CN102516396B - Induced reversible immortalized anterior odontoblast system and establishment method thereof - Google Patents

Abstract

The invention discloses an induced reversible immortalized anterior odontoblast system and an establishment method thereof. The induced reversible immortalized anterior odontoblast system introduces an immortalization gene. Two ends of the immortalization gene have LoxP sequences. The induced reversible immortalized anterior odontoblast system can express a fusion protein of a Cre enzyme and an estrogen receptor protein. The fusion protein has a large volume and thus the fusion protein cannot enter into a cell nucleus so that an LoxP locus of a genome cannot be knocked out by the fusion protein under normal conditions. After a cell is stimulated by 4-OH-Tomaxifen having certain concentration, estrogen acceptors of two ends of Cre fall off so that Cre can enter into a cell nucleus and then remove an immortalization gene coding sequence of the genome and an immortalization elimination purpose is realized. The induced reversible immortalized anterior odontoblast system provides an ideal model in vitro for further research on odontoblast biological characteristics and also provides an ideal material for regeneration and repair of dentin and research on mechanisms of diseases related to dentin development.

Description

Odontoblast cell line and establishment method thereof before induction reversibility immortalization

technical field:

The present invention relates to a kind of mammal cell line, relate to specifically a kind ofly utilize Odontoblast cell line before drug-induced immortalization reversibility mouse, and establishment method.

technical background:

Tooth, the digestion organs important as the mankind are mainly comprised of sclerous tissues, its structure is followed successively by enamel (crown portion from outside to inside, root of the tooth is dental cement), dentine and dental pulp, dentine has formed the sclerous tissues that surpasses tooth 2/3, become the important sclerous tissues's structure of tooth, and as contacting dental pulp soft tissue and important its inside of conjunctive tissue of enamel sclerous tissues, have the dentinal tubule of a large amount of rules arrangements on the other hand, the projection of odontoblast grows among these tubules, the effect of played nutrition, transmitting sensation.The symptoms such as the patient that major part suffers from hypoplasia of dentin symptom occurs that creamy white is partially yellow, out-of-shape and pulp cavity obturation, these have all affected these patients' normal diet nutrition intake greatly, have reduced its quality of life.These have all illustrated that this structure of dentine plays a part indispensable in tooth.

Dentinal can only being completed by odontoblast, the mesenchymal cell that odontoblast is originated by neural crest breaks up and forms under the induction of tooth enameloblast.Odontoblast polarizes in last atomization eventually gradually at it, is formed into odontoblastic process, and secretes mineralized tissue odontoblast process is risen and holds protection, the final Dentin Structure that contains dentinal tubule that forms.Except mineralization ability; odontoblast; as pulpodentinal complex's outermost one layer of cells be in tooth, accept the earliest external irritant can will comprise that most stimulus delivery such as cause of disease invasion are to people's dental pulp; thereby make people produce corresponding measure; therefore, it is considered to again one deck Cell protection.Yet, once playing the part of in people's tooth development and normal physiological processes that key player's like this cell broken up can not divided self but be positioned at outermost one deck of tooth always, follow all one's life of tooth, once that is to say that disease damage has appearred in this class cell, tooth can not again produce same cell and complete its function.Rare differentiation or the cellular stress of studying this cell for us of cell concentration brought huge obstacle, therefore how to facilitate a large amount of obtain this cell in odontoblast's research and even be just to seem particularly important aspect tooth correlative study.And one of solution to this problem is exactly to set up the stable Odontoblast cell line of immortalization.

Nearly ten years, a lot of teeth are grown scholars and are devoted to set up the mouse of immortalization or the immortalized cell line in people source, for tooth is grown research or the research of tooth relative disease provides the research model that can simulate normal physiological situation.If MacDougall in 1995 etc. are at document 1:MacDougall M, hiemann F, Ta H, et al (1995) Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures:establishment of clonal odontoblast cell line. Connect Tissue Res 33,97-103. has reported a kind of SV40 of use large T antigen transfected dental papilla cells and the odontoblast system that obtains.Sun ZL in 1998 etc. are at document 2:Sun ZL, Fang DN, Wu XY, et al (1998) Expression of dentin sialoprotein and other molecular determinants by a new cell line from dental papillae, MDPC-23.Connect Tissue Res 37,251-261. has reported a kind of odontoblast system that comes from little dental papilla cell, this clone can be expressed the distinctive albumen of some odontoblasts as DSPP, osteopontin etc.Galler K in 2006 etc. are at document 3:Galler K, Schweikl H, Thonemann B, et al (2006) Human pulp-derived cells immortalized with Simian Virus 40 T-antigen 114,138-146. has reported the immortalized cell line in a kind of people's pulp cells source.Through the transfection of SV40T antigen, cell is sustainable to go down to posterity.And express equally the characteristic albumen of multiple odontoblast.Because SV40T antigen itself is a kind of proto-oncogene, through the cell of its transfection, all there is potential one-tenth knurl.Increasing scholar just constantly finds the novel method that builds immortalized cell line.

Some scholars have set up the method for reversibility immortalized cells by the mode of slow virus in recent years, 2000, Salmon P etc. are at document 4:Salmon P, Oberholzer J, Occhiodoro T, Morel P, Lou J, Trono D(2000) Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes.Mol Ther 2, a kind of model of reversibility immortalized cells is proposed in 727-731.: this characteristic of utilizing slow virus infected cell is the slow virus infection sinusoidal endothelial cell with LoxP site by immutalizing gene two ends, thereby set up immortalized cell line, and then the clone of the slow virus infection immortalization of CreDNA recombinase is expressed in use, utilize Cre energy specific recognition LoxP site and the characteristic of the DNA excision between two LoxP sites is removed the DNA sequence dna of expressing immutalizing gene in immortalized cell line genome from genome, thereby successfully make clone lose immortalization ability and keep identical characteristic with primary cultured cells.Kowolik CM in 2004 etc. are at document 5:Kowolik CM, Liang S, Yu Y, Yee JK (2004) Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells Oncogene 23, the proximal renal tubular epithelial cells that utilized this model immortalization in 5950-5957., and obtained success.On the other hand, in experimenting, we find out that because the characteristic of slow virus infection can not reach absolutely, and virus infection repeatedly easily causes genomic unstable, therefore in going immortalization process, we need to will go immortal cell to screen, the cell of removing immutalizing gene to obtain Cre recombinase, removes immortalized cells.But some cell is such as its primary cultured cells of the odontoblast in eventually last differential period is grown very slowly and easily aging, simple drug screening meeting makes the cell of immortalization lose use value, thereby does not reach the object of preserving amplification primary cell by this system.Up to the present, also there is no the desirable method that immortalized cells is dedifferented.

