CN101948860A - Vector of uPA expression regulated by Tet-on system and application thereof - Google Patents

Abstract

The invention discloses an effect vector of uPA expression regulated by a Tet-on system. The nucleotide sequence of the effect vector comprises a bacteria tetracycline drug-resistant operon sequence, an immediate early promoter sequence of macrophages, a mice pro-urokinase activator expression cassette sequence and a sequence behind the last exon of the mice pro-urokinase activator expression cassette, the four sequences are serially connected in turn. The invention establishes a transgenic mice model of uPA expression regulated by the Tet-on system or tetracycline derivative inducible expression system, and lays solid foundation for further establishing a transgenic animal model of tissue specificity regulatable expression uPA for uPA function research in specific tissues.

Description

The carrier and the application thereof that expressed by tsiklomitsin abduction delivering system regulation uPA

Technical field

The present invention relates to a kind ofly be subjected to the carrier that tsiklomitsin abduction delivering system regulation uPA expresses and set up application in the transgene mouse model that tsiklomitsin abduction delivering system regulation uPA expresses.

Background technology

UPA (urokinase-plasminogen activator), promptly urokinase type plasminogen activator belongs to the serine proteinase enzyme family, and uPA catalysis Profibrinolysin is converted into plasmin, starts scleroproein and extracellular matrix protein dissolving cascade reaction.In general, uPA mainly participates in processes such as the differentiation, migration, tissue reconstruction, extracellular matrix degradation of cell, tumor-infiltrated and transfer under physiology, pathological conditions.The main component of uPA fibrinolytic approach comprises uPA, uPA acceptor (urokinase plasminogen activator receptor, uPAR) and I type inhibitor (plasminogen activator inhibitor-1, PAI-1) (Nicholl SM, Roztocil E, Davies MG.Plasminogen activator system and vascular disease.Curr Vasc Pharmacol 2006; 4:101-116).Discover, the uPA fibrinolytic is by way of not only relevant with the fibrosis pathogenesis, and (the Mondino A that in the formation of inherency immunity and acquired immune system, plays a role, Blasi F.uPA and uPAR in fibrinolysis, immunity and pathology.Trends in Immunology, 2004,25 (8): 450-455).In addition, uPA plays an important role in tissue injury and repair process, play a significant role in the liver reparation as uPA, but its crossing in transgenic mouse is expressed the severe liver injury that but causes newborn rat.

The Tet-On system is one of eukaryote foreign gene abduction delivering system of comparative maturity, it utilizes in the intestinal bacteria Tn10 transposon tetracycline operator principle to implement foreign gene and quantitatively and specifically expresses in eukaryotic cell, have characteristics such as efficient, nontoxic, that ON/OFF is tighter, successfully be applied to the research of cell and transgenic mice abduction delivering foreign gene.

Tetracycline operator (tet operon) abduction delivering system utilizes checking of bacterium tetracycline operator and derepression, realize the expression of foreign gene in eukaryotic cell, because this system regulation element is from prokaryotic organism, can be not influential to eukaryotic genetic expression, have advantages such as efficient and easy to operate.The Tet-On system is made up of two portions: 1. Tet reactive component (tetracycline responsive element, TRE), form by the operator gene sequence (TetO) of the 7 bacterium tetracycline resistant operons that copy and upright promotor morning (PminCMV) of human scavenger cell; 2. the transcriptional activators rtTA of Rong Heing (reverse tet-controlled transcriptional activator), be to merge a fusion rotein that forms by tsiklomitsin repressor TetR of bacterium and the virion protein VP16 transcription activating protein of human simple simplexvirus (HSV), can be induced and activate by doxycycline (Dox), and combine with TetO, by VP16 performance transcriptional activity.The interactional specificity of the existing tsiklomitsin repressor-operon of this system has the activation potential of VP16 transcriptional activators again, and they can make the expression of goal gene improve about 10 in cultured cell in vitro and transgenic mice ⁵(Urlinger S doubly, Baron U, Thellmann M, et al.Exploring the sequence space for tetracycline-dependent transcriptional activators:novel mutations yield expanded range and sensitivity[J] .Pro Natl Acad Sci USA, 2000,97:7963-7968).

Summary of the invention

The purpose of this invention is to provide a kind of effector plasmid and application thereof that expressed by tsiklomitsin abduction delivering system regulation uPA.

The effector plasmid that expressed by tsiklomitsin abduction delivering system regulation uPA provided by the present invention, its nucleotide sequence comprise the sequence behind upright morning of promoter sequence, mouse retention prokinase activator expression cassette and last exon of mouse retention prokinase activator expression cassette of bacterium tetracycline resistant operon and human scavenger cell; The nucleotides sequence of described mouse retention prokinase activator expression cassette is classified as from GENBANK number (Accession.Version) and is 5 of NM_008873.3 ' end 104-1405 position nucleotide sequence; The nucleotides sequence of the sequence of the 711bp behind described last exon of mouse retention prokinase activator expression cassette (the 11st exon) is classified as from GENBANK number (Accession.Version) and is 5 of NM_008873.3 ' end 1363-2074 position nucleotide sequence, and described bacterium tetracycline resistant operon sequence is 5 of sequence 1 ' end 7-300 position nucleotide sequence; Upright morning of the promoter sequence of described human scavenger cell is 5 of sequence 1 ' end 301-438 position nucleotide sequence.

It is described that to be subjected to the plasmid that sets out of the effector plasmid that tsiklomitsin abduction delivering system regulation uPA expresses be pTRE ₂Plasmid.The described nucleotides sequence of the effector plasmid of tsiklomitsin abduction delivering system regulation uPA expression that is subjected to is classified sequence 1 in the sequence table as.

