Biotechnology Co., Ltd, article No.: CD521-01) and extracting
(must dissolve in MilliQ) of 100~400ng TCTP BAC clone mixes and is transferred in the 0.1cM electricity revolving cup of pre-cooling, is placed in ice
Upper preparation conversion；BIORAD electroporation is set, and 1.75kV, 25 μ F, 200 Ω carry out electricity and turn；After electric shock, it is rapidly added 500 μ l
SOC simultaneously keeps the temperature 30 minutes Stbl3 recombinant bacteriums for obtaining the conversion of LRJ-072BAC AB recombinant vector in 32 DEG C；Bacterium solution is evenly coated in phase
On the Amb antibiotic resistance LB agar plate answered, in 32 DEG C keep the temperature 12~20 hours, after mention plasmid PCR and electrophoresis, gel it is ultraviolet at
As verifying.As a result as shown in figures 7 and 9.PCR verifying product electrophoretic image figure such as Figure 10's of LRJ-072 BAC AB recombinant vector
Shown in A and B.
Purpose band with XhoI/SnaBI and NotI digestion processing pDTA-Down-TCTP-C recycling is linearisation
PDTA-down-TCTP-C, electricity turn to have recombinated the BAC of B-Neo-A, have been recombinated beating for the attachment of segment containing TCTP-B-A-C
The BAC bacterium of targeting vector.
6. the digestion verification of targeting vector LRJ-072 B-A-C is analyzed
After converting Stbl3 competent cell, a small amount of upgrading grain and a large amount of upgrading grains, digestion TCTP- targeting vector verifying knot
Fruit is respectively as shown in figure 11 and shown in Figure 12
(1) HindIII digestion
It is expected that product: 471bp+5197bp+6342bp+9812bp；
(2) EcoRI+EcoRV digestion
It is expected that product 1251bp+2646bp+4078bp+4939bp+8908bp；
(3) SalI+ScaI digestion
It is expected that product 495bp+2717bp+4582bp+5626bp+7402bp.
7. the digestion verification analysis for largely preparing product of targeting vector LRJ-072B-A-C is as shown in figure 12.
PCR primer: LRJ-072-PCR-F1/PCR-R1；Neo-PCR-F/PCR-R2
Template: the BAC of transformation；Wild type gene group DNA
Product: 6016bp；6475bp
Neo-PCR-F/PCR-R2 primer can be used for next step PCR screening.PCR-F1/PCR-R1 primer cannot be used for next
Walk PCR screening.
Targeting vector pDTA-TCTP-A-B-C1 plasmid proposes greatly rear digestion detection, the result is shown in Figure 12:
Enzyme and digestion products list
SnaBI digestion products: 21822bp；
HindIII digestion products: 471bp+5197bp+6342bp+9812bp
EcoRI+EcoRV digestion products: 1251bp+2646bp+4078bp+4939bp+8908bp
SalI+ScaI digestion products: 1495bp+2717bp+4582bp+5626bp+7402bp.
8. the PCR primer optimal screening of targeting vector, the gel electrophoresis of PCR product primer as shown in figure 13 is adopted
Primer LRJ-072-PCR-F1/PCR-R1 used in homology arm with PCR amplification tctp gene；
Neo-PCR-F/PCR-R2 is primer,
The PCR primer optimal screening of 12 targeting vector of table
Primer size 6016bp；6475bp.The BAC and wild type gene group DNA of modification are template.
As a result, Neo-PCR-F/PCR-R2 primer can be used for next step PCR screening.PCR-F1/PCR-R1 primer cannot be used
It is screened in next step PCR.
Embodiment 2 is struck to ES cell microinjection tctp gene whole body subtracts targeting vector and middle target colony screening
(1) preparation of trophocyte and ES cell culture
Primary mouse embryonic fibroblast ES (GN-M127 covers peaceful biology) is taken, with final concentration of 10 μ g/Ml after amplification
Mitomycin C (Sangon Biotech (Shanghai) Co., Ltd.) handle 3 hours, finally packing freeze with -80 degree.It thaws
The processed culture dish of 0.1% gelatin is completed with trophoderm on the day before ES, the culture of ES cell is using the culture solution for adding LIF
(LIF concentration 1000U/Ml)
(2) electricity of ES cell turn and colony screening
Targeting vector MluI/EcoRV and NotI enzyme is linearized before transfection.Or ScaII is selected according to target practice policy map
With two kinds of enzymes of EcoRV.
It is resuspended in PBS after ES is cells trypsinised, the targeting vector DNA (45 μ g) of linearisation is thin with 1mL
Born of the same parents (2*107、/ mL) mix and be packed into electroporation slot, shock parameters 600V, 25 Μ f, after electric shock cell kind enter 4 cover with it is trophoblastic
Culture dish changes into afterwards for 24 hours containing G418 (280 μ g/Ml), the ES cell screening culture solution of GAN (2 μm of ol/L).(
3) targeted ES clones picking and amplification
Target is cloned in 7-8 days beginning pickings after transfection.Individually undifferentiated ES clone is placed in picking under low-powered microscope
96 orifice plates, each clone point is duplicate, and portion freezes, and the amplification of 24 well culture plates is gone to after portion culture.
Picking 200 apparent middle target clones are total in micro- sem observation.
(4) the Southern blot identification of the targeted ES cells of TCTP conditional gene targeting vector
By the linearisation of pDTA-LRJ-072 targeting vector and electroporation is transferred to ES cell
The preparation of Southern blot probe
With from mouse ES cell DNA measurement Southern blot probe (TCTP conditional gene strike the 5 ' probes that subtract and
3 ' probe)
By tctp gene targeting vector pDTA-LRJ-072 with Scal be digested with 5 ' probes ' afterwards electro-detection 5 ', use
With 3 ' probe in detecting 3 ' after EcoRV digestion.
Probe prepares primer
LRJ-072-5'Probe-F (Seq ID.No.25) gctatctatggggaaagaatat) and
LRJ-072-5'Probe-R ((Seq ID.No.26) gagcacttcatctgtcagca),
LRJ-072-3'Probe-F (Seq ID.No.27) agctagggagcaggttagaag) and
LRJ-072-3'Probe-R(Seq ID.No.28)ctctggtgagctcatctctg)；
PCR screens primer sets and includes 5 ' end screening primer pairs and hold screening primer pairs to 3 ',
Screen primer pairs in 5 ' ends
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat,
Screen primer pairs in 3 ' ends
LRJ-072-PCR-F2 (Seq ID.No.:3): Neo-PCR-F,
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
After tctp gene targeting vector pDTA-LRJ-072 is digested with ScaI, with 5, ' probe carries out Southern blot, such as
Shown in Figure 10 A, primer size 11.2bp.After tctp gene targeting vector is digested with EcRV, with 3, ' probe carries out Southern
Blot, primer size as shown in Figure 10 B are 21bp.' the probe and 3 ' probe can be used in target clone in then the result shows that 5
Southern blots screening "
With G418 (being purchased from Invitrogen (Gibco) company) screening positive clone, 200 clones are mentioned, it is cold with 96 orifice plates
Freeze, carries out PCR screening and Souther blot identification.
The anti-G418/GANC positive colony amplification of picking, extracts genome DNA, digestions is beaten with authenticated TCTP
Targeting vector 3 ' and 5 ' outside probe and 3 ' Neo probes carry out Southern blot, as a result as shown in figure 16, wild type and
TCTP targeted ES cells banding pattern has difference.
13 Souther blot of table verifying tctp gene conditionity strikes the targeted ES cells clone for subtracting targeting vector
14 are shown in Table with the DNA tests Southern blot probe probe sequence separated from ES cells.
5 ' probe (short homology arm) and 3 ' probes (long homology arm) can be used for following Southern screening
Table 14-1
LR-PCR screening positive clone primer and product clip size
Table 14-2 SR-PCR screening positive clone primer and product clip size
With 5, ' probe detects the segment of 11.2kb and 8.7Kb respectively in wild type and middle target clone, and with 3, ' probe exists
The segment of 21kb and 10.2Kb is detected respectively in wild type and middle target clone.It can be seen that being acted on respectively with Scal digestion and EcoRV
Afterwards, the fragment length of the homology arm generated respectively is different.9 positive colonies are obtained in total.#1 plate: C2, C5, C7, #2 plate: A5,
A11,B12,C7,E2,E4.C7, C2, C5 and No. B12 clones are selected to be counted.
Correctly clone C7, C2, C5 and B12 is finally chosen according to Southern blot testing result to be injected.
Probe 1 (Probe1) is located at the outside of the end 5` homology arm, and the restriction enzyme site introduced is held in B segment 3 '.5 ' probes
It is digested with ScaI, 3 ' probe is digested with EcoRV.Therefore, after wild type and saltant type ScaI and EcoRV digestion and probe combination sequence
The product of column is different, respectively as shown in Table 15.
9 positive colonies are obtained, these positive colonies inject the blastaea for being used for mouse embryo.
(5) the PCR detection of target positive colony in
200 clones of picking are subjected to LR-PCR and SR-PCR screening respectively.
The primer and clip size of table 15 LR-PCR and SR-PCR screening
LRJ-072 PCR-F2 and LRJ-072 PCR-R2 carry out LR-PCR screening, as a result as shown in Figure 14,6, screen altogether
17 positive colonies out, A1, B7, C2, C5, C7, C11, D9, E7,10 positive colonies of F11, H4 in plate 1, A5, A11, B12,
7 positive colonies such as C7, D11, E2, E4 carry out SR- in plate 2SR-LRJ-072-B1 Loxp-F and LRJ-072-B1 Loxp-R
As a result as shown in figure 15 PCR screening filters out 35 positive colonies altogether, and 21 of plate 1, A1, B7, C2, C5, C7, C11, D9,
E7, F11, H4, H2, H3,14 of plate 2, A5, A11, B12, C7, D11, E2, E4, B2, B7, C8, D5, E5.The core of positive colony
Type analysis is shown in
The karyotyping of 16. positive colony of table
Embodiment 3, middle target recombination embryonic cell blastaea injection and chimera culture
By microinjection (Microinjection) producer gene knock-out animal to blastaea, main process include with
Lower step:
The male mouse of 3.1 preparation infertility and false pregnancy female mice
Sterile hero mouse preparation
3.1.1 anesthesia prepares: 7 week old C57BL/6 hero mouse of selection are (in National Resource Center for Rodent Laboratory Animal Shanghai point
The heart) it weighs and 0.7% Nembutal sodium solution is injected intraperitoneally.
