CN103045606B - K14-VEGF transgenic mouse model, and construction method and application of model - Google Patents
K14-VEGF transgenic mouse model, and construction method and application of model Download PDFInfo
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Abstract
The invention belongs to the technical field of animal models, and provides a K14-VEGF (vascular endothelial growth factor) transgenic mouse model and a construction method thereof. The construction method comprises the steps that a human keratin 14 promoter and a mouse coding gene VEGF-A164 are inserted into an FVB mouse genome by a pronucleus microinjection technology, and expression of the mouse VEGF-A164 is controlled by the human keratin 14 promoter to ensure that a basal layer of skin of a transgenic mouse excessively expresses the VEGF-A164. The constructed K14-VEGF transgenic animal model can generate a phenotypic characteristic of psoriasis spontaneously. The K14-VEGF transgenic mouse model further screens, purifies and constructs a system. The animal model can be applied to research on pathogenesis of the psoriasis, and screening of a drug for preventing and treating the psoriasis.
Description
Technical field
The invention belongs to animal model technical field, be specifically related to a kind of K14-VEGF transgene mouse model and construction process thereof, and application in the research of onset of psoriasis mechanism and screening prophylactic treatment psoriasis.
Background technology
The exploration of Animal Models of Psoriasis has probably been experienced following three phases with structure.First stage, the spontaneous animal model stage.In early days due to few to the disease cause of disease, pathogenetic understanding, in addition the restriction of experiment condition, the mankind are difficult to build dermopathic animal model, but occurring in nature has some animals spontaneously to occur some pathological characters of disease, and people utilize these limited resources to do scientific research.Subordinate phase, the artificial constructed animal model stage.Along with the progress of science, basic medical subject, medical science related discipline are also developed accordingly, and especially immunology and molecular biological developing rapidly have been played huge pushing effect to the foundation of skin disease animal model.Now people can build coarse and even accurate animal model according to existing knowledge and laboratory facilities.Before the eighties in 20th century, people mainly become to assign to build some more coarse models by external application, subcutaneous injection some drugs.From the eighties in 20th century, due to immunologic development, patient's grafts is become to the powerful of study portion dermatosis to the model of Immune deficient mice.Phase III, under the background of Development of Molecular Biology, transgenic animal technology is more and more improved ripe.Transgenic animal almost relate to the every field of skin disease animal model.People even can accurately study the effect of certain factor in disease generating process.Psoriatic is dermatology common disease, and psoriatic animal model is also the emphasis of research.
Transgenic animal refer to the animal that is integrated with foreign gene in genome.People can use transgenic technology that foreign gene is imported to somatocyte, thus the animal strain that cultivation makes new advances or the animal model for specializing in.Nineteen ninety, Rothnagel takes the lead in proposing specific gene to be expressed in animal body by transgenic method, and provides valuable animal model for the mankind have the dermatosis of genetic predisposition.For example; some cytokine and somatomedin can be by turning because of means overexpression in animal body; thereby produce psoriasiform reaction (referring to document: Targeting Gene Expression to the Epidermis of Transgenic Mice:Potential Applications to Genetic Skin Disorders.Journal of InvestigativeDermatology (1990) 95,59S – 61S; Doi:10.1111/1523-1747.ep12505805)
Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a kind of vascular endothelial cell specificity mitogen of being secreted by angle albuminous cell.In Various Tissues, promote blood vessel hyperplasia; In psoriatic lesion, blood vessel hyperplasia is accelerated in overexpression; increase the perviousness of blood vessel on endotheliocyte; the migration of cell causes inflammation; stimulate adhesion molecule-1(ICAM-1) and endothelial cell adhesion molecule-1(VCAM-1) expression (referring to document: Yu-Ping Xia; Baosheng Li; Donna Hylton, etc.Transgenicdelivery of VEGF to mouse skin lead to an inflammatory condition resemblinghuman psoriasis.Blood2003; 102 (1): 161-168.).