The inventor improves the method for dedifferenting in the slow virus that utilizes this expression reversibility immutalizing gene, and the present invention has introduced estrogen receptor (ER), i.e. ER at the two ends of Cre ^t2creER ^t2fusion rotein, makes a kind of Cre construct of drug-induced property.This construct is expressed drug screening mark, thereby can filter out the cell of this fusion rotein of stably express.And this fusion rotein cannot enter nucleus because volume is excessive, therefore this albumen cannot be by knocking out between the LoxP site in genome under normal circumstances.And accept after certain density 4-OH-Tomaxifen stimulation when cell, the estrogen receptor at Cre two ends will come off, thus Cre is entered nucleus by the immutalizing gene encoding sequence of removing in genome, reaches the object of immortalization.

summary of the invention:

The object of the present invention is to provide a kind of clone of utilizing drug-induced immortalization reversibility, keeping front odontoblast's proterties and function, and establishment method.

For achieving the above object, first the present invention provides a kind of fusion rotein, it comprises at least one albumin A, described albumin A can act on material in nucleus, comprise nucleic acid or albumen, and at least one protein B combining with albumin A, described protein B cannot enter nucleus, and protein B is controlled comes off.Described controlled coming off comprises and is subject to drug regulation to come off, be subject to temperature adjusting to come off or light receiving area reason regulation and control come off.

Wherein, described albumin A can be Cre enzyme, its can specific recognition DNA sequence dna in LoxP site, and by two in the same way the DNA sequence dna between LoxP site from former sequence, remove.

Wherein, described protein B can be estrogen receptor, and this albumen can stop merged albumen to enter nucleus, and can come off from institute's fusion rotein two ends after being subject to his Moses's divisional processing.

The albumin A of above-mentioned fusion rotein is under normal circumstances owing to combining with protein B, thereby can not enter nucleus, cannot act on material (for example genome or albumen) in nucleus, and when being subject to corresponding conditions processing, protein B comes off from albumin A, thereby albumin A can normally enter nucleus, acts on corresponding site.

And then the invention provides the gene of the above-mentioned fusion rotein of coding, and the expression vector that contains genes involved.

Further the invention provides a kind of clone, it can express above-mentioned fusion rotein.This clone, contains said gene or expression vector.

Induction reversibility immortalized cell line of the present invention, it has been introduced and has caused immutalizing gene, at the two ends of this gene, has LoxP sequence, and this clone is also expressed above-mentioned fusion rotein simultaneously.Preferred described fusion rotein is the fusion rotein of Cre enzyme and estrogen receptor, and at the two ends of Cre enzyme each in conjunction with an estrogen receptor.

The present invention also provides a kind of method that builds above-mentioned clone, it is that the expression vector of the fusion rotein of above-mentioned expression Cre enzyme and estrogen receptor is imported in immortalized cell line, obtain reversibility immortalized cell line, described immortalized cell line has been introduced and has been caused immutalizing gene, at the two ends of this gene, has LoxP sequence.

In embodiments of the present invention, before the present invention, dentin cell (mouse) is the cell that sets out, and lentiviral vectors pLOX-tTagiresTK is imported to described cell, obtains immortalized cells, further by plasmid pCAGER ^t2creER ^t2-neo imports in described immortalized cell line, obtains inducing reversibility immortalization predentin cell.

Particularly, the structure of clone of the present invention can comprise the following steps:

I) make the lentiviral vectors of cellular immortalization, by Sweden Trono professor (document 4:Salmon P, Oberholzer J, Occhiodoro T, Morel P, Lou J, Trono D(2000) Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes.Mol Ther 2, lentiviral vectors 727-731.) providing: pLOX-tTagiresTK (Addgene) includes the encoding sequence of SV40 large T antigen and TK, and after slow virus infected cell, being integrated into genomic fragment two ends will be with LoxP site, by the slow virus of this carrier package after infecting target cell, cell is by stably express SV40 large T antigen and thymidine kinase element, the former makes being immortalized of cell ability, TK element can be used as the sign of negative sense screening, by the cell of virus infection after accepting Cymevan and stimulating by death.Meanwhile, all fragments two ends that whole slow virus is integrated into cellular genome will can be eliminated with LoxP site under the effect of Cre recombinase from genome.

Ii) making by i) cell of described viral immortalization can control down at medicine the carrier of immortalization: express ER ^t2creER ^t2with the carrier for expression of eukaryon of neomycin resistance, this plasmid transfection is infected to i) to state in the cell of slow virus, the cell clone that adopts G418 microbiotic forward to filter out may stably express ER ^t2creER ^t2albumen, and the cell ER after accepting 4-OH-Tamoxifen drug treating that expresses this fragment ^t2creER ^t2thereby by becoming Cre recombinase, enter LoxP site in nucleus identification genome the immutalizing gene between site is removed from genome, finally make cell lose the cell cycle that immortalization ability enters normal primary cultured cell.And now reuse Cymevan drug treating cell, will kill the cell that does not lose SV40T antigen immutalizing gene completely, the cell of the immortalization of making a return journey under residue.

I wherein) the described lentiviral vectors that makes cellular immortalization, can be used separately and set up immortalized cell line, and coordinates ii) described carrier, can be based upon the clone of the drug-induced immortalization that goes down.

Odontoblast cell line before derivable immortalization reversibility mouse provided by the invention, to set up acquisition by following steps: de-neck is put to death 18.5 days female mouse of healthy conceived embryo, obtain its embryo, and separation obtains dental papilla cells in mouse embryo mandibular first molar tooth bud under Stereo microscope; Therefrom filter out the front odontoblast in mesenchyme source; Pass through successively SV40T antigen and thymidine kinase encoding gene virus infection and ER ^t2creER ^t2gene transfection, filters out and has the front odontoblast of drug-induced lower reversibility immortalization mouse strain: mDPC6T.

Evaluation to mDPC6T cell proliferation and biochemical characteristic:

Cellular form is observed: under inverted phase contrast microscope, observe and find, mDPC6T presents monolayer growth in substratum, polygon, and cell is monokaryon, contains 1～3 kernel in each cell; In long-term cultivation process, extremely difficulty is observed syncyte; The morphological specificity of cell can remain to 100 from generation to generation, and can continue continuity.