3.1.2 make arrangements for surgery instrument: ophthalmic tweezers 3, eye scissors 1, and Shearing shears 1, alcolhol burner, one, alcohol watering can,
Three-edged needle, suture, sterilized filter paper piece (diameter 15cm or so).
3.1.3 C57BL/6 hero mouse will male mouse ligation: have been anaesthetized (in National Resource Center for Rodent Laboratory Animal Shanghai point
The heart) abdomen is away from cropping at genitals 2cm, and after 70% alcohol swab cleaning disinfection, be open skin layer and muscle layer respectively, uses ophthalmic tweezers
It clamps testis cellulite and pulls out testis, epididymis, vas deferens, choose vas deferens middle section, with suture by vas deferens and capillary
Blood vessel tightens, and is spaced at 1cm and tightens again, is cut with the middle section for simply tightening suture, the ligation operation of side is complete
At, ligature other side again with the same manner, two sides completion clamps cellulite with tweezers and backs into abdominal cavity, suture muscle layer and
Skin layer, single cage is raised after recovery.
3.1.4 Anabiosis after operation: ligation completes raising after two weeks, mates mating with 6 week old heat female mices, next day examines list after bolt
Solely raising whether confirming gestation after 15 days, checks ligation success or not.
False pregnancy female mice prepares:
3.1.5 heat female mice is selected: selecting proestrum and oestrus C57BL/6 female mice (national laboratory rodent
Kind of subcenter Shanghai branch center), tissue characterization is vagina crack, is organized as pale red to pink colour, relatively wet, vagina dorsal lip with
All occur many wrinkles in length and breadth on abdomen lip.
3.1.6 blastaea is taken with the male mouse post-coitum selection false pregnancy C57BL/6 hero mouse female mice of ligation: will publish feelings C57BL/6
Male mouse and female mice press 1:1, and secondary daily inspection female mice yin bolt, single cage raising is spare, are 0.5 day on the day of seeing bolt, and common oviduct transplantation is with being shown in
0.5 day false pregnancy mouse of bolt, uterine transplantation is with being shown in 3 days false pregnancy mouse of bolt.Uterus is extractd, with BMOC-3 culture solution (being purchased from GiBCO company)
Blastaea is flushed out, few drops of culture solutions are dripped on 60mm culture dish, are covered with mineral oil, blastaea is transferred in drop and is cultivated.
3.2 surpass ovulation
3.2.1 hormone prepares: with 0.9% normal saline dilution pregnant mare serum (PMSG) and Human ChorionicGo-nadotropin
(hCG), domestic hormone is diluted to 10IU/0.1ml, and import hormone is diluted to 5IU/0.1ml.
3.2.2 injection of hormone: preparing 4-6 week old female mice, is spaced 48 hours, every respectively intraperitoneal injection PMSG and
hCG10IU.It mates and mates with same strain sexal maturity hero mouse after hCG injection, secondary daily test bolt, single cage raising is spare, sees that the bolt same day is
Female mice is put to death after 0.5 day, 3.5 days acquires blastaea.
3.3 acquisition blastaeas
3.3.1 culture solution prepares: ((being purchased from GiBCO company) is put into 37 DEG C of water-baths and incubates M2 culture solution, KSOM culture solution
(be purchased from GiBCO company)) and 1mg/ml hyaluronidase (purchased from GiBCO company) culture drop is made into superclean bench,
It is spare to be put into 37 DEG C of carbon dioxide incubators (being purchased from, model) incubation.
3.3.2 it dissects animal: after cervical dislocation execution is shown in that 3.5 days female mices of bolt, the disinfection of 70% alcohol swab wipe abdomen, opening
Abdominal cavity takes its uterus, is put into the 35mm culture dish for having incubated 1ml M2 culture solution (being purchased from).
3.3.3 embryo collection: taking protokaryon embryo is put into fallopian tubal in 300 μ l hyaluronidase drops, micro- in entity
Under mirror, magnum tubae uterinae is found, is punctured with 1ml syringe needle, egg mother cell group is discharged, micro- after 3~4 minutes
Under the microscope, the granular cell around egg mother cell has digested, so that it may collect egg mother cell, KSOM training is put into after washes clean
Nutrient solution (being purchased from supplier Caisson product article No. IVL04-100ML) drop is put into carbon dioxide incubator (the silent winged water jacket of match
Formula CO2 incubator) in it is spare.Taking blastaea is put into uterus in 60mm culture dish, draws M2 culture solution with 1ml syringe,
Under stereomicroscope, syringe needle is inserted into uterus one end, uterus is rinsed, and collect blastaea under mirror, is put after washes clean
To KSOM cultivate drop, be put into carbon dioxide incubator cultivate it is spare.
The embryonic cell of recombination is imported blastaea by 3.4
Blastaea microinjection: ellipse injection drop is made on 60mm culture dish of M2 culture solution, paraffin oil is covered, will cultivate
Ware is put under micromanipulation instrument mirror (Olympus micromanipulation system-IX73IVF/ is at the micro- behaviour of cyclopentadienyl), and 30 blastaeas is taken to be put into note
It penetrates in drop, then draws stem cell and be added in injection drop, adjust micro objective, under suitable multiple, adjust injection needle, sucking 50
~100 stem cells find blastaea under mirror, and operation is held ovum needle (ICSI holds the ovum needle-U.S./Sunlight), fixed blastaea, behaviour
Make injection needle by 10~15 stem cell injections into 1 blastocoele, completes injection.Microinjection record is as shown in table 17.One
It criticizes blastaea injection to put back to after the completion in KSOM culture drop, in carbon dioxide incubator culture, select after restoring 30 minutes
Blastaea carries out uterine transplantation.
17 microinjection of table record is summarized
The transplanting of 3.5 fertilized eggs
Blastaea transplanting: coat at dorsal line of the false pregnancy mouse back far from 3~4cm of tail portion, after 70% alcohol swab cleaning disinfection
Skin layer is cut off, then finds ovary position, cuts off muscle layer, ovary, fallopian tubal and uterus are taken out, fixed with haemostatic clamp,
8 blastaeas are drawn under stereomicroscope, while the false pregnancy mouse for having fixed ovary, fallopian tubal being put under microscope, are found
Uterus and the few position of oviductal junction blood vessel pierce an osculum with 1ml syringe needle, the suction oviduct bead for having blastaea are inserted
Enter, embryo is blown into, transplants the other side with the same manner.Postoperative skin suture layer, single cage is raised after animal revival, is waited to be implanted
The fertilization egg implantation of embryonic cell is recombinated, female rat is become pregnant.
Embodiment 4, mating, breeding and tctp gene whole body knock-out mice germline obtain
ES cell (the C57BL/6ES cell TCTP that embodiment 3 is obtainedmut/+) (black) positive colony is through microinjection skill
Art is injected in the blastaea of Balb/C mouse (white) (being purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.), then is implanted into
The intrauterine of replace-conceive female rat (Kunming mouse, white), obtain the filial generation F0TCTP gene whole body that tctp gene whole body removes strike subtract it is chimeric
Body mouse.Chi-meric mice confirmation: the produce surviving of son after transplanting 17 days records produce surviving of son quantity, according to hair color was confirmed whether to have at 10~15 days embedding
Close mouse.Embryonic cell derives from C57BL/6, black；Blastaea donor Balb/C is white, so obtaining the piebald of black splotch
Allophenic mice.Tctp gene whole body knock-out mice germline: Balb/C (TCTPmut/+) is obtained by crossbreed
It is built using the mating of following scheme and is:
(1) the F1 heterozygote of antibiotics resistance gene is rejected
By female chimeras Balb/C (TCTPmut/+) mouse then with Cre recombinate enzyme positive hero mouse C57BL/6 (Cre+/
Antibiotics resistance gene is rejected in Cre, Neo-/Neo mating.It selects week old at 10 weeks, there is mating experience, healthy and strong Cre
The male mouse of recombination enzyme positive C57BL/6 (Cre+/Cre) mates therewith.Start to check Pregnancy after living together 1 week, if it find that bosom
It is pregnant individually to raise female mice, until production.It if it is Male chimeras body mouse, selects 8 weeks or more, healthy and strong Cre recombination
Enzyme positive C57BL/6 female mice C57BL/6 (Cre+/Cre, Neo-/Neo-) mates therewith, obtains and rejects antibiotics resistance gene
Tctp gene target practice F1 generation hybrid mice ((head builds mouse) (Balb/C ((Cre+/Cre, Neo-/Neo-)).F1 generation heterozygote is small
Mouse mates with wild type C57BL/6 again, is formed, is to treat as one.
What 15 TCTP whole body clpp gene of table subtracted chimera educates summary
Embodiment 4, targeted mice genotype identification
The exon 3 of 4.1 rejecting TCTP exon genes functional areas and 4 TCTP conditional gene strike and reduce purpose in mouse
The detection of recombinant DNA integration:
By wild-type mice Balb/C 6 as compareing, from the TCTP conditionity base for rejecting reporter gene box and resistant gene
Subtract heterozygote tctp gene target practice F1 generation hybrid mice (head builds mouse) because striking (Balb/C be ((Cre+/Cre, Neo-/Neo-'s)
Tail point tissue extraction DNA carries out the identification of genotype by the Partial Fragment of high-fidelity PCR amplification tctp gene.For gene
The design of primers of type identification is shown in B table 19-1 and table 19-2
Table 19-1 is used for the PCR primer of dna chimeric gene type identification
| Primer | Sequence | Tm(℃) | Primer size |
| LRJ-072-B1 Loxp-F | TCATTGAGACTGTTATCTGTAGACCAGACT | 60 | WT:289bp |
| LRJ-072-B2 Loxp-R | AGCAACCATACCATCTGGATTCATGT | 60 | Mut:341bp |
Its PCR reaction system and program are table 19-2
Tctp gene whole body, which strikes, subtracts mouse (Balb/C (the Genotyping PCR. amplification gene of (Cre+/Cre, Neo-/Neo-)
The electrophoretogram of segment is as shown in figure 18.