Summary of the invention
The object of the present invention is to provide a kind of K14-VEGF transgene mouse model, another object of the present invention is to provide the construction process of this transgene mouse model, and application in the research of onset of psoriasis mechanism and screening prophylactic treatment psoriasis.
Technical scheme of the present invention is, utilizing pronuclear microinjection technology will comprise human keratin 14(K14) promotor and mouse encoding gene VEGF-A164 be inserted in FVB musculus cdna group, by human keratin 14(K14) the mouse VEGF-A164 of promotor control expresses, render transgenic mouse basal layer of epidermis overexpression VEGF-A164.
A first aspect of the present invention, provides a kind of construction process of K14-VEGF transgene mouse model, and this construction process comprises the following steps:
A, pcr amplification attB1-mVEGF164-attB2(are as shown in SEQ ID NO:1) and attB2r-hGH-attB3(as shown in SEQ ID NO:2), BP(BP recombination) reaction builds intermediate carrier pDown-mVEGF164 and pTail-hGH;
B, utilize Gateway Technology(Gateway clone technology) pUp-K14, pDown-mVEGF164, pTail-hGH are carried out to LR with conventional plasmid pRP.Des3d react (LR recombination), build pRP.EX3d-K14>mVEGF164>hGH;
C, positive colony order-checking are reclaimed respective segments after confirming after linearizing, zygote male pronucleus is carried out to microinjection, prepare transgenic mice and progressively breed purifying.
The construction process of described a kind of K14-VEGF transgene mouse model, concrete steps are as follows:
I) structure of K14-VEGF164 transgenic fragment
I) structure of pRP.EX3d-K14>mVEGF164>hGH carrier
II) pcr amplification attB1-mVEGF164-attB2 and attB2r-hGH-attB3
Design and synthesize primer as follows:
III) BP reaction (BP recombination) structure intermediate carrier pDown-mVEGF164 and pTail-hGH
BP recombining reaction adds attB site by PCR method at goal gene two ends, the PCR product that comprises attB site and donor carrier pDONR
tMcarry out BP reaction, goal gene is building up in donor carrier, in reaction process, contain the donor carrier pDONR in attP site
tMand there is sequence exchange in the att site between the PCR product that contains attB site, after attB and the restructuring of attP site, cause the attP site sequence on the donor carrier that contains aim sequence after restructuring to produce variation, attP site becomes into attL site.
Donor carrier after restructuring is commonly referred to as entry clones (entry clone).
PUp-K14: K14 promotor is building up to entry clones by donor carrier pDONRP4P1r;
PDown-mVEGF164: donor carrier pDONR221 is that gene mVEGF164 is building up to entry clones;
PTail-hGH: with donor carrier pDONRP2rP3, another gene hGH is building up to entry clones in the time expressing two genes in same expression vector;
IV) LR reaction structure pRP.EX3d-K14>mVEGF164>hGH
LR recombining reaction (LR recombination) is by att site sequence exchange between the entry clones that contains attL site and the object carrier that contains attR site, after attL and the restructuring of attR site, sequence exchanges, cause the attR site sequence on the object carrier that contains aim sequence after restructuring to produce variation, reassemble into attB site sequence.Such object carrier is called General Expression clone or expression vector (expressionvector).
Utilize Gateway technology that pUp-K14, pDown-mVEGF164, pTail-hGH are carried out to LR with conventional plasmid pRP.Des3d and react, build pRP.EX3d-K14>mVEGF164>hGH.
PRP.EX3d-K14>mVEGF164>hGH complete sequence is entrusted the order-checking of match industry (Guangzhou) bio tech ltd, as shown in SEQ ID NO:7.
II) PCR bacterium colony screening positive clone
The object band that the theoretical big or small 1245bp of bacterium colony PCR screening pDown-mVEGF164:PCR product band conforms to;
Bacterium colony PCR screening pTail-hGH collection of illustrative plates: the object band that screening conforms to the theoretical big or small 2012bp of PCR product band;
Bacterium colony PCR screens pRP.EX3d-K14>mVEGF164>hGH: the object band that screening conforms to the theoretical big or small 1034bp of PCR product band.
