CN101748123B - Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof - Google Patents

Abstract

The invention discloses a manufacturing method of an adjustable liver damage animal model and a special DNA fragment thereof; a DNA fragment I is formed by sequentially connecting liver super promoters and antisense tetracycline activator coding genes of a Tet-on gene expression system from upstream to downstream; an DNI fragment II is formed by sequentially connecting tetracycline reaction factors, promoters and prourokinase activator coding genes of the Tet-off/Tet-on gene expression system from upstream to downstream; a recombination expression vector carrying the DNI fragment II is used for converting mammalian cells, the cells are cultivated to obtain recombinant adenovirus. The DNA fragment I is led in the animal to obtain a transgenosis first-construction animal; the transgenosis first-construction animal and scid/bg animal are back-crossed to obtain the animal carrying the DNA fragment I with the scid/bg background; the recombinant adenovirus is used for injecting the animal carrying the DNA fragment I with the scid/bg background, so as to obtain the adjustable liver damage animal model; in the model, the prourokinase activator expression is activated by doxycycline, the expression intensity is changed along with the change of dosage, the specificity is very high.

Description

The making method of adjustable liver damage animal model and special DNA fragment thereof

Technical field

The present invention relates to a kind of making method and special DNA fragment thereof of adjustable liver damage animal model.

Background technology

Hepatitis B virus (HBV) and hepatitis C virus (HCV) serious threat human beings'health, the whole world have 3.6 hundred million hepatitis B patients and 1.7 hundred million the third hepatopaths approximately, and 1,500,000 deaths are arranged every year approximately.In 10 years of past, making significant headway aspect these diseases of treatment, but still do not setting up effective healing means, and prior treatment method develops better antiviral and remains the task of top priority usually with spinoff.

At present, new antiviral mainly at cell levels to testing and estimating, be used for clinical before, must on animal model, test its activity and toxicity.The medicine of anti-HBV and HCV can only carry out studying in the body with chimpanzee; Because the reason of economic and moral aspect; The chimpanzee test has certain limitation, is used for HCV and HBV medicine and developing vaccines so people attempt to set up the humanized mouse model of liver, obtains some progress; As the liver of the source liver cell reconstruction A1b-uPA transgenic mice of choosing, such mouse can be infected by HBV and HCV.

In order to study intravital blood coagulation hemolytic system; People such as Janice L.Hechei have made up the transgenic mice of albumin promoter driving uPA; Overexpression uPA in the liver cell of this transgenic mice, the uPA in the blood plasma rises, and in newborn mice, causes internal hemorrhage easily and causes death.Have two lower strains of uPA expression level to be able to survival, but only about half of transgenic mouse birth back is dead among the offspring.The expression of the intravital uPA of transgenic mice should be stable; But the activity of the detected uPA in the blood of the mouse of two strains descends in time gradually; And the blood coagulation ability is also recovered gradually, and after about 1-2 month, mouse blood coagulation ability is recovered fully.The liver of the transgenic mice in one age in week is complete white; During age in week, the red area of some 1mm-1cm that are dispersed in has appearred in the liver to 3-5; 6-8 week can be observed the red area that is linked to be sheet.Two strains all are this situation, explain with genetically modified integration site irrelevant.These phenomenons show that the tissue of white is original, and red spot occurred afterwards.Some cells of further investigation discovery have been left out foreign gene through homologous recombination, finally can rebuild whole liver through increasing, and this explanation liver cell has powerful multiplication capacity, rebuilds mouse liver for the external source liver cell foundation is provided.The A1b-uPA mouse is backcrossed on the immunodeficient mouse scid/bg background; Be transplanted to A1b-uPA heterozygosis and the mouse liver that isozygotys from the liver cell of the isolating virus-free infection of hepatectomy patient; Finder source liver cell can reach the reconstruction of higher degree in the mouse body that transgenic is isozygotied; And can be infected by HBV and HCV, and in the mouse liver of transgenic heterozygosis, almost can't see the liver cell in people source.

Another important models that is used to transplant research be Fah-/-mouse; Tyrosinemia can take place in the mouse of Fah defective; Tyrosinemia is a kind of heredity amino acid metabolism disease, lacks the FAA lytic enzyme in patient's body, and tyrosine is in vivo during metabolism; Owing to FAA can not accumulate in hydrolysis, thereby cause the damage of liver cell.NTBC is a 4-hydroxyphenylpyruvate dioxygenase suppressor factor, can stop the accumulation of FAA, therefore takes the symptom that NTBC can improve tyrosinemia effectively.This mouse can the normal growth breeding through feeding NTBC.Because the FAH defective can cause hepatocellular death, so can be used as the model of hepatocyte transplantation on this model theory.Report 1000 normal mouse liver cells be transplanted to the FAH mouse, just can rebuild Fah-/-liver of mouse.But in very long following period of time, all do not use Fah-/-model carries out the humanized report of liver.