Above-mentioned carrier can be subjected to the rtTA abduction delivering in the tsiklomitsin abduction delivering system, above-mentioned carrier changes over to can make up the transgenic animal model that obtains being subjected to tsiklomitsin abduction delivering system regulation uPA to express in the animal, this transgenic animal model forms also to have made things convenient for as the animal model hybridization of animal model material and the specific expressed rtTA of different tissues and is subjected to the tsiklomitsin or derivatives thereof to induce structure at the animal model of different tissues expression uPA.

Above-mentioned carrier and the carrier for expression of eukaryon of expressing rtTA can be formed to be used to make up and be subjected to tsiklomitsin or tetracycline derivant to induce the combination carrier of the transgene mouse model that uPA expresses.

Above-mentioned carrier follows the carrier for expression of eukaryon of tissue specific expression rtTA to change over to jointly can be by tsiklomitsin or the specific expressed uPA of tetracycline derivant induced tissue in the animal.

In one embodiment of the invention, the carrier for expression of eukaryon of described expression rtTA is the carrier for expression of eukaryon of hepatic tissue specific expression rtTA.

The carrier for expression of eukaryon of described hepatic tissue specific expression rtTA, its nucleotide sequence comprise mice serum albumin enhancer sequence, mice serum albumin promoter sequence and the rtTA gene order of polyphone successively; The nucleotides sequence of described mice serum albumin enhanser is classified as from GENBANK number (Accession.Version) and is 5 of AC140220.4 ' end 104540-106528 position nucleotide sequence; The nucleotides sequence of described mice serum albumin promoter is classified as from GENBANK number (Accession.Version) and is 5 of AC140220.4 ' end 115631-115832 position nucleotide sequence; The nucleotides sequence of described rtTA gene is classified as from GENBANK number (Accession.Version) and is 5 of U89930.1 ' end 774-1781 position nucleotide sequence.

The plasmid that sets out of the carrier for expression of eukaryon of described hepatic tissue specific expression rtTA is the pTet-on plasmid.

The nucleotides sequence of the carrier for expression of eukaryon of described hepatic tissue specific expression rtTA is classified sequence 2 in the sequence table as.

Above-mentioned carrier and combination carrier are subjected to tsiklomitsin or tetracycline derivant to induce the application in the transgene mouse model of uPA expression also to belong to the scope of protection of the invention at structure.

In the described application, the transgene mouse model that described structure is subjected to tsiklomitsin or tetracycline derivant to induce uPA to express comprises the steps:

1) any described carrier among the claim 1-3 is imported the mouse screening and obtain transgenic mice;

2) carrier for expression of eukaryon with any described expression rtTA among the claim 4-8 imports mouse screening acquisition transgenic mice;

3) with step 1) and step 2) the mouse hybridization that obtains, screening changes the transgenic mice that obtains the carrier of any described carrier and described expression rtTA among the claim 1-3 over to, promptly obtains the transgene mouse model that is subjected to tsiklomitsin or tetracycline derivant to induce uPA to express;

Described tetracycline derivant can be doxycycline.

The present invention is subjected to tsiklomitsin abduction delivering system regulation to express the effector plasmid of uPA by structure, made up the transgene mouse model of tsiklomitsin abduction delivering system regulation expression uPA, this transgenic mice can obtain the transgenic mice of tissue specific expression uPA with the transgenic animal hybridization of tissue specific expression rtTA, thereby make things convenient for the structure of the animal model of the specific expressed uPA of different tissues, for making up tissue specificity controllable express uPA transgenic animal model, be used for particular organization's uPA functional study and established solid foundation.

Description of drawings

Fig. 1 is pTRE ₂-uPAcDNA plasmid BamH I/Sal I double digestion is identified

Fig. 2 is pTRE ₂-uPAcDNA-700 plasmid Hind III/Sma I I double digestion is identified

Fig. 3 is TRE ₂-uPA-711 transgene carrier element and transgenic animal primers designed Design Mode figure

Fig. 4 is that the mRNA level of uPA vivoexpression is identified

Fig. 5 is that the mRNA level and the biological activity of albumen of uPA vivoexpression identified

Fig. 6 identifies for tetO-uPA transgenosis head builds mouse PCR

Fig. 7 builds mouse primers designed design diagram for the Albumin:rtTA transgenosis head of hepatic tissue specific expression rtTA

Fig. 8 builds mouse PCR qualification result for the Albumin:rtTA transgenosis head of hepatic tissue specific expression rtTA

Fig. 9 is that the transgenic positive mouse different tissues rtTA and the GAPDH mRNA RT-PCR of No. 234 hepatic tissue specific expression rtTAs detects

Figure 10 is that negative mouse different tissues rtTA of the transgenosis of No. 232 hepatic tissue specific expression rtTAs and GAPDH mRNA RT-PCR detect

Figure 11 is that the transgenic positive mouse different tissues rtTA and the GAPDH mRNA RT-PCR of No. 222 hepatic tissue specific expression rtTAs detects

Figure 12 is that negative mouse different tissues rtTA of the transgenosis of No. 223 hepatic tissue specific expression rtTAs and GAPDH mRNA RT-PCR detect

Figure 13 is that the transgenic positive mouse different tissues rtTA and the GAPDH albumen Western blot of No. 234 hepatic tissue specific expression rtTAs detects

Figure 14 is that the transgenic positive mouse different tissues rtTA and the GAPDH albumen Western blot of No. 222 hepatic tissue specific expression rtTAs detects