The mDPC6T cell that the present invention sets up than the normal mouse of former culture before odontoblast's rate of propagation significantly improve, and the surface marker of odontoblast before expressing, can induce mineralising to form macroscopic mineralising tubercle in vitro; Tumorigenesis not in nude mice; Active growth, there is not aging sign in the cell of 100 generations, is a kind of front Odontoblast cell line of mouse of immortalization yet; And after adding 4-hydroxytamoxifen and Cymevan medicine, Growth of Cells almost stops, cell space presents the ripe odontoblast form of the polarity of extending to one end, almost presses identical completely with the front odontoblast proterties of former culture.In view of above characteristic, mDPC6T provides desirable external model for further research odontoblast biological characteristics, simultaneously also for dentine Regeneration and Repair and dentine development-related disorders Mechanism Study provide desirable material.On the other hand, the reversible Establishment of Cell Line method of this drug-induced immortalization is preserved some more difficult obtain or poky cell research provides new thinking for amplification.

accompanying drawing explanation:

The mDPC6T cell of Fig. 1 for observing under 200 times of inverted phase contrast microscopes; Odontoblast before the mouse that wherein (A) is former culture; (B) be immortalization 50 mDPC6T cell from generation to generation; (C) be immortalization 100 mDPC6T cell from generation to generation; (D) for adding 4-hydroxytamoxifen and Cymevan, cell processes the form of rear cell;

Fig. 2 is the external mineralising induction of mDPC6T photo; (A) gross examination of skeletal muscle mineralising tubercle after external evoked 14 days wherein; (B) be that under 200 times of mirrors of Alizarin red staining, showed cell is induced mineralising tubercle after 14 days; .

Fig. 3 is that protein immunoblot analysis shows the marker gene Dmp1 of odontoblast in mDPC6T, Dspp, and Nestin, and the protein expression of SV40Tag, using the odontoblast of former culture as positive control, and b-actin is as internal reference.

specific implementation method:

pCAGER ^t2 creER ^t2 -neo construct

By pCAGER ^t2creER ^t2(Addgene) adopt NotI and EcoRI restriction enzymes double zyme cutting, obtain the ER that length is 2951bp size ^t2creER ^t2complete encoding sequence, and pCAG2LMKSOimO (Addgene) is cut to obtain with NotI and EcoRI enzyme with neomycin resistance (neoR) fragment, using CAG as the eukaryotic expression skeleton of promotor, finally by ER ^t2creER ^t2complete encoding sequence inserts in this breach, obtains pCAGER ^t2creER ^t2-neo.

reversibility mouse Odontoblast cell line

According to supplier's specification sheets, the tooth bud odontoblast of 18.5 days separation and Culture Swiss mice embryonic phases of the present invention mandibular first molar, and by this cell of slow virus infection of pLOX-tTagiresTK packing, colony screening obtains the cell strain mDPC6T that simultaneously expresses SV40 large T antigen and thymidine kinase, this cell can rise in value fast under cellar culture mode condition, go down to posterity and do not occur aging phenomenon over 100 generations, and the significant gene of odontoblast can be stable the cell that is expressed in different algebraically in, cell has and can mineralising induce character, therefore be the odontoblast with immortalization characteristic.LoxP site is contained at this cellular immortalization gene SV40 large T antigen encoding sequence two ends on the other hand, under the effect of Cre recombinase, can from genome, depart from, and the ability of forfeiture immortalization.Therefore the immortalization performance of this cell has reversibility.

drug-induced property reversibility mouse Odontoblast cell line

On the basis of reversibility mouse Odontoblast cell line mDPC6T, the present invention adopts FuGene HD(Germany Roche company) transfection pCAGER ^t2creER ^t2-neo(SEQ ID NO:1), cell is placed in and contains 200ug/ml G418(U.S. Merck company) screening culture medium cultivate under, select Survival clone, and verify ER ^t2creER ^t2complete encoding sequence is integrated in cellular genome, by this strain cell called after mDPC6T-EC.Under conventional cell culture condition, in substratum, add the substratum 2ml/ hole of containing 10uM 4-OH-Tomaxifen, act on after 4 days, the substratum 2ml/ hole that replacing contains 20ug/ml GCV, kill the cell with SV40 large T antigen, remaining cell is the cell of immortalization, its growth characteristics and odontoblast's marker gene to be expressed in primary cell similar.

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.

the foundation of 1 one kinds of reversibility mouse Odontoblast cell lines of embodiment

First from embryo, within 18.5 days, Swiss mice embryonic, obtain the front odontoblast of former culture.From Animal Experimental Study center, Hubei Province, obtain the conceived 18.5 days female mouse of Swiss, take back Cell Lab, de-neck is put to death, and isolates 18.5 days mice embryonics of embryo immediately from pregnant mouse intrauterine; Below operation is all carried out on aseptic operating platform.With containing the dual anti-PBS(American I nvitrogen company of penicillin-Streptomycin sulphate) rinse the clot on embryo surface well, isolate roughly embryo's lower jaw, and be transferred in fresh PBS damping fluid, under Stereo microscope, find and isolate mandibular first molar tooth bud, the epithelium on mechanical separation tooth bud surface, acquisition dental papilla tissue, and with aseptic micro-tying tweezer, dental papilla tissue is crumbed to size 0.1～0.5mm ³the broken tissue block of size, is soaked in fresh containing in the dual anti-PBS damping fluid of penicillin-Streptomycin sulphate.To every hole in 6-well Tissue Culture Plate (Japanese IWAKI company), drip the DMEM in high glucose substratum that contains 20% Australia foetal calf serum (U.S. Thermo Fisher Scientific company) (U.S. Thermo Fisher Scientific company) of about 600uL, make smooth culture plate of substratum uniform spreading, density according to 9 tissue block in every hole is attached to dental papilla tissue on culture plate, in 37 ℃, 5%CO ₂overnight incubation in constant temperature cell culture incubator.Under inverted phase contrast microscope, can see that adherent tissue block has cell " to climb out of " next day around, and add to every hole the substratum that 1mL contains 20% foetal calf serum.

Then the cell that separating mesenchymal is originated from culturing cell, Ji Qian odontoblast.Under above-mentioned culture condition, within every two days, change a subculture.After about 1 week, Growth of Cells density is larger, and most cells are mesenchyme derived cell, and cell space is larger, and proterties is circle or polygon.And there is a few cell, be epithelial origin cell, cell space is little, is paving stone shape, and due to epithelial cell poor growth in this substratum, quantity is few.By the method for differential digestion, obtain the cell in mesenchyme source, concrete mode is as follows: draw the substratum in culture plate, and with PBS damping fluid, rinse cell twice by cell cultures, to dripping gently 1mL concentration in every porocyte, be 0.25% pancreatin (American I nvitrogen company), room temperature digestion about 2 minutes, under inverted phase contrast microscope, can find the cell that cell space is larger, be that mesenchyme derived cell departs from culture plate around gradually, epithelial origin cell is without any digested phenomenon.Suck pancreatin, to the DMEM in high glucose substratum that adds 2mL to contain 10% foetal calf serum in every hole, the mesenchyme derived cell that piping and druming digest, can find to blow and beat the cell that epithelium posterius originates and still be retained on former culture plate under mirror.The cell of blowing and beating is passaged on new culture plate according to the ratio of 1:2, and the dental papilla cells that obtains former culture is odontoblast.

With backward cell, import SV40 large T antigen.Divide two portions: the packing of 1 slow virus pLOX-tTagiresTK; 2 slow virus infected cells and screening.