Acquisition is shown in Figure 18, it is Balb/C ((Cre+/Cre, Neo-/Neo heterozygote that No. 3 and No. 42 heterozygotes, which are cloned,
Positive colony.
It is repeatedly bred, adds up to and obtain No. 4 as target hybrid mice in the positive, the breeding for homozygote mouse is bred.
After homozygote genotype identification, while ES cell screening is carried out using 5 ' Probe, 3 ' Probe and Neo Probe,.
Confirmation knocks out the correctness and stability of gene.Schematic diagram such as Figure 19 of Southern blot screening strategy, wherein with short-term table
The site of 5 ' Probe and 3 ' Probe are shown.The design of primers of shown probe 1 and probe 2 is the same as table 9；Specific design is as follows:
It is that Frt site upstream introduces 5 ' end Southern restriction enzyme sites at 5 ' ends, 3 ' the end site loxP downstreams introduce 3 ' ends
Southern restriction enzyme site, if correct recombination occurs, it will two bands of wild type and saltant type occur；If incorrect recombination,
Wild type band will only be will appear.
Tctp gene, which is detected, by the southern blot of 5 ' Probe and 3 ' Probe knocks out hybrid mice (Mut/WD)
Saltant type band only can be generated, (((Cre+/Cre, Neo-/Neo-) (Mut/WT) can generate wild type to Balb/C to hybrid mice
With saltant type band, and the wild-type mice (WT/WT) that tctp gene does not knock out only can generate wild type band.Each band it is big
It is small to be shown in Table 14.
Target knocks out the size of genetic fragment in table 14.Southern hybridization check heterozygote
| Restriction enzyme | Probe | WT | Hybridize target sequence |
| ScaI | 5’ | 11.2 | 8.7kb |
| EcorRV | 3’ | 21.0 | 15.4kb |
| NdeI | Neo(5’) | -- | 14.5kb |
| MfeI | Neo(3’) | -- | 10.2kb |
Southern Blot 30 mouse (heterozygote and each 15 of wild type) of total detection, extraction tctp gene, which strikes, to be subtracted
Zygote mouse (Mut/WT) and wild-type mice (WT/WT) rat-tail genomic DNA.Its Southern Blot qualification result point
Do not see such as shown in Figure 10 (5 ' Probe), it was confirmed that homozygote mouse has been obtained for stable heredity.
Embodiment 6, phenotypic evaluation
The tctp gene that the present invention obtains strikes reduction mouse, and (((Cre+/Cre, Neo-/Neo-) has new phenotype to Balb/C.
General vital signs are normal, and growth and development is normal.Due to tctp gene strike subtract animal model report it is few, phenotypic characteristic has
Wait further look at.
(1) the gene expression dose phenotypic evaluation of tctp gene
(2) TCTP whole body, which strikes, reduces the protein expression level phenotypic evaluation that mouse is respectively organized
The detection of TCTP protein expression is carried out using Westhern blot.
Tctp gene Westhern blot detection reagent and testing conditions used
TCTP protein antibodies used are anti-TCTP antibody ab58.
Remaining reagent, testing conditions, detection method is according to state of the art routine test handbook.
TCTP albumen TCTP whole body as shown in figure 19 strikes reduction mouse, and (((Cre+/Cre, Neo-/Neo-) whole body is each by Balb/C
Tissue T CTP expression reduces
Embodiment 7, gene pick out the life cycle of animal
| Mouse ID | Clone ID | Paternal ID | Maternal ID | DOB | Gender | Gene phenotype |
| 1D37-4 | 1-C7 | D37-3 | C57BL/6 | 2015-01-13 | ♂ | mut/+ |
| 2D37-2 | 1-C7 | 1D37-4 | C57BL/6 | 2015-04-10 | ♀ | mut/+ |
| 2D37-4 | 1-C7 | 1D37-4 | C57BL/6 | 2015-04-10 | ♀ | mut/+ |
| 2D37-6 | 1-C7 | 1D37-4 | C57BL/6 | 2015-04-10 | ♂ | mut/+ |
| 2D37-7 | 1-C7 | 1D37-4 | C57BL/6 | 2015-04-10 | ♂ | mut/+ |
| 2D37-10 | 1-C7 | 1D37-4 | C57BL/6 | 2015-04-10 | ♂ | mut/+ |
| 2D37-13 | 1-C7 | 1D37-4 | C57BL/6 | 2015-05-15 | ♀ | mut/+ |
| 2D37-14 | 1-C7 | 1D37-4 | C57BL/6 | 2015-05-15 | ♂ | mut/+ |
The life cycle and wild animal for the TCTP targeting vector animal that tctp gene is rejected have no raw without significant difference
Order period different report.Tctp gene rejects the mouse (Balb/C (Life Cycle of (Cre+/Cre, Neo-/Neo-) in the present invention
Phase has no shortening phenomenon, and general state is normal.
Above embodiments show that present inventor has been successfully established tctp gene whole body conditionity and has struck and reduce mouse
(Balb/C((Cre+/Cre,Neo-/Neo-)。
TCTP into it is complete deletion lead to mice embryonic Deaths, for further research tctp gene in vivo functionality,
TCTP molecular biology field needs tctp gene conditional gene target practice mouse.In the application, it is inserted into NEO gene to influence
Tctp gene normal expression, on the one hand, can realize that the whole body of gene is knocked out with TCTP, while utilize two side direction phase of NEO gene
Same LoxP sequence passes through the identification of Cre recombinase and deletes LoxP sequence proposition NEO sequence and eliminates when needed
The influence of NEO gene, to realize the foundation of tctp gene condition target practice mouse model.
Sequence table
<110>Dou Kefeng；Li Xiao
<120>people's tctp gene whole body strikes the animal model, preparation method and application subtracted
<130> 20180828
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> designed
<400> 1
cactactcta ccagctgtgg cac 23
<210> 2
<211> 23
<212> DNA
<213> designed
<400> 2
caactgacct tgggcaagaa cat 23
<210> 3
<211> 20
<212> DNA
<213> designed
<400> 3
gctcgactag agcttgcgga 20
<210> 4
<211> 26
<212> DNA
<213> designed
<400> 4
gttatggagc aaaggttact tagtgg 26
<210> 5
<211> 33
<212> DNA
<213> designed
<400> 5
cgatgaattc gtatacaaga tccgggagat cgc 33
<210> 6
<211> 36
<212> DNA
<213> designed
<400> 6
cgatagcgct agtactaagt tgctgtggcc tctctc 36
<210> 7
<211> 34
<212> DNA
<213> designed
<400> 7
cgatgcgatc gcggttccgc ttgatgagag tgac 34
<210> 8
<211> 30
<212> DNA
<213> designed
<400> 8
cgatgtcgac tagccatgtt ggcacacacc 30
<210> 9
<211> 43
<212> DNA
<213> designed
<400> 9
cgatacgcgt gatatcgcat atgtgttatt tttgagaact agg 43
<210> 10
<211> 34
<212> DNA
<213> designed
<400> 10
cgatccgcgg tatacttaca cacatgaccc tgag 34
<210> 11
<211> 34
<212> DNA
<213> designed
<400> 11
cgatccgcgg tatacttaca cacatgaccc tgag 34
<210> 12
<211> 46
<212> DNA
<213> designed
<400> 12
catggagaga ctgcttgaaa gatatctagc agttactcag tctcac 46
<210> 13
<211> 46
<212> DNA
<213> designed
<400> 13
gtgagactga gtaactgcta gatatctttc aagcagtctc tccatg 46
<210> 14
<211> 34
<212> DNA
<213> designed
<400> 14
cgatgcggcc gccttggtgt tcatagttat gccg 34
<210> 15
<211> 21
<212> DNA
<213> designed
<400> 15
gtcaccatga tcatctaccg g 21
<210> 16
<211> 20
<212> DNA
<213> designed
<400> 16
acgggttctt ctgttagtcc 20
<210> 17
<211> 22
<212> DNA
<213> designed
<400> 17
atgctatacg aagttatacg cg 22
<210> 18
<211> 23
<212> DNA
<213> designed
<400> 18
acactgaaac atcctattgc atg 23
<210> 19
<211> 20
<212> DNA
<213> designed
<400> 19
gctttccgat tgagcccagg 20
<210> 20
<211> 24
<212> DNA
<213> designed
<400> 20
tctctagagt cacagaactt atgg 24
<210> 21
<211> 20
<212> DNA
<213> designed
<400> 21
gctttccgat tgagcccagg 20
<210> 22
<211> 20
<212> DNA
<213> designed
<400> 22
agctagcttg gctggacgta 20
<210> 23
<211> 25
<212> DNA
<213> designed
<400> 23
gctagaagac attacctatt ggagc 25
<210> 24
<211> 22
<212> DNA
<213> designed
<400> 24
ggtttctgct cttcaagttt gc 22
<210> 25
<211> 22
<212> DNA
<213> designed
<400> 25
gctatctatg gggaaagaat at 22
<210> 26
<211> 20
<212> DNA
<213> designed
<400> 26
gagcacttca tctgtcagca 20
<210> 27
<211> 21
<212> DNA
<213> designed
<400> 27
agctagggag caggttagaa g 21
<210> 28
<211> 20
<212> DNA
<213> designed
<400> 28
ctctggtgag ctcatctctg 20
<210> 29
<211> 30
<212> DNA
<213> designed
<400> 29
tcattgagac tgttatctgt agaccagact 30
<210> 30
<211> 26
<212> DNA
<213> designed
<400> 30
agcaaccata ccatctggat tcatgt 26
<210> 31
<211> 391
<212> DNA
<213> designed
<400> 31
cgatgaattc gtatacaaga tccgggagat cgcggacggg ctgtgcctgg aggtggaggg 60
caaggtgagc ggggcgccgc gcgcggggag gctggccgcc tgcctgccgg gtcggccgag 120
ccgggccggg ctgggctggg cgccgcgggg aggccgctgg aactcgtgca atcctcgctg 180
ccgcctccag gcggaggaga cgctcttgcg gaccttgggt ttttctagaa aagtggaggc 240
ggagccgagc ctggaaatag gtccgcgaac tcagcgccat cctctttccg ggcgaacggg 300