III) linearization for enzyme restriction pRP.EX3d-K14>mVEGF164>hGH
The theoretical size of fragment that Mlu I/Sal I double digestion cuts out is 5267bp and 2823bp, and after positive colony order-checking is confirmed, the large fragment reclaiming wherein after linearizing is carried out transgenic mice injection.
IV) by FVB mouse zygote male pronucleus microinjection step III) plasmid that obtains, obtain positive K14-VEGF transgene mouse model.
A second aspect of the present invention, provides a kind of K14-VEGF transgene mouse model obtaining according to above-mentioned construction process.
The K14-VEGF transgenic animal model that the present invention builds can be spontaneous generation psoriatic phenotypic characteristic, as corium capillary blood vessel hyperplasia, epidermis perviousness increases, angle albuminous cell abnormal differentiation etc.Also can under the induction of other compounds, produce rapidly psoriatic phenotypic characteristic.
K14-VEGF transgene mouse model of the present invention, further screening, purifying and build and be:
When DNA pronuclear microinjection method is made transgenic mice, the goal gene proceeding to is only just incorporated on the wherein item chromosome of diploid animal, so be hemizygote, only some individuality is with the gene of integrating for its offspring, and what have does not have a goal gene.So transgenic mice needs a quite long screening and purge process, to obtaining homozygous transgenic mice.Usual method is to make after same approving and forwarding DNA murine, by a part wherein and normal wild-type mice hybridization, detect offspring's integration probability, by with a brood of positive male and female mouse mating, under the homologous recombination effect of gene, finally obtain homozygote mouse again.Because goal gene random integration is to genome, therefore head will have different integration sites for transgenic mice.The copy number of integrator gene may be also different in for transgenic mice at different head.Due to these reasons, each head for transgenic mice need to as one independently pedigree treat and separate and breed for transgenic mice with other head.Each head needs to carry out the detection of transgenosis Inheritance and expression for the filial generation of transgenic mice.Due to different integration sites and copy number, each head can have different expression levels for transgenic mice.Can adopt the method that classical breeding combines with PCR to screen transgenic mice homozygote.To be F1 for building offspring's (not brood newborn mouse is added up respectively) that primary (F0) transgenic mice that is and wild-type FVB mouse breeding breeding produce.The offspring that in F1, positive transgenic mice sib mating produces is F2.In F2, selecting several offsprings that positive mouse sib mating is produced is F3.According to the signal of the integration rate of each nest mouse and PCR-Southern, select and suppose the mouse of isozygotying.The homozygote mouse of supposition and wild-type mice breeding are produced to F4, according to offspring's integration rate, the homozygote transgenic mice of supposition is made to checking.Can continue to go down to posterity in the hope of setting up stable transgenic mice strain.Under normal circumstances, make transgenic mice, and through after Screening and Identification, obtain homozygous transgenic mice, and preserve breeding with homozygote.
A third aspect of the present invention, is to provide the application of above-mentioned K14-VEGF transgene mouse model in the research of onset of psoriasis mechanism and screening prophylactic treatment psoriasis.
(IMQ can increase the weight of psoriatic's the state of an illness to the medicines such as such as Imiquimod (IMQ), phorbol fat (TPA), part doctor is used for the treatment of skin carcinoma now, rodent cancer etc., TPA is a kind of tumor promoter) for after transgenic mouse model, there is similar psoriatic phenotype, can be used for the research of psoriasis pathology mechanism.
Brief description of the drawings
Fig. 1 is bacterium colony PCR screening pDown-mVEGF164 collection of illustrative plates
Wherein 1: negative control
2～8:pDown-mVEGF164 1.～7. number clone
PCR primers designed: pUpDo-flank-F:CGGCCAGTCTTAAGCTCGGG
pUpDo-flank-R：AATACGACTCACTATAGGGGA
Sequencing primer: pUpDo-flank-F:CGGCCAGTCTTAAGCTCGGG
pUpDo-flank-R：AATACGACTCACTATAGGGGA
The theoretical size of PCR product band is 1245bp, and the clone who conforms to object stripe size is positive colony.