Figure 15 is that negative mouse different tissues rtTA of the transgenosis of No. 232 and No. 223 hepatic tissue specific expression rtTAs and GAPDH albumen Western blot detect

Figure 16 detects for inducing the specific expressed uPA transgenic mice of hepatic tissue hepatic tissue to express the uPA immunohistochemical methods

The specific expressed uPA transgenic mice of hepatic tissue hepatocellular disease is of science to be detected Figure 17 in order inducing

Figure 18 for specificity induce hepatic tissue to express uPA after the transgenic mice Serum ALT levels detect

Embodiment

Method among the following embodiment if no special instructions, is ordinary method.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

Experiment material used among the following embodiment is as follows:

Huh7 clone is available from Wuhan Boster Biological Technology Co., Ltd., and foetal calf serum (FCS) is available from Gibco company, and Hepes, two resisting, glutamine etc. are all available from Clontech company.DNA extraction test kit, plasmid extraction kit, PCR purification kit are available from Promega company; RNeasy Mini Kit is available from Qiagen company; Lipofectamine box, Trizol reagent are the Invitrogen product.E.coli DH5 α competent cells and DNA standard Marker are available from Beijing Bo Maide Development Co., Ltd; QIAprep Spin Miniprep Kit and Gel Extraction Mini Kit are available from Qiagen company; T4 DNA polymerase, Pyrobest DNA Polymerase, T4 DNA Ligase, Takara RNA LA PCR Kit (AMV) Ver1.1, DNA Marker are available from Takara company.Proteinase inhibitor Aprotinin, Pepstatin A, Leupeptin, PMSF are all available from Amresco company; Restriction enzyme is available from NEB company and Takara company; It is synthetic that primer is given birth to the worker by Shanghai, and the anti-mouse uPA of rabbit monoclonal antibody is available from American diagnostica Inc, and Hematorylin, Yihong are available from Sigma company.Zymoplasm (Thrombin, T) and Fibrinogen (Fibrinogen is F) available from national biological product calibrating institute.The detection of ALT employing Fuji DRI-CHEM 7000 Biochemical Analyzers (FUJIFILM Corporation, Tokyo, Japan).

Te-on abduction delivering system (Cat.No.631108) is available from Clontech company; Anti-GAPDH polyclonal antibody (Cat.No.G9545) is available from Sigma company; DAB colour developing liquid, HRP mark goat anti-rabbit igg are available from company of middle China fir Golden Bridge; Wild-type C57BL/6J mouse is available from Military Medical Science Institute's Experimental Animal Center.Tsiklomitsin inducibility carrier for expression of eukaryon pTet-on and pTRE ₂(title or be pTRE2) plasmid is available from Clontech company.The microinjection of transgenic animal carrier DNA is finished by Shanghai model animals company limited.

Embodiment 1, transgene carrier pTRE ₂-uPA-711 vector construction and evaluation:

One, transgene carrier pTRE ₂-uPA-711 vector construction

Molecular cloning method extracts the total RNA of wild-type C57BL/6J mouse nephridial tissue routinely, reverse transcription PCR amplification mouse uPAcDNA coding region sequence 1302bp.Primer is uPAcDNA-F, uPAcDNA-R.UPAcDNA-F:cg GgatccAtgaaagtctggctggcgagcctg (introducing the BamH I);

UPAcDNA-R:cg GtcgacCatcagaaggccagacctttctcttc (introducing the Sal I).

The reverse transcription condition is:

PCR condition: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 30 seconds, 58 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute and 20 seconds, and 2-4 goes on foot 30 circulations; 72 ℃ 10 minutes, 4 ℃ 5 minutes.

Be cloned into pTRE ₂Carrier obtains recombinant vectors, this recombinant vectors is carried out enzyme is cut and sequence verification, will the correct recombinant vectors called after TRE of checking ₂-uPAcDNA.TRE ₂The BamH I of-uPAcDNA/Sal I double digestion is identified as shown in Figure 1.Swimming lane 1 is Marker2000 (Takara), and swimming lane 2 is BamH I/Sal I double digestion result, the visible uPAcDNA sequence of inserting.

Routinely molecular cloning method extract wild-type C57BL/6J mouse gene group DNA and behind last exons coding district of template pcr amplification uPA the 711bp sequence, for being NM_008873 1363-2074 position nucleotide sequence from GENBANK number (Accession.Version).Primer is uPA3 '-F2, uPA3 '-R2.

UPA3 '-F2 cg GtcgacGccctcaggtagctgagggaag (introducing the Sal I)

UPA3 '-R2 cg GtcgacGtgaaaccgacatttagtgctagtc (introducing the Sal I)

The PCR condition is: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute and 10 seconds, and 2-4 goes on foot 30 circulations; 72 ℃ 10 minutes, 4 ℃ 5 minutes.

After identifying the PCR product correctly, cut the PCR product and be connected into the TRE that cuts with Sal I enzyme with Sal I enzyme ₂3 of uPAcDNA ' end among the-uPAcDNA, Hind III/Sma I double digestion is judged direction of insertion, after cutting, clone's enzyme that forward connects to produce 158bp, 1027bp, 966bp, 4 bar segment of 3594bp, gel electrophoresis is visible 3 bands (Fig. 2) approximately, swimming lane 1 is Marker1kb (Takara), and swimming lane 2 is Hind III/Sma I double digestion result.Clone's called after pTRE with the forward connection ₂-uPA-711 selects this plasmid to carry out the transgenic animal preparation.PTRE ₂The structural representation of-uPA-711 as shown in Figure 3, as shown in Figure 3, pTRE ₂Comprise the sequence (the 1775-2485 position of sequence 1 in the sequence table) behind upright promoter sequence morning (the 301-438 position of sequence 1 in the sequence table), uPA cDNA (the 439-1774 position of sequence 1 in the sequence table), uPA the 11st exon of the bacterium tetracycline resistant operon of connecting successively (the 7-300 position of sequence 1 in the sequence table), human scavenger cell among the-uPA-711.