gacatattgt ctataagaca ggtttgcgct gtgcgcgctt aacccgtggc gactcgagag 360
aggccacagc aacttagtac tagcgctatc g 391
<210> 32
<211> 1120
<212> DNA
<213> designed
<400> 32
cgatgcgatc gcggttccgc ttgatgagag tgaccacctc tctcagtccg ggcagcgact 60
atttgggggg aggttaagat gtttcaggga gctgactgat cgttgccgga cctttttttt 120
tttttctctt tttttctttt tttttttttt tttttgcccc ttcgactaag ttgtattgtc 180
tttttgtttt gttttgtttt cttctaaatg tagatggtca gtagaacaga gggtgccatc 240
gatgactcgc tcatcggtgg aaatgcttcc gctgaaggtc cggagggcga aggtaccgaa 300
agcacagtag tcaccggtgt tgacattgtc atgaaccatc acttacaaga aaccagcttc 360
acaaaagagg cttacaaaaa gtacatcaaa gactacatga aatcgtaagt gacataaaca 420
ccccgttttg gtggtcagct tcctagaaga agttggttgc ttaggtagga agggcttaag 480
aaaggagggt cttttgttat acagtgaagg ttgattttca ataatgtgag caagccggta 540
gaaagttact ttaaaggtaa tataggaatt acattctttt aaatggtttc ttgtcaccta 600
gtaaactatc attttgctag aagacattac ctattggagc ttatattctt actttactga 660
aagattattc agtattttga tgacctgtta ctttactgtt ttagactcaa aggcaaactt 720
gaagagcaga aaccagaaag agtaaagcct tttatgactg gagctgcaga gcagattaag 780
cacatccttg ctaatttcaa taactaccag gtaaatggac caaagggttg tataataact 840
gtgggatccg aaaaagtctg tttgctgtct cgtacatggc tctggctgtc ctggaacacc 900
cttagaccca gttacctcag ccttctgaga tgaaagtctc agcccatttt gtgtagcacc 960
acaccaggca aggagctggt attaaaatga taggcattgc tttcttttca ttgagactgt 1020
tatctgtaga ccagactagc cttgaaccca gaggtctacc tgccagtgcc tcttaattgc 1080
tgggattaaa ggtgtgtgcc aacatggcta gtcgacatcg 1120
<210> 33
<211> 476
<212> DNA
<213> designed
<400> 33
cgatacgcgt gatatcgcat atgtgttatt tttgagaact aggggttttt cttgccaaat 60
tttggccagg ggttattctg gatctgcttt ttatagtttg attttatatt tgtgtgtata 120
agatagtcta attgtaggtt tttgttacgt gtttcttaag ttttttattg gtgaaaacat 180
gaatccagat ggtatggttg ctctcctgga ctaccgtgaa gatggtgtga ctccattcat 240
gattttcttt aaggatggct tagagatgga gaaatgtgta agtatcttta aattagtagt 300
gtcaagacag ggagtgcagc agtgattctt tgccatctgc aggtggcagg ccttgtagat 360
tgtgagatct ttatcctgtg ggagagtaga gccttgaaca tgaaaggggc ttgaagatga 420
gattagctgg cttgctagag tgctcagggt catgtgtgta agtataccgc ggatcg 476
<210> 34
<211> 467
<212> DNA
<213> designed
<400> 34
cgatctcgag tacgtaccac catgcccagt ttagagaaaa gtttttcagt caggtcttcc 60
tccctcagga aacactccct cagccggctg tagtggctat tcctggttgt caacttgaca 120
atatttggaa tgaactacaa tccggaattg gaaggctcac cagtgaccct tatctggagg 180
cttggagatc cttatctgga tcttggtttg aagatcttga gccatagtgg ctatggattc 240
cagaagattg aatctccgag ttaaggaaca cacctttaat ctgggctatg cctttcatct 300
gggattaaag gtgtggtgga acacaccttt aatctgggct acaccttctg ctggagacaa 360
tataaggaca ttggaagaag ggagtctagc tcttgctctt gctccttcgc ctgcttgctg 420
cgtgagactg agtaactgct agatatcttt caagcagtct ctccatg 467
<210> 35
<211> 462
<212> DNA
<213> designed
<400> 35
gtgagactga gtaactgcta gatatctttc aagcagtctc tccatgcgtc agcacttttg 60
tccttggtgt agtatcctct ctagatacct tttctgaacg gtttgagagg aggttgaaat 120
tatgatgcga ctttaccata aaatgtcagt atgtatagtt ttttttttaa tttttttttt 180
tatgtgtagg tgtgttttgc ctgcatttat gtatgtgcac catgtgtgtg cttggtgcca 240
gtggaggtca gaaagggcat ctggtcccct atgactggag tttcagacag ttgtgacctg 300
cattatgggt actatgagcc aaacccaggt cctcttccag agcagcaagt gctgctagcc 360
acagagtcat ctctctagcc ccatagtacg ttcttctgaa aaagcaaaag cagtttactt 420
tatagtcacg gcataactat gaacaccaag gcggccgcat cg 462
<210> 36
<211> 21780
<212> DNA
<213> designed
<400> 36
ggcgcgcctc gagtacgtac caccatgccc agtttagaga aaagtttttc agtcaggtct 60
tcctccctca ggaaacactc cctcagccgg ctgtagtggc tattcctggt tgtcaacttg 120
acaatatttg gaatgaacta caatccggaa ttggaaggct caccagtgac ccttatctgg 180
aggcttggag atccttatct ggatcttggt ttgaagatct tgagccatag tggctatgga 240
ttccagaaga ttgaatctcc gagttaagga acacaccttt aatctgggct atgcctttca 300
tctgggatta aaggtgtggt ggaacacacc tttaatctgg gctacacctt ctgctggaga 360
caatataagg acattggaag aagggagtct agctcttgct cttgctcctt cgcctgcttg 420
ctgcgtgaga ctgagtaact gctagatcct tggacttcca ttcacagctg cgactgaacc 480
attgttggga attgggctgc cgactgtaag tcatcaataa attcctttac tatttagaaa 540
ttatccataa gttctgtgac tctagagaac cctgactaat acagaaattg gtaccagcag 600
agtggggtat tcctgtgaca acctgaccat gttttgggga ggtctgtgga aggactttgg 660
aactttggtc ttgaagatcc atttgttgtt aagagctctg tgggatgttg tgtaggagct 720
tggaagataa tgttgagaac agtgcagaag atggaggcct ggcttgtgaa atttcagagg 780
gaaaattaaa gactcttttc agggccattg ctgttttgat tgtgaagatt ctgtagttct 840
ggttagctgg ggctgaagaa tcagctgtga ttaacaagat accagaacta ctaaagcaaa 900
aactttgcat tactgggact attgatgctg gttagctgga gctaagaaat tagcggtgat 960
taagaagaga ccagcatcat tgaggtgaca tcttctggga agtgttttct gaaagcacaa 1020
agaggctgtg ttccagagat agccaaggtt gcactcctgc tgcagcggga cttggtaata 1080
tgtaagggtc acccaggtgg tactggtttt gaaggcatgg acttacccgc ctttgagatg 1140
gatatctaca ggaacttggg cagtgtggac ttcccacgca ctgcggatgg tgacctggct 1200
ggcactgtgc accctcaact gcaggaccat gactttgagc cactgaggcc tggtgaaccc 1260
atcttcaagc ttttcagcgg agaagacgta ctgtatgagg gggactccat tgtgtaccct 1320
gtgttcatta atgaggcaag ccagtaaaga acatccctcc atggcctctg catcagctcc 1380
tgctccctga cctgcttgag ttccagtcct gacttccttt ggtgatgaac agcagcatgg 1440
aagtgtaagc cgaataaacc ctttcctccc caacttgctt cttggtcatg atgtttgtgc 1500
aggaatagaa accctgacta agacagccgg ctaacctgcc aaaagcaggc cattgagcaa 1560
cttcctgctc agggaaaatc agctgatctg ccagccccat ccacaagggc tggcttgagg 1620
ctttctctct tcagcacctg cactttgaaa gggtatcaag ttctttcttt tggttactat 1680
tttggtctct ctcaacctgg ctgcacatgg acattctctg aagatttaaa aaactcagat 1740
gcctggatcc cacccccaga atttctaact ctactgaact tgtgtgccgg cttgacatca 1800
ttttttcccc tcaagttccg actgccaggc aggtggacta cagatctatt gacctctaga 1860
ctgttcacca ctgtcaatgt gactttaaca gccctcaata ttaagttaga gctcagctca 1920
ccactagata agtcccgctg tccattagag taccggtcac tgatctgacc actacactac 1980
agtttcacct ggatcattat agttccaccg tgggccctta aactacagtc cagcccccct 2040
tgactactgg tgatgggcac gcttgcctgg aacttgctac tgccaggccg gaggccagtt 2100
tccccatact gtagcacaca ctgtttactt ttactatggt ccattgtctt ctgtgtcttt 2160
tttttttttc ctttgttttt tcaagacagg gtttttctgt gtagatcagg ttggtcttca 2220
actcagctct gcctgcttct acctccctag tgctgaaatt aaaggagtgc agtaccacca 2280
tgtccagctg gattcactgt attttttttt ttcattcaga gtttgagcca ggcagtgttg 2340
gcccaactca tttaatccca gcacttggga ggtagaggta ggtggatttc tgaattcaag 2400
gccagcctgg tctacaaagt gagttccagg acatccaggg ctacacagag aaatcctgtc 2460
tcgaacccca ccccccaaaa atgtgtattt gaacagagtc attaggcact ctcagaataa 2520
gggaataatc acagaaatgc tattctattt tttttattat ttatttaatt atttattttt 2580
gaaacaagct ctcacatagt ctagaacttg tgtggctgaa aagggcctca aactcctgct 2640
cctctggtct tcacctctgc atccctggga ttatgtgtgc tgcaatacag ctagtcctcc 2700
ccttcattca ttttcatagt tactcactag tcacttagtt ttctcaacct tgatttaaaa 2760
agcactgtgg tgtcagatgc tatgcatgtc tttgtcccag gactgagaaa gcagatgtgg 2820
ggggcggggg ggggggcggg agaatgagag aatatcacat tttctcaaag gaatagtcca 2880
ggttgcctgg ttctttatcc attttctgac ccaaggagtg ttccaaattc acctatgttc 2940
taatcacgag accacatcct gagtgatact ttctactcca gcctgtaagg cttttctcac 3000
tattaatggt attctgagat aagaatggtt ggctcacacc tgtaatccta gccctattga 3060
agctaaggca ggagaatttt gatttccagg cctgtctggc ctccgaaatc agttcaagac 3120
tggtttgggc tatgcaagca gaggaggtga gaggagaggt taactgaaca aaaggctttt 3180
ggacctgaca gtaataactt caggaatatt atgaaaaaaa atcgggaatt gcatacatat 3240
cccacatatt atttatcacc tgttaagtat gtcagcctct cactcatctt attaaattca 3300
cagaaattcc atttcatact aaagctcaaa gagggtgaac ttcctgagat cgtgggactg 3360
gtaacacaga caagatgcca gcactgctta gcttcaaatc ttcagctctc tttcaccatg 3420
gttttgtttt tttttttgtt tttttttttt gttttttttt gttttttaat tcttcaatga 3480
gccggacggt ggtggtgcac atctttaatc ccagcacttg ggaggcagag gcaggcggat 3540
ttctgagttc aaggccagtc tggtctataa agtgagttcc aggacagcca ggactataca 3600
gagaaaccct gtctcgaaaa aacaaaaaca aaaaaaaatt tttaatgatg ttaaaaaaat 3660
acatttgggc ataaaactgg agaaaataac agaaaaatcc aaaaagaata tgtttgggga 3720
tctatgtaaa tgagcatttt tgaataattt gaaaatgaat acctgggatg tgcaaataag 3780
attcacataa ctcagaaata actgagtttt gagactgcta agtcatagcc cctcggttct 3840
atgtctacgt tttctgatgt ccttcacttt tttactgtta ccccgtgtcc tttagagtat 3900
ctaggaaagc agcagaccac cgcatcttaa gtggacggtt tttcttctgc tctttggcta 3960
gagttttatt cctgaatggt ttactctgtg cttgtaacac tttgttagtg tgaactgcct 4020