Fig. 2 is bacterium colony PCR screening pTail-hGH collection of illustrative plates
Wherein 1: negative control
2～8:pTail-hGH 1.～7. number clone
PCR primers designed: pUpDo-flank-F:CGGCCAGTCTTAAGCTCGGG
pUpDo-flank-R：AATACGACTCACTATAGGGGA
Sequencing primer: pUpDo-flank-F:CGGCCAGTCTTAAGCTCGGG
pUpDo-flank-R：AATACGACTCACTATAGGGGA
hGH-F：CCAGCTGGCATATGACACCT
The theoretical size of PCR product band is 2012bp, and the clone who conforms to object stripe size is positive colony.
Fig. 3 is bacterium colony PCR screening pRP.EX3d-K14>mVEGF164>hGH
Wherein 1: negative control
2～7:pRP.EX3d-K14>mVEGF164>hGH 1.～6. number clone
PCR primers designed: pLV/Final-internal-f:CAAGTTTGTACAAAAAAGCAGGCT
pLV/Final-internal/tail-r:CACTTTGTACAA
Sequencing primer:
pLV-Final-internal-f:CAAGTTTGTACAAAAAAGCAGGCT
pLV-Final-internal-r:AGCCTGCTTTTTTGTACAAACTTG
pFinal-internal/tail-f:CCCAGCTTTCTTGTACAAAGTG
The theoretical size of PCR product band is 1034bp, and the clone who conforms to object stripe size is positive colony.
Fig. 4 is linearization for enzyme restriction pRP.EX3d-K14>mVEGF164>hGH
Wherein M:1kb DNA ladder
1:Mlu I/Sal I double digestion plasmid pRP.EX3d-K14>mVEGF164>hGH
The theoretical size of fragment that Mlu I/Sal I double digestion cuts out is 5267bp and 2823bp, and the large fragment reclaiming is wherein carried out transgenic mice injection.
Fig. 5 is agarose gel electrophoresis result
Wherein 1-water;
2-16 & 2-8-F1 is for mouse (2-12 is the filial generation of G00 for mouse, and 13-16 & 2-8 is the filial generation of FH00 for mouse);
17-DNA maker；
18-positive；
Numbering 2,4,6,7 mouse are transgenic positive mouse, other negative mouse.
Fig. 6 is the qualification of transgenosis generation mouse
Wherein 8 is DNA maker.
Fig. 7 is Real-time the selection result
Wherein G group VEGF164 genetic expression is the highest.
Fig. 8 is Western-blot the selection result
Wherein G histone is expressed the highest.
Fig. 9 is transgenosis homozygote mouse agarose gel electrophoresis result
Wherein 1 is DNA maker,
2-10 is the filial generation of supposition homozygote mouse, positive expression.
Figure 10 is carrier structure collection of illustrative plates.
Embodiment
Describe the present invention below in conjunction with embodiment and accompanying drawing.But the following example should not regarded limitation of the scope of the invention as.
Reagent:
bP Clonase
tMiI Enzyme Mix, invitrogen,
LR Clonase
TMII Plus Enzyme Mix，invitrogen，
QIAquick Gel Extraction Kit，QIAGEN,
The little extraction reagent kit of plasmid, TIANGEN,
Taq DNA Polymerase, U.S. Fermentas,
DNTP Mix, U.S. Fermentas,
GeneRuler
tM100bp DNA Ladder, U.S. Fermentas,
PrimeSTAR
TMHS DNA Polymerase，Takara，
GeneRuler
TM1kp DNA Ladder，Fermentas，
MluI restriction enzyme, R0198L, NEB;
SalI restriction enzyme, TAKARA,
DNA extraction test kit, TIANGEN
FVB mouse (Shanghai Slac Experimental Animal Co., Ltd.)