Two, cell levels is identified expression and the biologic activity thereof of transgene carrier uPA

With pTRE ₂-uPA-711 and Tet-on cotransfection Huh7 clone, Dox induces (1ug/ml), induce back 48 hours collecting cell and culture supernatant thereof respectively, RT-PCR identifies the expression (the inductive cell should amplify the purpose band of 316bp) of uPA, amplimer is: RT-uPA-F:cttcgtctgtagaccaacaag, RT-uPA-R:gtgcaagatgagctgctccac; Amplification such as Fig. 4: A and B.A is the detected result of uPA RT-PCR in the cell under the different condition among Fig. 4, and swimming lane 1 is Marker2000 (Takara), and swimming lane 2 is a template for inductive experimental group cell RNA with the Tet-on cotransfection but not; Swimming lane 3 is for being template with Tet-on cotransfection and inductive experimental group cell RNA; Swimming lane 4 is for being template PCR result with the total RNA of mouse nephridial tissue.B is the RT-PCR result with the corresponding internal reference GAPDH of figure A among Fig. 4.The amplimer of internal reference GAPDH is GAPDH-F:5 '-TTCACCACCATGGAGAAGGC-3 '; GAPDH-R:5 '-CCTCAGTGTAGCCCAAGATGC-3 '.This experiment shows, pTRE ₂-uPA-711 and Tet-on cotransfection Huh7 clone can detect the uPA rna level and express after Dox induces, promptly external Dox induces and realized that uPA mRNA expresses.

The solusphere method is identified the biologic activity of uPA.Be summarized as follows: induce back 48 hours collecting cell culture supernatant.Add in 10ml 1% sepharose zymoplasm (Thrombin, T) and Fibrinogen (Fibrinogen, F), rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices behind the mixing solidifies the back punching, adds the cells and supernatant of collecting, and places in 37 ℃ of wet boxes and spends the night.Fig. 5 is a solusphere method qualification result, and wherein 1 is uPA mark product (0.2IU), and 2 is normal cell culture supernatant group, and 3 are the cotransfection plasmid but do not induce group, and 4 cotransfection plasmids add Dox and induce group.This experiment shows, pTRE ₂-uPA-711 and Tet-on cotransfection Huh7 clone can detect the uPA protein expression, and have biologic activity after Dox induces.

The preparation of embodiment 2, tetO-uPA transgenic mice and evaluation

One, tetO-uPA transgenosis head builds preparation and the PCR evaluation of mouse

With pTRE ₂-uPA-711 plasmid is cut with Pvu I enzyme, sepharose reclaims 5739bp linearizing fragment, (maternal is CBA mouse to carry out C57 * CBA F1 mouse fertilized egg, male parent is the C57 mouse, all available from the abundant biotech inc of Beijing China) microinjection of cell procaryotic DNA, the injection ovum is transplanted to respectively in the female mouse uterine tube of false pregnancy, the about 0.5cm mouse of transgenic mice birth back the 2nd all clips tail, extract genomic dna, respectively design a pair of primer in the transgene carrier upstream and downstream and carry out the PCR evaluation, design of primers synoptic diagram and sequence as shown in Figure 6, primer sequence is as described below:

Upstream sequence primer: (positive mouse should be increased and be obtained the fragment of 465bp)

2-up-F：gtttagtgaaccgtcagatcgcctg；

2-up-R：ctaggct?aatagcat?caggtctg。

Downstream sequence primer: (positive mouse should be increased and be obtained the fragment of 498bp)

2-down-F：ggtagcttgaggagtagagacact；

2-down-R：gacaatggttgtaacagagtag。

The PCR reaction system is as follows: 2.5 μ l, 10 * Buffer (contains MgCl ₂), 2 μ l dNTP, 0.2 μ l Taq, 0.5 μ l (10 μ mol/L) primer (2-up-F and 2-up-R, perhaps 2-down-F and 2-down-R), the genomic dna of 1.5 μ l said extracted, 17.8 μ l ddH ₂O.The PCR reaction conditions is as follows: 5 ℃ of 5min of elder generation; 4 ℃ of 30S again, 54 ℃ of 30S, 72 ℃ of 30S carry out 34 circulations; Last 72 ℃ of 7min.The positive mouse of PCR is tetO-uPA transgenosis head and builds mouse.

The result shows, 73 mouse of microinjection birth are cut tail extracting genomic dna age in week in 2-3, find 5 transgenic positive mouse (2 ♀ of 3 ♂) (part PCR qualification result is seen Fig. 6) through the PCR screening, the overbit mark is as follows: yc-074 (♀), yc-075 (♀), yc-061 (♂), yc-055 (♂), yc-054 (♂).A is a upstream sequence primer qualification result among Fig. 6, and B is a downstream sequence primer qualification result.Among Fig. 6 A and the B, swimming lane 1 is molecular weight MBI GenenRuler 100bp DNA Ladder marker, and swimming lane 2-6 is that different head build mouse, and swimming lane 7 is a normal mice, and swimming lane 8 is a blank, 9 positive contrasts.