tctcctttga catactaacc taaccttctg tccgagggca ggcagggctg gctatgtcct 4080
tcttaagcga gaaaattgca acactagggt tacaacggac tataaccaaa tctggaaggg 4140
aagaaactgt gatttcatcc tgccatacat gtttacctga acggtgggtc tcggaagttg 4200
tatgttgtac ttcgtggtgg atgcgaaagc ccttcaagtg acccgggcaa gtgggaagct 4260
ccttattttg ggtctgttta gtcctgcatc gccaggaata aaagggataa aagtgacaat 4320
acagaactgt cctaacaaag gacaccgttt gcgaccaaga gcagaacagg ccagcgaaga 4380
gctgtccggc cagacccacc gcaggctttc actttcggaa cccactagag gaccagtccc 4440
caggacatct ccctccgcga gcaaaaccgg tgcagtctgt gggcagccaa ggaccggagg 4500
tcggagaccc gggacaccag cccggcgcca tccccgtagt ccccgcaggc tcagcggcgc 4560
gcccgacaac agtcatccga ttctgctgac attttctttc cgagaaaggg gtgggaactg 4620
aagcggcgtg gcgggaggcg gggcgcagtc acaccctggc cacgcccggg cggcgactca 4680
agcgtccggc catcggtcgg ccgcaagtcc cttcccgtcc cagcatgccc cgggcgcact 4740
atccgcacac cgcccccgtt gccccgcgca cccaggggca ctccgcattg tgtcccagca 4800
gagtccccgg atgccctccc ggggccggcc gggcgtagcc acgcccccgc accgccctgc 4860
gttcacgtca ccgctgacga cagttccggg ggagccgcgg acggtgacgt agccgagcgt 4920
gccctctata tgaggttggg gagcgcccgc gtcggccttt tccgcccgct cccccctccc 4980
cccgcgcgcc gctccggctg caccgcgctc gcttccgcgc tgtcaggcta gcgccgccgt 5040
ccccagccgt caccatgatc atctaccggg acctcatcag ccgtaagtcc cggcgcccgc 5100
gggcctgggt gcgggtgggc accggggagg ccggggacac gagcgcagag cttgggccgg 5160
gagccgccgc gtgcgccgag cccggcgcgg gaaatggcgg gccttcgctc gctcacgggc 5220
ggctctctct gttcgctttc agatgacgag ctgttctccg acatctacaa gatccgggag 5280
atcgcggacg ggctgtgcct ggaggtggag ggcaaggtga gcggggcgcc gcgcgcgggg 5340
aggctggccg cctgcctgcc gggtcggccg agccgggccg ggctgggctg ggcgccgcgg 5400
ggaggccgct ggaactcgtg caatcctcgc tgccgcctcc aggcggagga gacgctcttg 5460
cggaccttgg gtttttctag aaaagtggag gcggagccga gcctggaaat aggtccgcga 5520
actcagcgcc atcctctttc cgggcgaacg gggacatatt gtctataaga caggtttgcg 5580
ctgtgcgcgc ttaacccgtg gcgactcgag agaggccaca gcaacttagt actagcgctg 5640
aagttcctat tctctagaaa gtataggaac ttcgaaccct ttcccacacc accctccaca 5700
cttgccccaa acactgccaa ctatgtagga ggaaggggtt gggactaaca gaagaacccg 5760
ttgtggggaa gctgttggga gggtcacttt atgttcttgc ccaaggtcag ttgggtggcc 5820
tgcttctgat gaggtggtcc caaggtctgg ggtagaaggt gagagggaca ggccaccaag 5880
gtcagccccc ccccccctat cccataggag ccaggtccct ctcctggaca ggaagactga 5940
aggggagatg ccagagactc agtgaagcct ggggtaccct attggagtcc ttcaaggaaa 6000
caaacttggc ctcaccaggc ctcagccttg gctcctcctg ggaactctac tgcccttggg 6060
atccccttgt agttgtgggt tacataggaa gggggacggg attccccttg actggctagc 6120
ctactctttt cttcagtctt ctccatctcc tctcacctgt ctctcgaccc tttccctagg 6180
atagacttgg aaaaagataa ggggagaaaa caaatgcaaa cgaggccaga aagattttgg 6240
ctgggcattc cttccgctag cttttattgg gatcccctag tttgtgatag gccttttagc 6300
tacatctgcc aatccatctc attttcacac acacacacca ctttccttct ggtcagtggg 6360
cacatgccca gcctcaagtt tatatcacca cccccaatgc ccaacacttg tatggccttg 6420
ggcgggtcat cccccccccc ccacccccag tatctgcaac ctcaagcttg ggtgcgtggg 6480
ttgtggataa gtagctagac tccagcaacc agtaacctct gccctttctc ctccatgaca 6540
accaggtccc aggtcccgaa aaccaaagaa gaagaaccct aacaaagagg acaagcggcc 6600
tcgcacagcc ttcactgctg agcagctcca gaggctcaag gctgagtttc agaccaacag 6660
gtacctgaca gagcagcggc gccagagtct ggcacaggag ctcggtaccg cccctctccc 6720
tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc 6780
tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 6840
cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 6900
ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct 6960
gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa 7020
aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt 7080
tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag 7140
gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt 7200
acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt 7260
ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg gcgaggagct 7320
gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 7380
cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 7440
ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 7500
cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 7560
catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 7620
gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 7680
catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 7740
ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 7800
ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 7860
catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 7920
gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 7980
cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagatcata 8040
atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 8100
ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 8160
aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 8220
cattctagtt gtggtttgtc caaactcatc aatgtatctt aataacttcg tataatgtat 8280
gctatacgaa gttataggtc tgaagaggag tttacgtcca gccaagctag cttggctgca 8340
ggtcgtcgaa attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg 8400
ctttagcagc cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt 8460
ccacatccac cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt 8520
ctactcctcc cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt 8580
gacaaatgga agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat 8640
ggaagcgggt aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg 8700
ctcagaggct gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg 8760
cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg 8820
ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac ctgcagcctg ttgacaatta 8880
atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggg 8940
atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 9000
attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 9060
gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 9120
actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 9180
tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 9240
gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 9300
aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 9360
tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 9420
cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc 9480
cgacggcgat gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 9540
aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 9600
ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 9660
cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 9720
tcttgacgag ttcttctgag gggatcaatt ctctagagct cgctgatcag cctcgactgt 9780
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 9840
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 9900
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 9960
agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 10020
cagctggggc tcgactagag cttgcggaac ccttcgaagt tcctattctc tagaaagtat 10080
aggaacttca tcagtcaggt acataatata acttcgtata atgtatgcta tacgaagtta 10140
tatcgatgcg atcgcggttc cgcttgatga gagtgaccac ctctctcagt ccgggcagcg 10200
actatttggg gggaggttaa gatgtttcag ggagctgact gatcgttgcc ggaccttttt 10260
ttttttttct ctttttttct tttttttttt ttttttttgc cccttcgact aagttgtatt 10320
gtctttttgt tttgttttgt tttcttctaa atgtagatgg tcagtagaac agagggtgcc 10380
atcgatgact cgctcatcgg tggaaatgct tccgctgaag gtccggaggg cgaaggtacc 10440
gaaagcacag tagtcaccgg tgttgacatt gtcatgaacc atcacttaca agaaaccagc 10500
ttcacaaaag aggcttacaa aaagtacatc aaagactaca tgaaatcgta agtgacataa 10560
acaccccgtt ttggtggtca gcttcctaga agaagttggt tgcttaggta ggaagggctt 10620
aagaaaggag ggtcttttgt tatacagtga aggttgattt tcaataatgt gagcaagccg 10680
gtagaaagtt actttaaagg taatatagga attacattct tttaaatggt ttcttgtcac 10740
ctagtaaact atcattttgc tagaagacat tacctattgg agcttatatt cttactttac 10800
tgaaagatta ttcagtattt tgatgacctg ttactttact gttttagact caaaggcaaa 10860
cttgaagagc agaaaccaga aagagtaaag ccttttatga ctggagctgc agagcagatt 10920
aagcacatcc ttgctaattt caataactac caggtaaatg gaccaaaggg ttgtataata 10980
actgtgggat ccgaaaaagt ctgtttgctg tctcgtacat ggctctggct gtcctggaac 11040
acccttagac ccagttacct cagccttctg agatgaaagt ctcagcccat tttgtgtagc 11100
accacaccag gcaaggagct ggtattaaaa tgataggcat tgctttcttt tcattgagac 11160
tgttatctgt agaccagact agccttgaac ccagaggtct acctgccagt gcctcttaat 11220