Embodiment 1:
One, the structure of VEGF164 transgenic fragment
1.PCR amplification attB1-mVEGF164-attB2 and attB2r-hGH-attB3
Design and synthesize primer as follows:
2. utilize Gateway Technology to build pDown-mVEGF164 and pTail-hGH
1) 25 DEG C, BP reacts 1h.Reaction system: attB1-mVEGF164-attB2100ng, pDONR
tM221100ng, BP clonase1ul, TE buffer up to5 μ l.
2) 25 DEG C, BP reacts 1h.Reaction system: attB2r-hGH-attB3100ng, pDONR
tMp2R-P3100ng, BP clonase1 μ l, TE buffer up to5 μ l.
3) add 0.5ul Proteinase K more than termination after two reaction 10min, to transform BP reaction product to intestinal bacteria Stbl3 in 37 DEG C.Dissolve 1 pipe competence on ice, 2 μ LBP reactants are joined in One Shot stb13Chemicallly Competent Cells, hatch on ice 30 minutes, then under 42 DEG C of conditions, thermal shock cell was transferred to immediately and is hatched 2 minutes on ice after 90 seconds, added 300 μ L S.O.C.medium in hatching 1 hour in 37 DEG C, the shaking table of 225RPM..100 μ L conversion products are coated onto on the LB flat board that contains 50 μ g/mL Kan to 37 DEG C of overnight incubation.
3.PCR screening positive clone bacterium colony.
PCR reaction system: adopt 30ul PCR reaction system, comprising:
Aqua sterilisa 16.1 μ l,
10×Taq Buffer with(NH
4)
2SO
4 3μl，
dNTP Mixture(10μM) 3μl，
MgCl
2 2μl，
Taq DNA polymerase 1.5μl，
Template DNA 2 μ l,
Forward direction primer (10 μ M) 1.2 μ l,
Reverse primer (10uM) 1.2 μ l.
Cycling condition: denaturation: 94 DEG C, 3min; Sex change: 94 DEG C, 30S; 60 DEG C, 30S; 72 DEG C, 1min(2kb/min); 29 circulations; Finally extend: 72 DEG C, 5min.PCR reaction finishes rear picking positive colony plasmid, carries out cloning and sequencing.(Fig. 1).
4. utilize Gateway Technology to build pRP.EX3d-K14>mVEGF164>hGH(Fig. 5)
25 DEG C, LR reacts 16h, reaction system: pUp-K1416.21ng, pDown-mVEGF164 11.65ng, pTail-hGH(Fig. 2) 14.16ng, maternal carrier pRP.Des3d 15.83ng, LR clonase 1 μ l, TE buffer up to 5 μ l.Add 0.5ul Proteinase K in 37 DEG C of termination reaction 10min, transform LR reaction product to Stbl3.Utilize bacterium colony PCR screening positive clone, picking positive colony plasmid, carries out cloning and sequencing (Fig. 3).After positive colony linearization for enzyme restriction (Fig. 4), reclaim large fragment and carry out microinjection.
Two, obtain transgenosis head and build mouse
Donor zygote is taken from FVB (♀) and FVB(
) a purifying generation, by zygote male pronucleus injection target gene, the transgenic mice of preparation in this way, its Insert Fragment is random integration, the transgenic positive mouse of acquisition is built mouse, (common FVB type small white mouse) headed by.
Transgenosis head builds mouse after for some time, and compared with wild-type, ear occurs obviously to redden, thicken, blood vessel hyperplasia, and epidermis film projection, has oedema generation isophenous when serious, is significantly different from wild-type of the same age.