The packing of 1 slow virus pLOX-Ttag-iresTK

Concrete grammar is as follows:

1) cotransfection 293FT(American I nvitrogen company) cell is first 24 hours, by cell according to 1X10 ⁶amount be inoculated in a hole in six orifice plates, volume is 1.5mL;

2) in the Eprendorf of sterilization pipe, with 170uL serum-free DMEM in high glucose substratum, dissolve 2ug pLOX-Ttag-iresTK (Addgene:12246) 1.6ug psPAX2 (Addgene:12260) and 0.6ug pMD2.G(Addgene:12259) plasmid, mix;

3) to 2) add 8.5uL FuGene HD(Germany Roche company in the mixed solution of configuration), soft mixes with micropipet piping and druming, standing 15 minutes of room temperature;

4) by 3) in the reagent that mixes, be added dropwise to uniformly 1) in the 293FT cell culture medium prepared, shake gently even, in 37 ℃, 5%CO ₂overnight incubation in constant temperature cell culture incubator;

5) transfection is after 24 hours, changes 4) state the cell culture medium of overnight incubation, in 37 ℃, 5%CO ₂in constant temperature cell culture incubator, cultivate;

6) transfection is 48 hours, within 72 hours, collects 5) cell culture fluid of culturing cell, and change fresh culture;

7) 6) collected cell culture fluid is the liquid that contains slow virus, and the filter that is 0.4um by aperture by the slow virus liquid of collection is removed wherein dead cell composition, and the liquid obtaining can be used as pLOX-tTagiresTK slow virus and use.

2 slow virus infected cells and screening

Concrete grammar is as follows:

1) Polybrene (U.S. Sigma company) that is 8ug/uL with nuclease free ultrapure water (American I nvitrogen company) configuration concentration, ((U.S. Merck Millipore company) the suction filtration degerming of the filter in 0.22um aperture;

2) first-generation odontoblast of the above-mentioned cultivation of trysinization, becomes cell suspension, and counting, is adjusted into 1X10 by concentration ⁵/ mL, gets 0.5mL cell suspension, mix, and to add inwards 1uL concentration is the Polybrene(U.S. Sigma company of 8ug/uL with the slow virus liquid 0.5mL of above-mentioned making), all join in a hole of 6 orifice plates, in 37 ℃, 5%CO ₂overnight incubation in constant temperature cell culture incubator;

3) slow virus infection is after 12 hours, by 2) the fresh DMEM in high glucose substratum that contains 10% foetal calf serum of cultured cells replacing;

4) because the Growth of Cells speed of expression SV40 large T antigen is very fast, and odontoblast's speed of growth of former culture is very slow and very difficult vitro culture was difficult to surpass 5 generations, therefore along with continuing subculture, the cell that feeling of success is caught SV40 large T antigen slow virus will account for the overwhelming majority of all cells; Metainfective cell is continued to cultivate, when cell is opened to 80% density, according to the ratio of 1:3 ~ 1:5, go down to posterity, and in 37 ℃, 5% CO ₂in constant temperature cell culture incubator, cultivate;

5) go down to posterity after 5 generations, by cell dissociation counting, concentration is adjusted into 1X10 ³/ mL, draws 7mL and is inoculated in the Tissue Culture Dish of diameter 10cm, in 37 ℃, and 5 %CO ₂in constant temperature cell culture incubator, cultivate, within every 3 days, change a subculture, until there is obvious cell clone under mirror;

6) by 5) in the cell clone that obtains adopt clone's ring digestion had digestive transfer culture amplification from culture dish, the cell that each clonal expansion goes out is all frozen in generation morning: after cell dissociation, move into 15mL centrifuge tube, centrifugal 5 minutes of 300g, abandon supernatant, add the DMEM in high glucose substratum that contains 50% foetal calf serum 10% methyl-sulphoxide, concentration is adjusted into 10 ⁷above, be distributed in 1.5mL cell cryopreservation tube, cryopreservation tube is put into cell cryopreservation program temperature reduction box (U.S. Nalgene company), be then transferred to-80 ℃ of refrigerator overnight, be transferred to liquid nitrogen cryopreservation by freeze-stored cell next day;

7) with the DMEM in high glucose substratum configuration that contains 10% foetal calf serum, contain GCV(U.S. Sigma company) the concentration screening culture medium that is 10uM;

8) same clone is inoculated in respectively in two holes of 24 orifice plates according to isoconcentration, treat that cell grows to 80% density, one hole changes the substratum that contains GCV into, and another Kong Ze is the DMEM in high glucose substratum of fresh 10% foetal calf serum, the former is as experimental group, the latter as a control group, by cell in 37 ℃, 5%CO ₂in constant temperature cell culture incubator, cultivate;

9) after 3 days, at Microscopic observation 8) cultured cells, if add the cell of GCV substratum to have mortality, control group does not have ANOMALOUS VARIATIONS, the cellular genome that so just proves this clone is the stable encode fragment that contains coding SV40 large T antigen and TK that is integrated into, and this clone is left to follow-up evaluation and screening;

Through above process, obtain altogether 12 stable clonal cell lines, be named as respectively mDPC1T～mDPC12T, to be wherein expressed as dentin cell's marker gene the most remarkable for mDPC6T, gone down to posterity so far and surpassed for 100 generations, do not occur any old and feeble feature.Cytobiology thinks, passage surpassed for 100 generations and is considered to have immortalization level, and the mDPC6T that we set up has been the cell of immortalization.

the 2 one kinds of foundation that can induce reversibility mouse Odontoblast cell line of embodiment

1) pCAGER ^t2creER ^t2-neo plasmid construction

By pCAGER ^t2creER ^t2(Addgene:13777) adopt noti(U.S. NEB company) and ecoRi(U.S. NEB company) restriction enzymes double zyme cutting, obtains the ER that length is 2951bp size ^t2creER ^t2complete encoding sequence, and pCAG2LMKOSimO (Addgene:20866) is used noti and ecoRi enzyme is cut to obtain and is usingd CAG as the eukaryotic expression skeleton of promotor, finally by ER with neomycin resistance (neoR) fragment ^t2creER ^t2complete encoding sequence inserts in this breach, obtains pCAGER ^t2creER ^t2-neo (SEQ ID NO:1), and the plasmid skeleton that does not insert encoding sequence directly adopts flush end to connect as blank plasmid: pCAG-neo.