tgctgggatt aaaggtgtgt gccaacatgg ctagtcgaca taacttcgta taatgtatgc 11280
tatacgaagt tatacgcgtg atatcgcata tgtgttattt ttgagaacta ggggtttttc 11340
ttgccaaatt ttggccaggg gttattctgg atctgctttt tatagtttga ttttatattt 11400
gtgtgtataa gatagtctaa ttgtaggttt ttgttacgtg tttcttaagt tttttattgg 11460
tgaaaacatg aatccagatg gtatggttgc tctcctggac taccgtgaag atggtgtgac 11520
tccattcatg attttcttta aggatggctt agagatggag aaatgtgtaa gtatctttaa 11580
attagtagtg tcaagacagg gagtgcagca gtgattcttt gccatctgca ggtggcaggc 11640
cttgtagatt gtgagatctt tatcctgtgg gagagtagag ccttgaacat gaaaggggct 11700
tgaagatgag attagctggc ttgctagagt gctcagggtc atgtgtgtaa gtagtttgac 11760
actggcctgg gataaactca cgtagaatga gtgtggtctc tgccccacgg tagttcaagt 11820
ccatttcata ttacatgcaa taggatgttt cagtgtttac tgaggtcagt aagaaggaat 11880
acagaaatgt ctgttttata agggactggt ggagacagtg ttccttgtcc tagattttgg 11940
aggctttttt tgtaagcaca gtagaaggtg agtttagaga tgtactggag aaagtgggtg 12000
accgcctggg acagtggggt aggagtgtta ttcagacaac agctggtgtt tgtcagtaga 12060
gcactggagt gggcaggaag atgggtgagt gctgccaact cggtgagggt ctgcatccac 12120
tgatagacct cgaacagttt gtggttgttc ttctggtttg cactaggatg caaaaggaaa 12180
ctctccctgc gcttcctgcc tgcctttgtg gcagttcaga ttgaattagg gagtacatct 12240
acatgctagg acagttataa gctcaggctg gggcagttgt taatgccatc tctttgtttt 12300
gcagtaacaa attggatcta tcacctgtca ccataattgg ctgctgctta ccatccatac 12360
aacaccagga cttaggacaa atgggactga tgtcatcttg agcttttatt ttgaccgtga 12420
tttatttgga gtggtggcat tgttttttta aggaaaaaaa acatgtcatg tgggttgtct 12480
aaaaataaag tgcatttaaa tccacttaag aactctttgc tgtgattgtg ctgaccctgt 12540
agtctgagaa gctagagcct ggttgagcat cgctagaaag ctaagcctct tcagttaact 12600
gctgcagtgg ggactctaca agactggagt gtgtgaactc aagaatctca gttacagtga 12660
agggagaggt gagaagtgga ccctgacttt catcacctcc agtggaagag cttaccaaaa 12720
gcacaaaaag accaactagt tggagacaag tactgtctgc tcctggacta atgtcagatt 12780
cctggtaact agaatagagg ttgccaagcc tggcctgtgc cactttagag ttgtacaaac 12840
agggcacaga tccatcgggg agttcggtac ctggtggggt gggatacagt gaatacagcg 12900
tagttccttc ccgggagtgc cagcagccca tgcttactgg ggtaaggtta gatgacccag 12960
tcacctgcgg gaattgggtc ttggaaagcc tagaactgca tagtctgctg taggagtatt 13020
aacatgcttt gctgtctaag gtttgtccag tgatagaagc cttaaaaagg tactaaactt 13080
aagagggtac tagcacctct gcaatatgaa gcccaaataa ctggatacta ggtattgtga 13140
tgaaggggaa gtgttgctag gtctaggaag ccacttagct gtctttgaat tttaatgggt 13200
cttgaggtca ctggtcagaa agaaacttga gaaaagttct agtgctagcc ctagagaagt 13260
gaacacaaga cagcagaagc ttggtagggg aggaagacca gaactcaata gtatacacat 13320
ttcagattat gagtaagcat atatgtgttt gtgcacgtgg ctgcagatgc ccattgtcaa 13380
aggagagatt tcctggagat ggacataagt atagggtacc aaagggccct gctgagcctt 13440
ccttgactgg tttgggttgt ttgttgagag tttctctgta ttcctgtctt ggaacttgtt 13500
ttgtaatctc ttgagctcaa ggcctcccaa gtgctgggat taaagtgaac aacaaacagg 13560
ctgggaagat tggtttttga gacagctttt acatccaagg ttgaccttaa attgctgagg 13620
atactttgcc gtcaccaccc atgacaatag gcacgtgact cttgcttagt tgatttggtc 13680
ctgaagttga atctagggat tatggggtta gcggcacagc accaaacctt gagcttcgtt 13740
ctcaaacctt ggcttttttt tttttttgcc ttgatagacc ttgaactcct caaccttagt 13800
gctaggaccg gaggcttgcc ctaccatacc aggttcctaa cttttgaaga caacttttcc 13860
taattgtctc tgcagttttc tgcagtcctg ttgagtccta gtgtgtgcct tgtgttttat 13920
atatggcctt atttcagaag ggcaggaatt catgaattaa gttgtcttga gtgtgctatt 13980
ttattataac ttactttcta gttattaagc acccatgctt tctgcatagg tcaggagagt 14040
gcagcacttg atctttctga tgtttggtga aaacaggttt tgagtaactg tgttctgggc 14100
tttgttgtat gtgtggaaca atggggtgat gagaagaaag gttttggtgt ttgtgaaagc 14160
cttcgcagtg tgccacttga atgtaggtgt gaaggagcag ctatggggct atctgaaagc 14220
agaccgtgca agccgcaaga gtgaacatct tcatcaacgt gttgtgggta tccagacaga 14280
ggcttccagc tgtacctagg aactcttgaa cctgtcttag tctttggagt taggatctga 14340
aggatggcag caatcctact ctaagatgag ctaactttgc ctttgctcag ctggtagcca 14400
agataggcat ccttgctgtg ttggttgctt ttgtcagctt gtcagttgga cacaccctag 14460
tgttgccaca actgagaaaa tgccaccttg agattgatcg gtaaacaagg atataagaca 14520
ttttcttggg aagtgggagg catagctcat tgtgaggggt gtcacctgtg ggcaggtgtc 14580
ctggtgtgta ttagaaagca aactgagaag ccatggaaag caagcaagcc tgtgggcagc 14640
actcctccat ggcatctgct tcagttcctc caggttcctg tgttgagtca cagctctaac 14700
ctctctccat gtgtgatgtg gaggtggaag ctaaataagc cctttctccc caggttcttc 14760
atcgtggtgt ttcatcacat tgtgttgtta gtcaggcctc tctagattta ttctagagaa 14820
tgaatctgtc tcgtgtattt tatattttgc ttacataaca taattatgtt cattgatata 14880
attataaaat atataatgag acaaattata catcatatgc tataacacag ggtttcaaac 14940
aatggaaaca gcatttaatg atctttcaca tgctgggcca tgaggacctc tggaaaggat 15000
gtgggattgg gggcgggccg tgtcaatgtt tcagttaaaa aaaccatcga ctgtgcacct 15060
agaggattct tttcaagtag cttgctcagt gatggctcct cggctctctt ctccagcttg 15120
tgagacttct ctgtatttat gtgcacactg tgcttccctg ggtgtcacag cagagagcgc 15180
aacagacccc tctgcagaac tgtggccctg agatagtcag tggctcactg gagggcacag 15240
cccagagacc acataaagcc actattgtta gtcagcaatt aaaatgcagg gatttgggtt 15300
cgttttgtat gtgtgctgcg atgaacccag ggctccatgt caatcacctc tcagtgagca 15360
acgcccccag ccctgagtcc aatgttttta attataacat tgagtcctgg gtttctgtca 15420
gttctttctt gtctcttttt tgtttatttt tttctttttg ttttttcttc tttggggaca 15480
cagtttctct gtgtagcctg cctgtcctcg gaatcactct attgatcagg gtagcctcaa 15540
actcagagat ccacctgtct ccgcctcctg agtgctgggt taaaagtatg tgcttcccag 15600
cttgagtttc tgccttgggt tatcattgct gtgatgacca tggccaaaaa caagatggag 15660
aggaaaaggt ttatttattt ggtttatgct tacacatcca tcactgaagg atgtcagggc 15720
agcagcctgc tgatgcagag gccatggatg ggtgctgctt actggcttgc tccccatggc 15780
ttgctcagcc tgctttccga ttgagcccag gaccaccagc ccatttatgt caatattcac 15840
aagtacaaag ttattttttt ggactttgga attaatgatt tagggggaaa aaaatcacaa 15900
aaatagcaca aaacacaaat ctttcaagca gtctctccat gcgtcagcac ttttgtcctt 15960
ggtgtagtat cctctctaga taccttttct gaacggtttg agaggaggtt gaaattatga 16020
tgcgacttta ccataaaatg tcagtatgta tagttttttt tttaattttt ttttttatgt 16080
gtaggtgtgt tttgcctgca tttatgtatg tgcaccatgt gtgtgcttgg tgccagtgga 16140
ggtcagaaag ggcatctggt cccctatgac tggagtttca gacagttgtg acctgcatta 16200
tgggtactat gagccaaacc caggtcctct tccagagcag caagtgctgc tagccacaga 16260
gtcatctctc tagccccata gtacgttctt ctgaaaaagc aaaagcagtt tactttatag 16320
tcacggcata actatgaaca ccaaggcggc cgccaggtct gaagaggagt ttacgtccag 16380
ccaagctagc ttggctgcag gtcgtcgaaa ttctaccggg taggggaggc gcttttccca 16440
aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc 16500
ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt 16560
ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag 16620
ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg 16680
gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt 16740
tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg 16800
ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg 16860
cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc 16920
tgcagcctgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 16980
tgaggaacta aaccatggga gtcaaagttc tgtttgccct gatctgcatc gctgtggccg 17040
aggccaagcc caccgagaac aacgaagact tcaacatcgt ggccgtggcc agcaacttcg 17100
cgaccacgga tctcgatgct gaccgcggga agttgcccgg caagaagctg ccgctggagg 17160
tgctcaaaga gatggaagcc aatgcccgga aagctggctg caccaggggc tgtctgatct 17220
gcctgtccca catcaagtgc acgcccaaga tgaagaagtt catcccagga cgctgccaca 17280
cctacgaagg cgacaaagag tccgcacagg gcggcatagg cgaggcgatc gtcgacattc 17340
ctgagattcc tgggttcaag gacttggagc ccatggagca gttcatcgca caggtcgatc 17400
tgtgtgtgga ctgcacaact ggctgcctca aagggcttgc caacgtgcag tgttctgacc 17460
tgctcaagaa gtggctgccg caacgctgtg cgacctttgc cagcaagatc cagggccagg 17520
tggacaagat caagggggcc ggtggtgact aagcggcacg tgctacgaga tttcgattcc 17580
accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 17640
atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 17700
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 17760
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 17820
ccgtccaatt gtttctagag gccggccgtc gagcagtgtg gttttcaaga ggaagcaaaa 17880
agcctctcca cccaggcctg gaatgtttcc acccaatgtc gagcagtgtg gttttgcaag 17940
aggaagcaaa aagcctctcc acccaggcct ggaatgtttc cacccaatgt cgagcaaacc 18000
ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtcggg gcggcgcggt 18060