Three, the screening of transgenic mice, purifying and build and be
When DNA pronuclear microinjection method is made transgenic mice, the goal gene proceeding to is only just incorporated on the wherein item chromosome of diploid animal, so be hemizygote, only some individuality is with the gene of integrating for its offspring, and what have does not have a goal gene.So transgenic mice needs a quite long screening and purge process, to obtaining homozygous transgenic mice.Usual method is to make after same approving and forwarding DNA murine, by a part wherein and normal wild-type mice hybridization, detect offspring's integration probability, by with a brood of positive male and female mouse mating, under the homologous recombination effect of gene, finally obtain homozygote mouse again.Because goal gene random integration is to genome, therefore head will have different integration sites for transgenic mice.The copy number of integrator gene may be also different in for transgenic mice at different head.Due to these reasons, each head for transgenic mice need to as one independently pedigree treat and separate and breed for transgenic mice with other head.Each head needs to carry out the detection of transgenosis Inheritance and expression for the filial generation of transgenic mice.Due to different integration sites and copy number, each head can have different expression levels for transgenic mice.Can adopt the method that classical breeding combines with PCR to screen transgenic mice homozygote.To be F1 for building offspring's (not brood newborn mouse is added up respectively) that primary (F0) transgenic mice that is and wild-type FVB mouse breeding breeding produce.The offspring that in F1, positive transgenic mice sib mating produces is F2.In F2, selecting several offsprings that positive mouse sib mating is produced is F3.According to the signal of the integration rate of each nest mouse and PCR-Southern, select and suppose the mouse of isozygotying.The homozygote mouse of supposition and wild-type mice breeding are produced to F4, according to offspring's integration rate, the homozygote transgenic mice of supposition is made to checking.Can continue to go down to posterity in the hope of setting up stable transgenic mice strain.Under normal circumstances, make transgenic mice, and through after Screening and Identification, obtain homozygous transgenic mice, and preserve breeding with homozygote.
The vivoexpression qualification of embodiment 2 transgenic fragments
In the PCR of 20ul reaction system, add two pairs of primers, after pcr amplification reaction, as shown in Figure 5, No. 2, No. 4, No. 6, No. 7 is transgenic positive to 1% agarose gel electrophoresis result, other are transgenosis feminine gender.
Embodiment 3: the expression identification of transgenosis F1 generation mouse VEGF164 gene
Choose above transgenic mouse to be measured in 4 week age, cut mouse toe or one section of mouse tail and manage to EP, require to extract DNA according to DNA extraction test kit, the two primers of PCR are identified.
1) PCR primer:
Goal gene primer
Forward direction primer: GAC CCA AGA GAA CTC ACC GTA;
Reverse primer: GAG TAG CAC CTT CCA CGA CCA;
Internal reference primer
Forward direction primer: CAC TGG CTG AGG AAG GAG AC;
Reverse primer: TCT TAG CTC TGC TCT CCG GT;
2) PCR reaction system: adopt 20ulPCR reaction system, comprising:
Premix Taq
TM 10ul，
DNA template 1ul，
Forward direction primer 0.6ul,
Reverse primer 0.6ul,
Internal reference forward direction primer 0.3ul,
Reverse primer 0.3ul,
Distilled water 7.2ul;
3) PCR reaction conditions: denaturation: 94 DEG C, 3min; Sex change: 94 DEG C, 30S; 59 DEG C, 30S; 72 DEG C, 35S; 35 circulations; Finally extend: 12 DEG C;
4) two primer qualifications, wherein have goal gene and internal reference 2 bands, and wherein internal reference band is mainly used in monitoring the mouse genome quality of extracting, and result as shown in Figure 6.
Embodiment 4: the each pedigree VEGF164 genetic expression qualification of transgenic mouse
The each pedigree of transgenosis (A, B, D, E, F, G, H) mouse is after going down to posterity, choose immediately the filial generation of each pedigree mouse, clip mouse ear, extracting RNA, in conjunction with Real-time and Western-blot qualification result, filter out the G pedigree that VEGF expression is higher and carry out purifying (w is for blank), result as Figure 7-8.
Embodiment 5:
The homozygote mouse of supposition and wild-type mice breeding are produced to F4, according to offspring's integration rate, the homozygote transgenic mice of supposition is made to checking, if offspring mouse qualification result is entirely positive, this mouse is homozygote, and result as shown in Figure 9.