2) build the reversibility mouse Odontoblast cell line of Drug induction

The mDPC6T clone in generation morning is passaged in 6 porocyte culture plates, when cell is paved with Tissue Culture Dish 50% left and right, carries out gene transfection, concrete grammar is as follows:

(1) at least prepare two porocytes;

(2), in two aseptic 1.5mLEppendorf pipes, with the OptiMEM of 200uL, subtract the pCAGER that blood serum medium (American I nvitrogen company) dissolves respectively 2ug ^t2creER ^t2-neo plasmid or pCAG-neo plasmid, the former is as experimental group, and the latter is as blank group;

(3) in two pipes, add respectively 8uL FuGene HP(Germany Roche company), piping and druming mixes gently, standing 15 minutes of room temperature;

(4) two pipe transfection mixtures are evenly added dropwise to respectively in two orifice plate cells, shake up gently, in 37 ℃, 5%CO ₂in constant temperature cell culture incubator, cultivate;

(5) after transfection two days, experimental group and blank group cell culture medium all changed the DMEM in high glucose substratum containing 10% foetal calf serum of 200ug/ml G418 into, in 37 ℃, and 5% CO ₂in constant temperature cell culture incubator, cultured continuously is 10 days, and discards dead cell by the G418 substratum more renewing;

Carry out afterwards cell clone and propagation work.Cell is after above-mentioned screening experiment, and the cell of blank group is almost all dead, and experimental group truly has 10% cell survival, and has and present on a small quantity little clonal growth, by microscopic examination, and marker clone.Clone ring digestion, each clone amplification of going down to posterity separately, cell is according to the amplification of going down to posterity of 1:3 ratio, and each clone is frozen according to method described in application example one.

Extract the genomic dna of each clone cell, adopt PCR checking ER ^t2creER ^t2whether complete encoding sequence is integrated in genomic dna, and concrete grammar is as follows:

(1) each clone's cell is gone down to posterity respectively in a hole of 6 orifice plates and cultivate;

(2) when Growth of Cells to 90% density, with 0.25% pancreatin, every porocyte is digested, the every porocyte of centrifugal collection, in every tube cell precipitation, add 200uL to be dissolved with the TE damping fluid (pH=8.0 of 2mg/mL Proteinase K (American I nvitrogen company), China Tian Gen company), in 60 ℃, hatch 1 hour;

After (3) 1 hours, to the phenol that adds 200uL in (2) pipe, chloroform, primary isoamyl alcohol solution (25:24:1, pH=8.0), firmly shakes 15 seconds, centrifugal 10 minutes of 4 ℃ of 15000g;

(4) extract supernatant, preserve, get 1uL as pcr template, adopt Cre Auele Specific Primer (forward: 5 '-ATGTCCAATTTACTGACC-3 '; Negative sense: 5 '-CTAATCGCCATCTTCCAG-3 '; Product size: 1032bp; Synthetic by American I nvitrogen company) according to following reaction conditions, carry out PCR checking, selecting has the clone of positive band as ER ^t2creER ^t2complete encoding sequence is integrated into genomic clone, and these clones be can drug-induced property reversibility mouse Odontoblast cell line.

PCR reaction system mixes by following composition on ice:

10 * PCR damping fluid (Japanese TAKARA company) 5uL

MgCl2(25mmol/L, Japanese TAKARA company) 2uL

DNTP (each 10mmol/L) 1uL

Forward primer (10umol/L) 1uL

Negative sense primer (10umol/L) 1uL

Nuclease free water (U.S. GIBICO company) 37uL

Supernatant 1uL

Taq DNA polymerase(Japan TAKARA company) 1uL

After mixing, following program amplification on PCR instrument: 94 ℃/3min → [94 ℃/30sec → 58 ℃/30sec → 72 ℃/1min] * → 72 ℃/10min of 33 circulations.Primer is handed over the order-checking of American I nvitrogen company, by Cre recombinase sequence B LAST comparison in product sequence and Genebank.

Through above-mentioned experiment screening checking, obtain three stable clonal cell lines, called after mDPC6TEC, mDPC7TEC, mDPC8TEC.The mDPC6TEC of take below carries out the description of related experiment as example.

the 3 one kinds of CHARACTERISTICS IDENTIFICATION that can induce reversibility mouse Odontoblast cell line of embodiment

1) cell proliferation rate analysis

1*10 is counted in odontoblast before the former culture in the 3rd generation and the every porocyte of mDPC6T(to be identified ⁵) being passaged to 6 orifice plates, every kind of cell is done 3 groups of secondary holes.Use EdU cell proliferation detecting kit (Chinese Rui Bo company) to detect ability of cell proliferation.Concrete grammar is as follows:

(1) use containing the DMEM culture medium culturing of 1%FBS 24 hours EdU1:1000 culture medium culturing 4 hours after going down to posterity 24 hours.

(2) 4% paraformaldehydes are fixed 15 minutes.

(3) 0.2% glycine incubated at room 10 minutes.

(4) PBS rinses 2 times, each 5 minutes

(5) use containing saturatingization of PBS of 0.5% triton-100 10 minutes

(6) PBS rinses one time 5 minutes.

(7) use the every hole 1ml of Apoolo staining fluid, lucifuge incubated at room 30 minutes.

(8) containing the PBS of 0.5% triton-100 3 times each 5 '

(9) Hochest 1:100 lucifuge is hatched 10 minutes.

(10) PBS rinses ten minutes.

(11) EdU shows red fluorescence under 550nm exciting light.Hochest shows blue-fluorescence under ultraviolet excitation.

(12) choosing at random every hole, 5 visuals field counts.EdU stained positive cell count accounts for the ratio reflection ability of cell proliferation of total cell count.

Experimental result shows: mDPC6T ability of cell proliferation is significantly higher than primary cultured cells.

) cell aging detection

Odontoblast and the mDPC6T in the 50th generation before the former culture in the 6th generation are passaged to respectively to six orifice plate (cell count 1*10 ⁵) after 24 hours, use cell aging detection kit (Chinese green the skies company) identification of cell aging index.Concrete grammar is as follows:

(1) absorb cell culture fluid, with PBS washing 1 time, every hole adds 1 milliliter of beta-galactosidase enzymes dyeing stationary liquid, and room temperature is fixed 15 minutes.

(2) absorb cell stationary liquid, use PBS washed cell 3 times, each 3 minutes.

Absorb PBS or HBSS, every hole adds 1 milliliter of dyeing work.Working fluid compound method: beta-galactosidase enzymes staining fluid A:10 μ l, beta-galactosidase enzymes staining fluid B:10 μ l, beta-galactosidase enzymes staining fluid C:930 μ l, X-Gal solution: 50 μ l.

(3) 37 ℃ of overnight incubation, seal 6 orifice plates with preservative film and avoid evaporating.

(4) under inverted microscope, observe.

Senile cell endochylema can be dyed for blueness, and 5 visuals field are chosen in every hole at random, carries out statistical analysis after counting.

Experimental result shows: before the former culture in 6 generations, aging occurs the existing part cell of odontoblast, and the mDPC6T cell in the 50th generation has no old and feeble positive index.

) research of cell mineralization ability.