Two, the structure of the proteic transgenic mice of hepatic tissue specific expression rtTA and evaluation thereof

1, the structure of the carrier pTet-on-albumin of hepatic tissue specific expression rtTA

1) the pTet-on plasmid changes structure

For mice serum albumin gene promotor and enhancer sequence are inserted pTet-on promotor position, to replace the CMV promotor in the pTet-on carrier, synthetic link is followed successively by SpeI, EcoR V, Spe I, Kpn I, Apa I, Not I, EcoR I to introduce proper restriction site; Concrete grammar is as described below:

Synthetic link primer, sequence is tet-on-linker F:5 '-ctaggatatcactagtggtaccgggcccgcggccgcg-3 ', tet-on-linker R:5 '-aattcgcggccgcgggcccggtaccactagtgatatc-3 ', 95 ℃ of annealing 10min in the PCR instrument with tet-on-linker F and tet-on-linker R obtain the PCR product.

The PCR product of above-mentioned acquisition is used EcoR I and Spe I double digestion equally, after reclaiming purifying, be inserted between the EcoR I and Spe I restriction enzyme site of pTet-on, obtain recombinant vectors, the recombinant vectors that obtains is identified with EcoR V/Sal I double digestion, EcoR V/BamH I double digestion, order-checking identifies that the result shows that linker has inserted in the pTet-on carrier then; The recombinant vectors called after pTet-on-link that evaluation is shown the correct link sequence that contains above-mentioned PCR acquisition.

2) structure of the carrier pTet-on-albumin plasmid of hepatic tissue specific expression rtTA:

Change in the later pTet-on carrier of structure cloning and identify that correct enhancer sequence and promoter sequence insert, make up the pTet-on-albumin carrier, concrete grammar is as described below:

Method according to molecular cloning is extracted C57BL/6J mouse (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) hepatic tissue genomic dna, as template pcr amplification mice serum albumin enhancer sequence (is 5 of AC140220.4 ' end 104540-106528 position nucleotide sequence from GENBANK number (Accession.Version)), upstream primer F 5 '-gccgagctcctgccggctagcttccttagcatg-3 ' (introducing Sac I site), downstream primer R 5 '-gggttaaggatcccaagctggag-3 ' (having BamH I site), pcr amplification obtains the fragment of 1993bp, show that through order-checking it is 5 of AC140220.4 ' end 104540-106528 position nucleotide sequence that this fragment has from GENBANK number (Accession.Version), is mice serum albumin enhancer sequence; Fragment Sac I and BamH I double digestion with the PCR acquisition, be cloned between pGEM-7ZF carrier S ac I and the BamH I restriction enzyme site, obtain recombinant vectors, this recombinant vectors carried out enzyme is cut and sequence verification, will the correct carrier called after p7ZF-Up-promoter that contains mice serum albumin enhancer sequence of checking.

With mouse C57BL/6J mouse (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) genomic dna is template pcr amplification mice serum albumin promoter sequence (is 5 of AC140220.4 ' end 115631-115832 position nucleotide sequence from GENBANK number (Accession.Version)), upstream primer F 5 '-cgggatccacagctccagatggcaaacatac-3 ' (introducing BamH I site), downstream primer R 5 '-tttgccagaggctagtggggttg-3 ', amplified production is cut with BamH I enzyme, reclaims the test kit purifying through PCR product sepharose and obtains the mice serum albumin promoter.

With p7ZF-up-promoter BamH I and Sma I double digestion, cutting glue reclaims as carrier, mice serum albumin promoter behind the purifying is connected in the carrier, obtain recombinant vectors, the recombinant vectors that obtains cut with BamH I enzyme identify and order-checking, enzyme is cut identified and check order and show correct recombinant vectors called after p7ZF-up-albumin.P7ZF-up-albumin is cut with Sac I enzyme, T4 DNA Polymerase disappears flat, Kpn I enzyme is cut, cut the fragment of glue recovery 2233bp, the fragment that reclaims is connected in the pTet-on-link carrier behind EcoR V and the Kpn I double digestion, obtain recombinant vectors, the band appearance that recombinant vectors is cut visible 5022bp, 1284bp, 2689bp with BamH I enzyme conforms to predicting the outcome; Check order in enhanser and promotor joint to recombinant vectors, the result conforms to expection.This carrier is carried out the enzyme evaluation of cutting and check order, evaluation is shown correct carrier called after pTet-on-albumin.The medium and small mouse serum albumin enhanser of the sequence of pTet-on-albumin, mice serum albumin promoter and rt TA gene (is 5 of U89930.1 ' end 774-1781 position nucleotide sequence from GENBANK number (Accession.Version)) be polyphone successively, and the sequence of this pTet-on-albumin is seen sequence 2 in the sequence table.Wherein mice serum albumin enhanser, mice serum albumin promoter and rt TA gene are respectively 115-2103 position, 2109-2310 position, 2370-3377 position nucleotide sequences in the sequence 2.