cccaggtcca cttcgcatat taaggtgacg cgtgtggcct cgaacaccga gcgaccctgc 18120
aggtcctcgc catggatcct gatgatgttg ttgattcttc taaatctttt gtgatggaaa 18180
acttttcttc gtaccacggg actaaacctg gttatgtaga ttccattcaa aaaggtatac 18240
aaaagccaaa atctggtaca caaggaaatt atgacgatga ttggaaaggg ttttatagta 18300
ccgacaataa atacgacgct gcgggatact ctgtagataa tgaaaacccg ctctctggaa 18360
aagctggagg cgtggtcaaa gtgacgtatc caggactgac gaaggttctc gcactaaaag 18420
tggataatgc cgaaactatt aagaaagagt taggtttaag tctcactgaa ccgttgatgg 18480
agcaagtcgg aacggaagag tttatcaaaa ggttcggtga tggtgcttcg cgtgtagtgc 18540
tcagccttcc cttcgctgag gggagttcta gcgttgaata tattaataac tgggaacagg 18600
cgaaagcgtt aagcgtagaa cttgagatta attttgaaac ccgtggaaaa cgtggccaag 18660
atgcgatgta tgagtatatg gctcaagcct gtgcaggaaa tcgtgtcgcg atgtatgagt 18720
atatggctca agcctgtgca ggaaatcgtg tcaggcgatc tctttgtgaa ggaaccttac 18780
ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat 18840
ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag 18900
attccaacct atggaactga tgaatgggag cagtggtgga atgcagatcc cgcggtggag 18960
ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg 19020
tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 19080
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 19140
cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 19200
tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 19260
cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 19320
tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 19380
tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 19440
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 19500
ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 19560
gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc 19620
acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 19680
atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 19740
agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 19800
cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 19860
gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 19920
cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 19980
tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 20040
ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 20100
ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 20160
atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 20220
cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 20280
atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 20340
gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 20400
cgctcggcgc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 20460
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 20520
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 20580
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 20640
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 20700
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 20760
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 20820
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 20880
gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 20940
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 21000
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 21060
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 21120
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 21180
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 21240
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 21300
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 21360
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 21420
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 21480
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 21540
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 21600
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 21660
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 21720
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 21780
Claims (12)
1. the tctp gene whole body based on recombinase identifying system strikes the non-human mammal model subtracted and constructs kit, feature
It is, the non-human mammal model constructs kit and includes
Tctp gene whole body, which strikes, subtracts the cloning primer that targeting vector constitutes segment, and the targeting vector element cloning primer includes:
LRJ-072-A-F (Seq ID.No.5): cgatGAATTCGTATACAAGATCCGGGAGATCGC,
LRJ-072-A-R (Seq ID.No.6): cgatAGCGCTAGTACTAAGTTGCTGTGGCCTCTCTC,
LRJ-072-B1-F (Seq ID.No.7): cgatGCGATCGCGGTTCCGCTTGATGAGAGTGAC,
LRJ-072-B1-R (Seq ID.No.:8): cgatGTCGACTAGCCATGTTGGCACACACC,
LRJ-072-B2-F (Seq ID.No.:9): cgatACGCGTGATATCGCATATGTGTTATTTTTGAGAACTAGG,
LRJ-072-B2-R (Seq ID.No.:10): cgatCCGCGGTATACTTACACACATGACCCTGAG；
LRJ-072-C-F (Seq ID.No.:11): cgatCTCGAGTACGTACCACCATGCCCAGTTTAGA,
LRJ-072-C-R (in) (Seq ID.No.:12): CATGGAGAGACTGCTTGAAAGATATCTAGCAGTTACTCAGT
CTCAC
LRJ-072-C-F(in)(Seq ID.No.:13):GTGAGACTGAGTAACTGCTAGATATCTTTCAAGCAGTCTCT
CCATG
LRJ-072-C-R(Seq ID.No.:14):cgatGCGGCCGCCTTGGTGTTCATAGTTATGCCG。
2. kit as described in claim 1, which is characterized in that the non-human mammal model constructs also to wrap in kit
It includes tctp gene whole body and strikes and subtract that targeting vector constitutes the verifying primer of segment, detection probe prepares primer, sequencing primer, practicing shooting carries
The PCR of body screens primer, genotyping primer,
The verifying primer
LRJ-072-Atest-F (Seq ID.No.:15): GTCACCATGATCATCTACCGG,
LRJ-072-Atest-R (Seq ID.No.:16): acgggttcttctgttagtcc,
LRJ-072-Btest-F (Seq ID.No.:17): atgctatacgaagttatacgcg,
LRJ-072-Btest-R:(Seq ID.No.:18): acactgaaacatcctattgcatg,
LRJ-072-C1test-F(Seq ID.No.:19:gctttccgattgagcccagg,
LRJ-072-C1test-R (Seq ID.No.:20): tctctagagtcacagaacttatgg,
LRJ-072-C2test-F (Seq ID.No.:21): gctttccgattgagcccagg,
LRJ-072-C2test-R (Seq ID.No.:22): agctagcttggctggacgta,
The sequencing primer includes:
LRJ-072-B1-616F (Seq ID.No.:23): gctagaagacattacctattggagc and
LRJ-072-B1-713R (Seq ID.No.:24): ggtttctgctcttcaagtttgc；
The probe prepares primer
LRJ-072-5'Probe-F (Seq ID.No.25) gctatctatggggaaagaatat) and
LRJ-072-5'Probe-R ((Seq ID.No.26) gagcacttcatctgtcagca),
LRJ-072-3'Probe-F (Seq ID.No.27) agctagggagcaggttagaag) and
LRJ-072-3'Probe-R(Seq ID.No.28)ctctggtgagctcatctctg)；
The genotyping primer is to including:
LRJ-072-B1Loxp-F (Seq ID.No.29): tcattgagactgttatctgtagaccagact,
LRJ-072-B1Loxp-R (Seq ID.No.30): agcaaccataccatctggattcatgt；
The PCR screening primer sets include 5 ' end screening primer pairs and hold screening primer pairs to 3 ',
It screens primer pair and includes in the 5 ' end
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat,
It screens primer pair and includes in the 3 ' end
LRJ-072-PCR-F2 (Seq ID.No.:3): GCTCGACTAGAGCTTGCGGA,
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
3. a kind of tctp gene whole body based on recombinase identification strikes the non-human mammal targeting vector subtracted, which is characterized in that
The whole body of the tctp gene strike subtract be based on exon 2 frameshift mutation caused by homologous recombination generate exon 3 and
4 product of exon is truncated protein；
The targeting vector of the non-human mammal of the tctp gene whole body from 5 ' to 3 ' sequentially comprising sequentially connected B1 segment,
B2 segment, B3 segment and C1 and C2 segment it is warm at the B2-B1-A-C1-C2 sequence attachment that is formed by connecting of C segment；It is described
B2 fragment sequence is as shown in SEQ ID NO:33, and the B1 fragment sequence is as shown in SEQ ID NO:32, and the A fragment sequence is such as
Shown in SEQ ID NO:31, the C1 fragment sequence such as SEQ ID NO:34 institute, the C2 segment as shown in SEQ ID NO:35,
The tctp gene whole body, which strikes, subtracts targeting vector LRJ-072 sequence as shown in SEQ ID NO:36,
The B1 segment includes positive screening element, wherein the positive screening element includes sequentially connected 5 ' recombinase identification sequence
It arranges the positive riddled basins box -3 ' of 1- reporter gene box-and recombinates enzyme recognition sequence 1.