The psoriatic phenotypic characteristic that embodiment 6:K14-VEGF transgenic animal model produces
The present invention build K14-VEGF transgenic animal model can be spontaneous generation psoriatic phenotypic characteristic, as: epidermis thickens, perviousness increases, corium capillary blood vessel hyperplasia, inflammatory cell infiltration, angle albuminous cell abnormal differentiation, vegf expression increases, and key cytokines is expressed to be increased, Deng, without T cellular infiltration.Can produce rapidly psoriatic phenotypic characteristic under as the induction of TPA, IMQ at other compounds, and occur T cellular infiltration.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (3)
1. the construction process of a K14-VEGF transgene mouse model, it is characterized in that, the method is to utilize pronuclear microinjection technology will comprise human keratin 14 promotors and mouse encoding gene VEGF-A164 is inserted in FVB musculus cdna group, mouse VEGF-A164 by human keratin 14 promotor controls expresses, render transgenic mouse basal layer of epidermis overexpression VEGF-A164, the method comprises the following steps:
A, the attB1-mVEGF164-attB2 of pcr amplification as shown in SEQ ID NO:1 and the attB2r-hGH-attB3 as shown in SEQ IDNO:2, BP reaction builds intermediate carrier pDown-mVEGF164 and pTail-hGH;
B, utilize Gateway clone technology that pUp-K14, pDown-mVEGF164, pTail-hGH are carried out to LR with conventional plasmid pRP.Des3d to react, build pRP.EX3d-K14>mVEGF164>hGH, complete sequence is as shown in SEQ ID NO:7;
C, positive colony order-checking are reclaimed respective segments after confirming after linearizing, zygote male pronucleus is carried out to microinjection, prepare transgenic mice and progressively breed purifying.
2. the construction process of a kind of K14-VEGF transgene mouse model according to claim 1, is characterized in that, the method concrete steps are as follows:
I) structure of pRP.EX3d-K14>mVEGF164>hGH carrier
I) to design and synthesize primer as follows for pcr amplification attB1-mVEGF164-attB2 and attB2r-hGH-attB3:
II) BP reaction structure intermediate carrier pDown-mVEGF164 and pTail-hGH
III) LR reaction structure pRP.EX3d-K14>mVEGF164>hGH
IV) PCR bacterium colony screening positive clone
The object band that the theoretical big or small 1245bp of bacterium colony PCR screening pDown-mVEGF164:PCR product band conforms to;
Bacterium colony PCR screening pTail-hGH collection of illustrative plates: the object band that screening conforms to the theoretical big or small 2012bp of PCR product band;
Bacterium colony PCR screens pRP.EX3d-K14>mVEGF164>hGH: the object band that screening conforms to the theoretical big or small 1034bp of PCR product band;
II) linearization for enzyme restriction pRP.EX3d-K14>mVEGF164>hGH
The theoretical size of fragment that Mlu I/Sal I double digestion cuts out is 5267bp and 2823bp, and after positive colony order-checking is confirmed, the large fragment reclaiming wherein after linearizing is carried out transgenic mice injection;
III) by FVB mouse zygote male pronucleus microinjection step II) plasmid that obtains, obtain positive K14-VEGF transgene mouse model.
3. the application of the mouse model that the construction process of a K14-VEGF transgene mouse model as claimed in claim 1 or 2 obtains in screening prevention or treatment psoriasis.
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| Title |
|---|
| H Hvid等.TPA induction leads to a Th17-like response in transgenic K14/VEGF mice: a novel in vivo screening model of psoriasis.《Jpn Soc Immunol》.2008,第20卷1097-1106. |
| TPA induction leads to a Th17-like response in transgenic K14/VEGF mice: a novel in vivo screening model of psoriasis;H Hvid等;《Jpn Soc Immunol》;20081231;第20卷;1097-1106 * |
| 小鼠survivin基因慢病毒表达载体的构建及鉴定;杨平等;《中国组织工程研究与临床康复》;20111231;第15 卷(第49 期);9244-9248 * |
| 杨平等.小鼠survivin基因慢病毒表达载体的构建及鉴定.《中国组织工程研究与临床康复》.2011,第15 卷(第49 期),9244-9248. |
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