2, the live body of the albumin promoter transcriptional activity of the carrier pTet-on-albumin of hepatic tissue specific expression rtTA is identified

1) Albumin:rtTA transgenosis head builds mouse preparation and PCR evaluation

The pTet-on-albumin plasmid is cut with the XhoI enzyme, sepharose reclaims the 6034bp fragment, (maternal is CBA mouse to carry out C57 * CBA F1 mouse fertilized egg, male parent is the C57 mouse, all available from the abundant biotech inc of Beijing China) microinjection of cell procaryotic DNA, the injection ovum is transplanted to respectively in the female mouse uterine tube of false pregnancy, the about 0.5cm mouse of transgenic mice birth back the 2nd all clips tail, extract genomic dna, respectively design a pair of primer in the transgene carrier upstream and downstream and carry out the PCR evaluation, design of primers synoptic diagram and sequence as shown in Figure 7, primer sequence is as described below:

The upstream sequence primer:

1-up-F：GTGCAGCTTGGCTTGAACTCGTTC；

1-up-R：GAGTATGGTGCCTATCTAACATCTC。

The downstream sequence primer:

1-down-F：GACGCGCTAGACGAFTTCGATCTG；

1-down-R：ACCTTGCACAGATAGCGTGGTC。

The PCR reaction system is as follows: 2.5 μ l, 10 * Buffer (contains MgCl ₂), 2 μ l dNTP, 0.2 μ l Taq, 0.5 μ l (10 μ mol/L) primer (1-up-F and 1-up-R, perhaps 1-down-F and 1-down-R), the genomic dna of 1.5 μ l said extracted, 17.8 μ l ddH ₂O.

The PCR reaction conditions is as follows: 5 ℃ of 5min of elder generation; 4 ℃ of 30S again, 54 ℃ of 30S, 72 ℃ of 30S carry out 34 circulations; Last 72 ℃ of 7min.

The positive mouse of PCR is Albumin:rtTA transgenosis head and builds mouse.

The result shows, 49 mouse of microinjection birth are cut tail extracting genomic dna in 2-3 age in week, find 9 transgenic positive mouse (part PCR qualification result is seen Fig. 8) through the PCR screening, and the overbit mark is as follows: yc-063 (♀), yc-051 (♀), yc-071 (♀), yc-073 (♀), yc-065 (♀), yc-068 (♂), yc-064 (♂), yc-056 (♂), yc-059 (♂).A is a upstream sequence primer qualification result among Fig. 8, and B is a downstream sequence primer qualification result.Among A and the B, swimming lane 1-9 is that different head build mouse among Fig. 8, and swimming lane 10 is molecular weight marker, and swimming lane 11 is a normal mice, swimming lane 12 positive contrasts.

2) Albumin:rtTA transgenosis F1 is for acquisition and the evaluation of mouse

9 head that get the step 1) acquisition build mouse, be cultured to the ripe head of 8 all rheological properties and build mouse, with itself and 8 the week ages wild-type mice C57BL/6J mating, 2 age in week newborn mouse carry out the overbit mark, wherein 4 head build young 8 nests of mouse common property, yc-063 and yc-065 produce 3 nests respectively, yc-064 and yc-056 produce 1 nest respectively, cut tail during 3 ages in week and extract genomic dna by molecular cloning, PCR detects positive F1 for mouse, the same step 1) of PCR system and reaction conditions.

3, transgenic mice F1 identifies for the rtTA transcriptional level

Get different head and build F1 that mouse gives birth to for transgenic positive mouse (yc-063 and F1 that yc-065 gives birth to are for transgenic positive mouse 234 and 222) and negative mouse (the negative mouse 232 and 223 of yc-063 and F that yc-065 gives birth to 1 generation transgenosis), disconnected neck is put to death back clip brain, thymus gland, the heart, lung, kidney, intestines, spleen, different tissues such as liver a little (being no more than 30mg), in 1.5ml EP pipe, shred, total RNA extracts and uses RNeasy Mini Kit (Qiagen, Cat.No.74106), the total RNA that extracts is through Nanodrop 1000 spectrophotometric determination concentration and purity, various total tissue RNA are got 400ng, use Takara RNA LA PCR Kit (AMV) Ver1.1 to carry out reverse transcription reaction, system is: 2 μ l MgCl ₂(25mmol/L), 1 μ l, 10 * RNA PCR Buffer, 1 μ l dNTP Mixture, 0.25 μ l RNase Inhibitor, 0.5 μ l AMV Reverse Transcriptase, 0.5 μ l Random 9mers, 0.5 μ l Oligo dT-Adaptor Primer, 400ng RNA is with RNase Free dH ₂O complements to 10 μ l systems, and reaction conditions is: 30 ℃ of 10min, and 42 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min finish the back in the RT reaction and add following PCR system: 3 μ l MgCl ₂(25mmol/L), 4 μ l10 * LA PCR Buffer II, 31.75 μ l dH ₂O, 0.25 μ l Takara LA Taq, 0.5 μ l primer (10 μ mol/L, respectively with primer to rtTA-F and rtTA-R, perhaps GAPDH-F and GAPDH-R increase) reaction conditions is as follows: 94 ℃ of 2min, (94 ℃ of 30S, 56 ℃ of 30S, 72 ℃ of 30S) * and 32,72 ℃ of 10min, the RT-PCR product is identified with 1% agarose gel electrophoresis.Homogenate is manipulated 5 grades of homogenate 40S of T10 basic ULTRA-TURRAX refiner and is got final product (available from IKA company) in total RNA leaching process.Primer sequence is as follows:

rtTA-F：5`-GACGCGCTAGACGATTTCGATCTG-3`

rtTA-R：5`-ACCTTGCACAGATAGCGTGGTC-3`

GAPDH-F：5`-ACC?ACC?ATG?GAG?AAG?GCT?GC-3`

GAPDH-R：5`-CTC?AGT?GTA?GCC?CAG?GAT?GC-3`

Albumin:rtTA transgenosis F1 expresses qualification result such as Fig. 9-shown in Figure 12 for positive mouse rtTA mRNA, the result shows that No. 234 positive mouse rtTA mRNA are specific expressed at hepatic tissue, No. 222 positive mouse are except that hepatic tissue is expressed, at lung and kidney a small amount of expression is arranged also, No. 232 and No. 223 various the organizing all of negative mouse are not expressed.Left figure is transgenic mouse different tissues rtTA RT-PCR detection among Fig. 9-Figure 12, and right figure is transgenic mouse different tissues GAPDH mRNA RT-PCR and detects.The PC of left figure represents the total RNA RT-PCR result of Huh7 cell extraction behind the transient transfection pTet-on plasmid 48h among Fig. 9-Figure 12.