4. tctp gene whole body according to claim 3 strikes the non-human mammal targeting vector subtracted, which is characterized in that
The B1 segment includes from 5 ' to 5 ' recombination enzyme recognition sequence 1, positive screening elements, ' recombination enzyme recognition sequence of 3 ' connections
2, resistance screening box gene, 3 recombination enzyme recognition sequences 1,5- ' recombination enzyme recognition sequence 2, outside T non-human mammal tctp gene
Show sub- 3-4, the B1 segment 5 ' and 3 ' be ScaI and EcoV restriction enzyme site respectively；
The B2 segment includes from 5 ' to 3- ' recombination enzyme recognition sequence 2 (3 '-lox P) non-human mammal TCTP of 3 ' connections
Gene extron 5-6,3 ' homology arms；The B12 segment 5 ' and 3 ' be ScaI and EcoV restriction enzyme site respectively,；
The A segment includes from 5 ' to 3 ' connections, 5 ' homology arms, non-human mammal tctp gene exons 1-2, the A
Segment 5 ' and 3 ' be EcoI and ScaI and restriction enzyme site respectively,
5 the 5 of the C2 segment ' and 3 ' are SnaBI and EcoV restriction enzyme site respectively, ' of the C1 segment and 3 ' are EcoV respectively
With EcoI restriction enzyme site, the C1 and C2 fusion segment include negative selection marker gene,
The 5 of the positive screening element ' are connect with the downstream introne of the exon 2 of the non-human mammal tctp gene；Institute
State 3 ends of positive screening element with described 5 ' recombination enzyme sequence 2 is connect, 3 ' in the non-human mammal tctp gene are homologous
Negative selection marker gene is connected on the outside of arm.
5. the tctp gene whole body according to claim 3 based on recombinase identification strikes the non-human mammal target practice subtracted and carries
Body, which is characterized in that the reporter gene tape has a composing type shearing receptor, can mediate the reporter gene mRNA's
Shearing is forced, so that the reporter gene is expressed under the driving of target practice gene promoter to tctp gene described in tracer
Expression；The transcription of this target practice gene can be terminated in advance comprising polyadenylic acid (Poly A) in the reporter gene box, to strike
Except tctp gene；
' and 3 ' be respectively 5 ' recombination enzyme recognition sequences 1 for the 5 of the reporter gene box and the antibiotics resistance gene box dyad
' recombination enzyme recognition sequence 1, the reporter gene box and antibiotics resistance gene box centre are recombination enzyme recognition sequence with 3
2, the recombination enzyme recognition sequence 1 can be identified by recombinase, to realize the reporter gene box and the antibiotic resistance
The deletion of box gene.
6. the tctp gene whole body based on recombinase identification described in claim any one of 3-5, which strikes to subtract, plays non-human mammal target practice
The construction method of carrier, which is characterized in that comprising steps of
(1) it is template from normal non-human mammal separation genomic DNA, expands the segment A, the segment with PCR method
B1, the segment B2 and segment C；
(2) by digestion identification, be sequenced correct B1 or B2 segment and A segment is consecutively connected to positive screening starting vector 1 and obtains
Intermediate vector；Meanwhile correct C1, C2 segment composition will be sequenced to be connected to negative selection base after C segment by Overlap PCR
Because starting vector 2 obtains negative selection gene starting vector 2-TCTP-C；
(3) correct intermediate vector 1-TCTP-B1-B2-A will be connected, cut out with restriction enzyme and recycles recycling purpose piece
Section A-NeoR-B, BAC bacterium of the conversion containing pBCTG are recombinated to obtain TCTP-NeoR BAC；Bacterium colony PCR detects TCTP-NeoR
BAC, identification amplified band are correct；
(4) linearization process of negative selection gene starting vector 2-TCTP-C, i.e. digestion handle negative selection gene starting vector 2-
TCTP-C, recycles purpose band negative selection gene-TCTP-C, and electricity turns to have recombinated the BAC bacterium of NeoR；Negative selection gene-
TCTP-C saves TCTP-NeoR BAC, obtains the recombinant bacterium of the targeting vector conversion of B, A and the C segment containing TCTP；After rescue
The recombinant bacterium of the targeting vector-TCTP-BAC conversion of acquisition drops into row PCR Testing and appraisal, and correct recombinant clone is expanded,
Digestion verification after purified.
7. based on recombinase identification non-human mammal tctp gene whole body strike subtract non-human mammal model, its embryo,
Filial generation, tissue or cell, sperm, egg cell, fertilized eggs.
8. the non-human mammal tctp gene whole body as claimed in claim 7 based on recombinase identification, which strikes, subtracts animal model, embryo
The preparation method of tire, filial generation, tissue or cell, sperm, egg cell, fertilized eggs, which is characterized in that the method includes walking as follows
It is rapid:
(1) the described in any item TCTP targeting vectors of claim 3-5 are transferred to embryonic stem cell；
(2) embryonic stem cell screening and identification；
(3) chimeric non-human mammal is prepared；Recombination embryonic cell microinjection is entered in foster animal embryo, false pregnancy is transplanted to
In animal body, mate with intact animal；
(4) genotype identification and tctp gene strike the screening for subtracting positive chimeric animal: carrying out base to obtained chimeric animal
Because of type verifying and protein expression level verifying, screens positive tctp gene and strike the chimeric animal subtracted；
(5) the chimeric non-human mammal F1 generation heterozygote non-human mammal for rejecting reporter gene and positive selection markers is obtained；
(6) the F1 generation heterozygote non-human mammal model that breeding tctp gene whole body clpp gene subtracts.
9. method according to claim 8, which is characterized in that
The step (1) is to construct targeting vector TCTP-final vector, the targeting vector that will be built using BAC carrier
TCTP-final vector identifies that the primer that PCR is identified is that the step (2) is by targeting vector TCTP- with PCR and digestion
Final vector is gone in embryonic stem cell and is practiced shooting using electroporation, brings it about homologous recombination, by PCR and
Southern blot filters out middle target clone；Wherein the PCR of 5 ' end screening reacts primer pair are as follows:
LRJ-072-PCR-F1 (Seq ID.No.:1): cactactctaccagctgtggcac and
LRJ-072-PCR-R1 (Seq ID.No.:2): caactgaccttgggcaagaacat
It screens primer pair and includes in the 3 ' end
LRJ-072-PCR-F2 (Seq ID.No.:3): Neo-PCR-F
LRJ-072-PCR-R2 (Seq ID.No.:4): gttatggagcaaaggttacttagtgg.
In the step (3), injection blastaea is derived from C57BL/6N inbred strais non-human mammal, by superfecundation, it is natural by
It is pregnant, it is developed to blastocyst stage in embryoid body, for injecting；Pseudo-pregnant non-human's mammalian receptors uterus, false pregnancy are implanted into after injection
Receptor is the first-filial generation of C57BL/6N and CBA, gives birth to and is fitted into chimeric male mouse of the rate greater than 50% out.
The step (4) be the high chimeric male mouse of the chimeric rate that will obtain successively with Flper non-human mammal and wild type
C57BL/6N mating non-human mammals, the inhuman lactation of chimera that reporter gene and Neo resistant gene are left out in acquisition completely are dynamic
Object.
The step (5) is the chimera non-human mammal and whole body that will leave out reporter gene and Neo resistant gene completely again
Property, which is struck, to be subtracted cre breeding line mating and obtains the systemic chimera non-human mammal for striking and subtracting TCTP, and systemic strike subtracts TCTP's
Chimera non-human mammal and wild type C57BL/6N background female mice are returned, obtain it is systemic strike subtract tctp gene F1 generation it is miscellaneous
Zygote；Extracted coda gene group DNA carries out PCR reaction identification genotype, judges wild type, heterozygote or pure according to electrophoresis result
Zygote.
10. a kind of tctp gene whole body strikes the non-human mammal neural progenitor cell line subtracted, which is characterized in that use claim 3-5
Any one targeting vector transfects non-human mammalian embryo stem cell, and the middle target positive cell clone of acquisition is as inhuman
Mammal tctp gene knocks out neural progenitor cell line；Wherein, for identifying that the specific PCR of the middle target positive cell clone draws
Object includes
Probe prepares primer
LRJ-072-5'Probe-F (Seq ID.No.25) gctatctatggggaaagaatat) and
LRJ-072-5'Probe-R((Seq ID.No.26)gagcacttcatctgtcagca)；
LRJ-072-3'Probe-F (Seq ID.No.27) agctagggagcaggttagaag) and
LRJ-072-3'Probe-R((Seq ID.No.28)ctctggtgagctcatctctg)。
11. the described in any item targeting vectors of kit described in claim 1, claim 3~5, as claimed in claim 7
Tctp gene whole body based on recombinase identification strikes non-human mammal sperm, egg cell, fertilized eggs, the embryo for subtracting targeting vector
Tire, filial generation, tissue or cell, the construction method of targeting vector according to any one of claims 8, tctp gene is complete described in claim 9
Body strike the preparation method of the non-human mammal model subtracted, filial generation, embryo or cell in immunological investigation, immunotoxicology, exempt from
Application in the research of epidemic disease rejection and its mechanism, safety evaluatio, the safety evaluatio includes biogenic material
Safety evaluatio before clinical test.
12. the described in any item targeting vectors of kit described in claim 1, claim 3~5, as claimed in claim 7
Tctp gene whole body based on recombinase identification strikes non-human mammal sperm, egg cell, fertilized eggs, the embryo of the targeting vector subtracted
Tire, filial generation, tissue or cell, the construction method of targeting vector according to any one of claims 8, tctp gene is complete described in claim 9
Body strike the preparation method of the non-human mammal model subtracted, filial generation, embryo or cell in terms of pharmacodynamic evaluation purposes,
Purposes, the purposes in the medicine preparation for the treatment of disease for making cancer pathology model.
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