Odontoblast before the former culture in the 6th generation and the 6th generation mDPC6T are passaged to respectively to six orifice plate (cell count 2*10 ⁵), after 48 hours, add mineralising inducing culture.Its composition is: contain 10% foetal calf serum, 10 nmol/L dexamethasone (U.S. Sigma company), 50ug/ml vitamins C (U.S. Sigma company), 10 nmol/L β Sodium Glycerophosphates (U.S. Sigma company), 100u/ml penicillin, the DMEM substratum of 100u/ml Streptomycin sulphate (American I nvitrogen company).

Add induced liquid to be designated as 0 day the same day.Within every 72 hours, change a mineralising induction.0,1, within 5,7,11,14 days, extract cell protein and RNA respectively.0,7, within 14 days, fixed cell is done Alizarin red staining.Concrete grammar is:

(1) proteins extraction:

1. suck cell culture medium, use the PBS of precooling to rinse cell.

2. add protein lysate M-PERTM(U.S. Thermo Fisher Scientific company), 400ul/ hole.Hatch on ice 5 minutes.

3. use cell to scrape adherent cell and form cell suspension, cell suspension is transferred to centrifuge tube.

4. on vortex vibrator, vibrate 15 seconds, hatch on ice ten minutes.Repeat 3 times.

5. 4 ℃, centrifugal 15 minutes of 13000rpf

6. draw supernatant ,-80 ℃ of preservations.

(2) RNA extracts

1. suck cell culture medium, use the PBS after DEPC processes to rinse cell.

2. add TriZol(American I nvitrogen company), 1ml/ hole.Incubated at room 5 minutes.

3. blow and beat cell and form cell suspension, be transferred to 1.5ml centrifuge tube.

4. add 200ul chloroform, concuss 15 seconds, room temperature is placed 10 minutes.

5. 4 ℃, centrifugal 15 minutes of 13000rpf

6. the aqueous phase liquid on careful sucking-off upper strata 300ul roughly, is transferred to new EP pipe.

7. at Guan Zhongzai, add 0.5ml Virahol.Careful shakes up, and room temperature is placed 10 minutes.

8. 4 ℃, centrifugal 10 minutes of 13000rpf.

9. abandon supernatant, add 75% ethanol rinsing, 4 ℃, centrifugal 5 minutes of 7500rpf.

10. drying at room temperature is 10 minutes, adds 20ulDEPC water dissolution precipitation.Survey concentration.

(3) mineralising tubercle dyeing.

1. 0.5g sodium alizarinsulfonate pulvis (U.S. Sigma company) is dissolved in 100ml Trish-HCl, is configured to 0.5% Alizarin red staining liquid.Adjust PH to 7.2,4 ℃ of lucifuges save backup.

2. suck cell culture medium, use PBS to rinse cell.

3. 95% ethanol is fixed 10 minutes.

4. PBS rinses 5 minutes, in triplicate.

5. add the every hole 1ml of Alizarin red staining liquid, hatch 1 hour for 37 ℃.

6. use ddH ₂o washes away background color.

7. under inverted microscope, take a picture, carry out tubercle counting.

Experimental result demonstration, similar with the front odontoblast of former culture, at mineralising inducing culture, cultivate after 14 days, mDPC6T also can produce normal mineralising tubercle.

) odontoblast's marker gene expression checking

(1) by 3) protein that obtains carries out protein blot experiment.

Key step is:

1. 20ul protein extract adds albumen sample-loading buffer 5ul, 95 ℃ of sex change 5 minutes.

2. every hole loading 20ul on 10%SDS PAGE glue.

3. electrophoresis after 30 minutes under 60V voltage, 100v1 electrophoresis 1 hour.

4. use pvdf membrane (German Roche company) transfer printing, under 75V voltage, transfer printing is 2.5 hours.

5. transfer printing success is confirmed in ponceau dyeing.

6. by pvdf membrane with sealing under confining liquid room temperature 1 hour;

7. in confining liquid, add primary antibodie: NESTIN(U.S. eBioscience company), DMP1(U.S. Biovision company), DSP(U.S. Santa Cruz company), β-actin(U.S. Santa Cruz company), 4 ℃ of overnight incubation;

8. primary antibodie is used TBST rinsing, 10 minutes * 5 times after hatching and finishing;

9. the IgG bis-anti-(U.S. Santa Cruz company) (being diluted to proper concn with TBST) that adds HRP mark, is placed in shaking table incubated at room 1.5 hours, hatches and finishes to use TBST rinsing, 10 minutes * 5 times afterwards;

10. ECL chemoluminescence colour developing, development, photographic fixing: pvdf membrane is put into freshly prepared ECL reagent (U.S. Thermo Fisher Scientific company) in darkroom luminous, film (Kodak) develops, photographic fixing is taken a picture.

(2) by 3) RNA that obtains carries out Realtime RT-PCR experiment.

(A) using reverse transcription test kit (Japanese Takara company) is cDNA by RNA reverse transcription.

Reaction system is as follows:

Reagent	Volume (μ l)
		RNase Free H2O	7
5×RT Buffer	4
		dNTP Mixture	2
RNase inhibitor	1
		OligdT	1
Total RNA	5
		Total Volume	20

Reverse transcription (RT) temperature cycle is as follows:

54 ℃, 20 minutes; 99 ℃, 5 minutes; 4 ℃, 5 minutes, termination reaction.

(B) DMP1, DSPP, ALP and GAPDH primer sequence (American I nvitrogen company) see the following form

(C) Realtime RT-PCR reaction (Japanese Takara company).System is as following table:

Being formulated on ice of reaction solution carried out.

Reaction conditions is as follows:

95 ℃ of denaturations 30 seconds;

95 ℃ of sex change 5 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended 30 seconds, 40 circulations of increasing;

72 ℃ are extended 5 minutes.

Experimental result shows: mDPC6T cell can be expressed as dentin cell's characteristic gene as Dmp1, Dsp, and Nestin, these marker gene, along with mineralising is induced, are expressed gradually and are strengthened.

embodiment 4 can induce the safety analysis of reversibility mouse Odontoblast cell line

1) cell tumorigenicity is analyzed

aCC cell and mDPC6T to be identified are passaged in T175 culturing bottle, after cell covers with, digest centrifugal.

Use DMEM substratum re-suspended cell, reach density 1*10 ⁷individual/Animal Experimental Study center, ml.Cong Hubei Province obtains nude mice, and pallium cell injection is entered to nude mice by subcutaneous; Left side injection ACC cell, mDPC6T cell to be identified is injected on right side.Each position injection 200ul, slowly injection, on skin mound of subcutaneous formation.After 14 days, check lump formational situation.De-neck method is put to death nude mice, takes off lump, take pictures, and HE dyeing.

Experimental result shows: ACC cell injection zone forms obvious lump, and section is shown as tumour cell, and does not have DPC6T cell injection zone not see lump, and tumour cell is not seen in section.Presentation of results mDPC6T is without becoming knurl or low one-tenth knurl.