4, Albumin:rtTA transgenic positive mouse F1 identifies for the rtTA protein expression

F1 is for the extraction of transgenic positive mouse (yc-063 and F1 that yc-065 gives birth to are for transgenic positive mouse 234 and 222) and negative mouse (yc-063 and F1 that yc-065 gives birth to are for the negative mouse 232 and 223 of transgenosis) different tissues total protein, use the tissue protein lysate of following prescription: 10mM 1M Tris-HCl, 1% (V/V) Triton X-100,1% (V/V) sodium deoxycholate, 0.1% (V/V) 10% (g/ml) SDS, 0.4% (V/V) NP-40,150mM NaCl, 5mM EDTA, 0.2mM sodium orthovanadate, the distilled water constant volume.In the tissue protein lysate, add proteinase inhibitor Aprotinin, Pepstatin A, Leupeptin, PMSF before the extraction respectively, make its final concentration be respectively 2 μ g/ml, 0.7 μ g/ml, 0.5 μ g/ml, 1mM.

The concrete grammar that tissue protein extracts is: get and be no more than the various tissues of 30mg, add above-mentioned tissue protein lysate and 4 kinds of proteinase inhibitor, behind 5 grades of homogenate 40S of T10 basic ULTRA-TURRAX dispersion machine, ice bath 30min, every 5min shakes even 1 time, 4 ℃ of centrifugal 15min of 13000rpm, supernatant is total protein solution.

Total protein quantitatively uses Bradford determination of protein concentration test kit (green skies company, P0006), respectively get 50 μ g total protein solution, add 5 * SDS sample-loading buffer and 1/20 volume beta-mercaptoethanol, boil 10min, the centrifugal 5min of 10000rpm, supernatant are used for the proteic Western of rtTA and detect, and all the other place-80 ℃ of preservations.The rtTA Protein Detection one anti-TetR monoclonal antibody (Clontech that uses, Cat.No.631108), extent of dilution 1: 1000, the GAPDH albumen one anti-Anti-GAPDH polyclonal antibody (Sigma that uses, Cat.No.G9545), two anti-HRP mark goat anti-mouse IgG and the HRP mark goat anti-rabbit igg (companies of middle China fir Golden Bridge that use respectively, Lot.No.80259), extent of dilution 1: 5000, developing solution uses SuperSignal WestDura Trial Kit (Thermo SCIENTIFIC, Code #37071, Lot #KE132546).

Albumin:rtTA transgenosis F1 is for positive mouse rtTA protein expression qualification result such as Figure 13-shown in Figure 15, and the result shows, No. 234 and No. 222 positive mouse rtTA albumen are specific expressed at hepatic tissue, and No. 232 and No. 223 negative mouse are not then expressed respectively organizing all.Left figure is respectively transgenic positive mouse 234 and 222 different tissues rtTA Western blot detected results among Figure 13-14, and right figure is respective organization GAPDH albumen Western blot detected result among the left figure.Figure 15 is the negative mouse 232 (left figure) of transgenosis and 223 (right figure) different tissues rtTA Western blot detected result, and PC all represents Huh7 cell extraction total protein Western blot result behind the transient transfection pTet-on plasmid 48h among Figure 13-15.

Get identify rtTA albumen at the specific expressed brood F1 of hepatic tissue for the selfing of transgenic positive mouse male and female, the F2 that gives birth to be used for protecting for the transgenic positive mouse and kind breed.The transgenic mice that is used for obtaining with step 1 is hybridized.

But the preparation and the evaluation of the specific expressed uPA transgenic mice of three inducing hepatocytes

5 head that get step 1 acquisition build mouse, be cultured to the ripe head of 8 all rheological properties and build mouse, with itself and 8 the week ages wild-type mice C57BL/6J mating, 2 the week age newborn mouse carry out the overbit mark, cut tail and extract genomic dna by molecular cloning, PCR detects uPA transgenic positive F1 for mouse, the same step 1 of PCR system and reaction conditions.With 8 age in week uPA transgenic positive F1 for mouse and step 2 make up 8 age in week rtTA albumen hybridize at the specific expressed Albumin-rtTA transgenic mice of hepatic tissue, give birth to 2 the week age newborn mouse carry out the overbit mark, cut tail and extract genomic dna by molecular cloning, PCR detects the positive mouse of double transgenic, the same step 1 of tetO-uPA PCR system and reaction conditions, it is as described below that Albumin-rtTA PCR detects primer:

1-up-F：GTGCAGCTTGGCTTGAACTCGTTC；

1-up-R：GAGTATGGTGCCTATCTAACATCTC。

The PCR reaction system is as follows: 2.5 μ l, 10 * Buffer (contains MgCl ₂), 2 μ l dNTP, 0.2 μ l Taq, 0.5 μ l (10 μ mol/L) primer (1-up-F and 1-up-R), the genomic dna of 1.5 μ l said extracted, 17.8 μ lddH ₂O.

The PCR reaction conditions is as follows: 5 ℃ of 5min of elder generation; 4 ℃ of 30S again, 54 ℃ of 30S, 72 ℃ of 30S carry out 34 circulations; Last 72 ℃ of 7min.

It is the positive mouse (albumin-rtTA:TetO-uPA) of double transgenic that tetO-uPA and Albumin-rtTA PCR are the male mouse.

This experimental principle is: the Albumin-rtTA transgenic mice is at hepatic tissue specific expression rtTA albumen, the hybridization of this mouse and tetO-uPA transgenic mouse the offspring rat of giving birth to also can specific expressed rtTA albumen at hepatic tissue, when adding Dox induces, rtTA shows as activity form, combine with operator gene sequence TetO among the tetO-uPA, the performance transcriptional activity realizes that hepatic tissue uPA is specific expressed.

3, double transgenic murine liver tissue uPA expresses and identifies

Identify the expression of feeding male double transgenic mouse (albumin-rtTA:TetO-uPA) drinking-water that contains Dox (0.5mg/ml) inducing uPA, induce ten week the back collect serum and carry out the ALT level and identify; The execution mouse is also got and respectively organizes internal organs to carry out the specific expressed detection of uPA.With the drinking-water that does not add Dox is contrast.The visible uPA of experimental result utilizes the anti-mouse uPA of rabbit monoclonal antibody immunity group detection necrosis of liver tissue kitchen range to have uPA specific expressed at hepatic tissue specific expressed (Figure 16); The liver cell endochylema is loose in the histopathology inducing mouse hepatic tissue, the necrosis of hepatic tissue kitchen range shape, and Kupffer Cell increases (Figure 17).Detect the serum alt level and increase, hint that further hepatic tissue is subjected to certain damage (Figure 18).A represents the negative mouse group of double transgenic among Figure 18, and B represents the negative mouse group of single transgene, and C represents the positive mouse group of double transgenic.

Claims (10)

1. carrier, its nucleotide sequence comprise the sequence behind upright morning of promoter sequence, mouse retention prokinase activator expression cassette and last exon of mouse retention prokinase activator expression cassette of the bacterium tetracycline resistant operon sequence of polyphone successively, human scavenger cell; The nucleotides sequence of described mouse retention prokinase activator expression cassette is classified as from GENBANK number and is 5 of NM_008873.3 ' end 104-1405 position nucleotide sequence; The nucleotides sequence of the sequence behind described last exon of mouse retention prokinase activator expression cassette is classified white GENBANK number as and is 5 of NM_008873.3 ' end 1363-2074 position nucleotide sequence, and described bacterium tetracycline resistant operon sequence is 5 of sequence 1 ' end 7-300 position nucleotide sequence; Upright morning of the promoter sequence of described human scavenger cell is 5 of sequence 1 ' end 301-438 position nucleotide sequence.

2. carrier according to claim 1 is characterized in that: the plasmid that sets out that makes up described carrier is pTRE ₂Plasmid.

3. carrier according to claim 2 is characterized in that: the nucleotides sequence of described carrier is classified sequence 1 in the sequence table as.

4. be used to make up and be subjected to tsiklomitsin or tetracycline derivant to induce the combination carrier of the transgene mouse model that uPA expresses, form by the carrier for expression of eukaryon of any described carrier and tissue specific expression rtTA among the claim 1-3.

5. combination carrier according to claim 4 is characterized in that: the carrier for expression of eukaryon of tissue specific expression rtTA is the carrier for expression of eukaryon of hepatic tissue specific expression rtTA; The carrier for expression of eukaryon of described hepatic tissue specific expression rtTA, its nucleotide sequence comprise mice serum albumin enhancer sequence, mice serum albumin promoter sequence and the rtTA gene order of polyphone successively; The nucleotides sequence of described mice serum albumin enhanser is classified as from GENBANK number and is 5 of AC140220.4 ' end 104540-106528 position nucleotide sequence; The nucleotides sequence of described mice serum albumin promoter is classified as from GENBANK number and is 5 of AC140220.4 ' end 115631-115832 position nucleotide sequence; The nucleotides sequence of described rtTA gene is classified as from GENBANK number and is 5 of U89930.1 ' end 774-1781 position nucleotide sequence.

6. combination carrier according to claim 5 is characterized in that: the plasmid that sets out of the carrier for expression of eukaryon of described hepatic tissue specific expression rtTA is the pTet-on plasmid.

7. combination carrier according to claim 6 is characterized in that: the nucleotides sequence of the carrier for expression of eukaryon of described hepatic tissue specific expression rtTA is classified sequence 2 in the sequence table as.

8. any described carrier is subjected to tsiklomitsin abduction delivering system regulation to express application in the transgenic animal of uPA at structure among the claim 1-3, and described animal is preferably mouse.

Among the claim 1-3 among any described carrier or the claim 4-9 any one described one group carrier be subjected to tsiklomitsin or tetracycline derivant to induce application in the transgenic animal model that uPA expresses at structure, described animal is preferably mouse.

10. application according to claim 9 is characterized in that: the transgene mouse model that described structure is subjected to tsiklomitsin or tetracycline derivant to induce uPA to express comprises the steps:

1) any described carrier among the claim 1-3 is imported the mouse screening and obtain transgenic mice;

2) carrier for expression of eukaryon with any described expression rtTA among the claim 4-8 imports mouse screening acquisition transgenic mice;

3) with step 1) and step 2) the mouse hybridization that obtains, screening changes the transgenic mice that obtains the carrier of any described carrier and described expression rtTA among the claim 1-3 over to, promptly obtains the transgene mouse model that is subjected to tsiklomitsin or tetracycline derivant to induce uPA to express;

Described tetracycline derivant is a doxycycline.

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