) chromosome karyotype analysis

(1) before test cell line, reach 50% degrees of fusion, experiment cell the day before yesterday changes liquid.

(2) colchicine (U.S. Sigma company) is processed cell, hatches 2 hours for 37 ℃.

(3) culturing cell is mixed with 0.25% trysinization piping and druming, divide and install in centrifuge tube, 1500rpm horizontal centrifugal 10 minutes, abandons supernatant.

(4) in precipitation, add 37 ℃ of warm 0.075MKCL 5ml that bathe, mix gently, after 5 minutes, add immediately Kano stationary liquid 2ml, mix.

(5) the centrifugal 10min of horizontal centrifuge 1500rpm, abandons supernatant.

(6) in precipitation, add 37 ℃ of warm Kano stationary liquid 7ml that bathe, mix gently.

(7) the centrifugal 10min of horizontal centrifuge 1500rpm, (if find have jaundice material again to abandon supernatant with stationary liquid washing methods with (6) in cell, stays 0.5ml-1ml liquid, resuspended precipitation.

(8) draw cell suspension and drip sheet (it is freezing that slide is put into 4 ℃ of refrigerators in advance), put 3 hours dry plates in 75 ℃ of baking boxs.

(9) get 2.5% pancreatin storage liquid, with the phosphoric acid buffer of PH7.4, be diluted to 0.025% working fluid, be placed in 37 ℃ of water-bath temperature and bathe.

(10) getting karyomit(e) sheet is placed in pancreatin and digests 55 seconds-2min.

(11) get karyomit(e) sheet, remove pancreatin, put into the phosphoric acid buffer configuring and rinse twice.

(12) get rid of liquid, then put into Gimsa(U.S. Sigma company) the dye liquor 2-3min that dyes.

(13) distilled water rinses 2 minutes, dries up.

(14) microscopic examination mapping.

The demonstration of chromosome karyotype analysis result, mDPC6T cell is 40 karyomit(e)s.Do not find numerical chromosome aberration.

embodiment 5 mouse Odontoblast cell lines go immortalization experiment

This case introduction makes the ER that can induce reversibility mouse Odontoblast cell line only to express in kytoplasm by medicine irritation ^t2creER ^t2enter nucleus, the LoxP site at identification immutalizing gene SV40 large T antigen encoding sequence two ends removes immutalizing gene, thereby makes cell lose immortalization ability from genome.Under drug screening, obtain the cell that there is no immutalizing gene completely.Concrete operations are as follows:

1) will be cultured to the mDPC6TEC cell in 50 generations, in had digestive transfer culture to six orifice plate, treat cell open to density be 30% ~ 50%, add the DMEM in high glucose substratum that contains 10uM 4-OH-Tomaxifen and 10% foetal calf serum, 37 ℃, 5%CO ₂in constant temperature cell culture incubator, cultivate 4 days, within two days, change the substratum that once contains 4-OH-Tomaxifen;

2) be 1) in cultured cells be replaced with the DMEM in high glucose substratum that contains 10uM GCV and 10% foetal calf serum, 37 ℃, 5%CO ₂in constant temperature cell culture incubator, cultivate 3 days, dead cell great majority are still to express the cell that SV40 large T antigen has immortalization character, and remaining basic for not having the cell of immortalization ability;

3) identification of cell removes immortalization:

This step is divided into two portions, respectively from DNA and protein angle checking 2) cell of surviving do not express SV40 large T antigen, and concrete steps are as follows:

(1) in PCR checking cellular genome, do not contain SV40 large T antigen encoding sequence:

1. according to method described in example 2, extract survivaling cell and mDPC6TEC cell genomic dna, the former DNA is as experimental group, the positive contrast of the latter;

2. adopt SV40 large T antigen Auele Specific Primer (forward: 5 '-AGCAGACACTCTATGCCTGTGTGGAGTAAG-3 '; Negative sense: 5 '-GACTTTGGAGGCTTCTGGATGCAACTGAG-3 '; Product size: 751bp; Synthetic by American I nvitrogen company) carry out PCR, water is as negative control;

3. product carries out agarose gel electrophoresis detection, and visible experimental group and negative control group are showed no object band, and unique obvious band appears in positive controls near 800bp

By above experiment and result, we learn that the mDPC6TEC cell of processing successively through 4-OH-Tomaxifen and GCV lost the DNA sequence dna of coding SV40 large T antigen.

(2) protein blot experiment

1. according to extracting survivaling cell and mDPC6TEC total protein of cell in example 3, the former DNA is as experimental group, the positive contrast of the latter.

2. according to method in example 3, adopt SV40 large T antigen monoclonal antibody (U.S. Biovision company) to carry out protein blot experiment (primary antibodie concentration: 1:800, two anti-concentration are 1:2000); Use ACTIN is internal reference antibody.

3. protein blot experiment result shows that experimental group has no object band, and unique obvious band appears in positive controls near 95KD.Above result confirms: the mDPC6TEC cell that process 4-OH-Tomaxifen and GCV process successively has no longer been expressed SV40 large T antigen.

With mDPC7TEC and mDPC8TEC cell, carry out related experiment, also obtained above-mentioned similar result.

Sequence table

<110> Wuhan University

Odontoblast cell line and establishment method thereof before <120> induction reversibility immortalization

<130>

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 9385

<212> DNA

<213> artificial sequence

<400> 1

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catgggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 660

ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg 720

gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 780

gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 840

ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 900

tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg 960

actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 1020

ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg 1080

gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt 1140

gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg 1200

cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg 1260

cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 1320

gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga 1380

gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc 1440

gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg 1500

gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc tgtcgaggcg 1560

cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 1620

tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc 1680

gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt 1740

cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct 1800

gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta 1860

gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc 1920

tggttattgt gctgtctcat cattttggca aagaattccc gggtgagccg ccaccatggc 1980

tggagacatg agagctgcca acctttggcc aagcccgctc atgatcaaac gctctaagaa 2040

gaacagcctg gccttgtccc tgacggccga ccagatggtc agtgccttgt tggatgctga 2100

gccccccata ctctattccg agtatgatcc taccagaccc ttcagtgaag cttcgatgat 2160

gggcttactg accaacctgg cagacaggga gctggttcac atgatcaact gggcgaagag 2220

ggtgccaggc tttgtggatt tgaccctcca tgatcaggtc caccttctag aatgtgcctg 2280

gctagagatc ctgatgattg gtctcgtctg gcgctccatg gagcacccag tgaagctact 2340

gtttgctcct aacttgctct tggacaggaa ccagggaaaa tgtgtagagg gcatggtgga 2400

gatcttcgac atgctgctgg ctacatcatc tcggttccgc atgatgaatc tgcagggaga 2460

ggagtttgtg tgcctcaaat ctattatttt gcttaattct ggagtgtaca catttctgtc 2520

cagcaccctg aagtctctgg aagagaagga ccatatccac cgagtcctgg acaagatcac 2580

agacactttg atccacctga tggccaaggc aggcctgacc ctgcagcagc agcaccagcg 2640

gctggcccag ctcctcctca tcctctccca catcaggcac atgagtaaca aaggcatgga 2700

gcatctgtac agcatgaagt gcaagaacgt ggtgcccctc tatgacctgc tgctggaggc 2760

ggcggacgcc caccgcctac atgcgcccac tagccgtgga ggggcatccg tggaggagac 2820

ggaccaaagc cacttggcca ctgcgggctc tacttcatcg cattccttgc aaaagtatta 2880

catcacgggg gaggcagagg gtttccctgc cacagctgtc gacaatttac tgaccgtaca 2940

ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca agaacctgat 3000

ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt 3060

ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc 3120

tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag taaaaactat 3180

ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc cacgaccaag 3240

tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg ttgatgccgg 3300

tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg ttcgttcact 3360

catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg ggattgctta 3420

taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata tctcacgtac 3480

tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta gcaccgcagg 3540

tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga tttccgtctc 3600

tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa atggtgttgc 3660

cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt ttgaagcaac 3720

tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg cctggtctgg 3780

acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt caataccgga 3840

gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata tccgtaacct 3900

ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgatctcg agccatctgc 3960

tggagacatg agagctgcca acctttggcc aagcccgctc atgatcaaac gctctaagaa 4020

gaacagcctg gccttgtccc tgacggccga ccagatggtc agtgccttgt tggatgctga 4080

gccccccata ctctattccg agtatgatcc taccagaccc ttcagtgaag cttcgatgat 4140

gggcttactg accaacctgg cagacaggga gctggttcac atgatcaact gggcgaagag 4200

ggtgccaggc tttgtggatt tgaccctcca tgatcaggtc caccttctag aatgtgcctg 4260

gctagagatc ctgatgattg gtctcgtctg gcgctccatg gagcacccag tgaagctact 4320

gtttgctcct aacttgctct tggacaggaa ccagggaaaa tgtgtagagg gcatggtgga 4380

gatcttcgac atgctgctgg ctacatcatc tcggttccgc atgatgaatc tgcagggaga 4440

ggagtttgtg tgcctcaaat ctattatttt gcttaattct ggagtgtaca catttctgtc 4500

cagcaccctg aagtctctgg aagagaagga ccatatccac cgagtcctgg acaagatcac 4560

agacactttg atccacctga tggccaaggc aggcctgacc ctgcagcagc agcaccagcg 4620

gctggcccag ctcctcctca tcctctccca catcaggcac atgagtaaca aaggcatgga 4680

gcatctgtac agcatgaagt gcaagaacgt ggtgcccctc tatgacctgc tgctggaggc 4740

ggcggacgcc caccgcctac atgcgcccac tagccgtgga ggggcatccg tggaggagac 4800

ggaccaaagc cacttggcca ctgcgggctc tacttcatcg cattccttgc aaaagtatta 4860

catcacgggg gaggcagagg gtttccctgc cacagcttga tagcggccgc tcgagcatgc 4920

atctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga 4980

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 5040

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 5100

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 5160

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 5220

gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 5280

cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 5340

ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 5400

atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 5460

ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 5520

tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 5580

accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg gcctattggt 5640

taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 5700

gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa agcatgcatc 5760

tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 5820

aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 5880

ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 5940

atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt 6000

ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg 6060

atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt 6120

ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 6180

gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga 6240

ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg 6300

ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact 6360

ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg 6420

agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct 6480

gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 6540

gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt 6600

tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg 6660

cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc 6720

ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag 6780

agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 6840

cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 6900

cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 6960

cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 7020

gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 7080

tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 7140

ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 7200

ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 7260

gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 7320

atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 7380

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 7440

tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 7500

agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 7560

aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 7620

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 7680

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 7740

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 7800

ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 7860

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 7920

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 7980

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 8040

gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 8100

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 8160

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 8220

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 8280

gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 8340

aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 8400

aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 8460

ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 8520

gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 8580

aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 8640

ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 8700

tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 8760

ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 8820

cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 8880

agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 8940

gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 9000

gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 9060

acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 9120

acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 9180

agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 9240

aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 9300

gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 9360

tccccgaaaa gtgccacctg acgtc 9385

<210> 2

<211> 16

<212> DNA

<213> artificial sequence

<400> 2

tgctggagcc acaaac 16

<210> 3

<211> 18

<212> DNA

<213> artificial sequence

<400> 3

aaaccctatg caaccttc 18

<210> 4

<211> 18

<212> DNA

<213> artificial sequence

<400> 4

acaggcaaat gaagaccc 18

<210> 5

<211> 17

<212> DNA

<213> artificial sequence

<400> 5

ttcactggct tgtatgg 17

<210> 6

<211> 16

<212> DNA

<213> artificial sequence

<400> 6

ctgatgtgga gtatga 16

<210> 7

<211> 16

<212> DNA

<213> artificial sequence

<400> 7

tgtatctcgg tttgaa 16

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

tgcaccacca actgcttagc 20

<210> 9

<211> 21

<212> DNA

<213> artificial sequence

<400> 9

ggcatggact gtggtcatga g 21

<210> 10

<211> 30

<212> DNA

<213> artificial sequence

<400> 10

agcagacact ctatgcctgt gtggagtaag 30

<210> 11

<211> 29

<212> DNA

<213> artificial sequence

<400> 11

gactttggag gcttctggat gcaactgag 29

Claims (2)

1. one kind builds the method for inducing reversibility immortalized cell line, it is characterized in that, with mouse predentin cell, for the cell that sets out, lentiviral vectors pLOX-tTagiresTK is imported to described cell, obtain immortalized cells, further by the plasmid pCAGER of nucleotide sequence shown in SEQ ID No.1 ^t2creER ^t2-neo imports in described immortalized cell line, obtains inducing reversibility immortalization predentin clone.

2. a method of preparing immortalized cells, is characterized in that, adopting the German Roche FuGene HD of company transfection sequence is the pCAGER of SEQ ID No.1 ^t2creER ^t2-neo, is placed in immortalized cell line under the screening culture medium cultivation that contains 200 μ g/ml G418, selects Survival clone, and verifies ER ^t2creER ^t2complete encoding sequence is integrated in cellular genome, by this strain cell called after mDPC6T-EC, under conventional cell culture condition, in substratum, add the substratum 2ml/ hole of containing 10uM4-OH-Tomaxifen, act on after 4 days, the substratum 2ml/ hole that replacing contains 20 μ g/ml GCV, kill the cell with SV40 large T antigen, remaining cell is the cell of immortalization.

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