Though people source liver cell can be good at rebuilding the Alb-uPA mouse, this mouse has several shortcomings to hinder its application: 1) breeding difficulty: after the mouse birth of transgenic heterozygosis, have half to cause death approximately; The homozygote mouse is all dead in one month; The mouse that can only obtain to isozygoty through the mating of heterozygosis mouse, according to genetic development, it is homozygote that the offspring has 1/4 mouse approximately; It is lethal to remove new life, is difficult to obtain the mouse of isozygotying.2) the transplanting window is little; Homozygous mouse can be dead about one month, so must rebuild the liver of mouse as early as possible, transplant time after birth 7-14 days usually.3) operation is inconvenient, and 7-14 days mouse is not easy to operation technique.

In the intestinal bacteria E.coli, TetR (Tet repressor) is a negative regulation tetracyclin resistance operon.TetR is attached to the expression of blocking gene on the Tet operon sequence when not having tsiklomitsin.When tsiklomitsin was arranged, TetR discharged from operon sequence, and gene begins to express Tet-off system that Here it is.Different with the Tet-Off system; The Tet-0n system uses the trans Tet that has two sudden changes to suppress sub-rTetRs (reverse Tetrepressors); Merge the land of rTetRs and the active region of VP16, and what senior tet-on system used is minimum VP16 active region.The land of rTetRs combines with the tet operon sequence when vibra-(Doxycycline tetracycline derivant) or tsiklomitsin are arranged, and starts the expression of goal gene.The expression that the senior tet-On system that obtains through the optimization to the tet-on system has improved goal gene, toxicity reduces, and obtains the proteic clone of stably express more easily.Senior tet-On system has following advantage mutually: 1) very tight regulation and control: because the rtTA and the TRE-Tight that optimize, the background expression level of goal gene is low.2) have no side effect: because do not have corresponding sequence, the tetO sequence that acts on TRE-Tight that the land of rtTA-Adavanced is very special in mammalian cell in the eukaryotic gene group.3) efficiently fast inducing action: Dox can be in 30 minutes the expression of goal gene be improved 1000 times, and other inducible system speed is slow, can not induce fully.4) absolute expression levels is high: the high expression level of Tet system even be higher than the direct startup of CMV promotor has the Tet-Off transient expression of reporting in the HeLa cell stronger 35 times than CMV.5) induced drug: Tc and Dox price are relatively cheap, nontoxic, and result's repeatability is high.Based on above advantage, we use senior tet-on system to set up model.

Adenovirus carrier is a kind of carrier that is used for therapeutic transgene of development in recent years, and it is low that it has toxicity, the high and unconformable characteristics of efficient.Discover, import the intravital adenovirus of mouse, main infected liver cell through blood.Therefore can use adenovirus carrier that foreign gene is imported mouse liver.

Summary of the invention

The making method and the special DNA fragment thereof that the purpose of this invention is to provide a kind of adjustable liver damage animal model.

Dna fragmentation I provided by the invention comprises to downstream from the upper reaches successively: the antisense tetracycline of liver specific promoter, Tet-on gene expression system activates sub-encoding sox.

Said liver specific promoter can adopt multiple promotor, like the mouse albumin promoter.

Said dna fragmentation I also comprises the enhanser that is positioned at the mouse albumin promoter upper reaches, and said enhanser and said mouse albumin promoter form following 1) or 2) or 3) or 4) fusion dna:

1) in the sequence table sequence 3 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 29-2364 position;

2) its nucleotide sequence is the dna molecular shown in the sequence 3 in the sequence table;

3) under stringent condition with 1) or 2) dna sequence dna hybridization that limits and dna molecular with identical function;

4) with sequence table in the dna sequence dna that limits of sequence 3 have 90% above homology, and have the dna molecular of identical function.

It is following 5 that said antisense tetracycline activates son) or 6) protein:

5) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

6) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have antisense tetracycline activate subfunction by sequence 2 deutero-protein.

Said dna fragmentation I also comprise the position be positioned at the polyA that antisense tetracycline activates sub-encoding sox downstream, it is following 7 that the antisense tetracycline of said connection polyA activates sub-encoding sox) or 8) or 9) dna molecular:

7) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

8) under stringent condition with 7) the dna sequence dna hybridization that limits and the said antisense tetracycline of encoding activate the dna molecular of son;

9) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and coding has the protein DNA molecule of antisense tetracycline activation subfunction;

The recombinant expression vector that contains said dna fragmentation I also belongs to protection scope of the present invention.

The present invention also protects by the tsiklomitsin response factor (TRE) of Tet-off/Tet-on gene expression system, promotor and uPA activator encoding sox by the order in the upper reaches to the downstream dna fragmentation II that is connected in sequence.

Said uPA activator is following 10) or 11) protein:

10) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 5;

11) with the aminoacid sequence of sequence 5 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have uPA activator function by sequence 5 deutero-protein.

The encoding sox of said uPA activator is following 12) or 13) or 14) or 15) dna molecular:

12) in the sequence table sequence 4 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 44-1346 position;

13) its nucleotide sequence is the dna molecular shown in the sequence 4 in the sequence table;

14) under stringent condition with 12) or 13) the dna sequence dna hybridization that limits and the dna molecular of the said uPA activator of encoding;

15) dna sequence dna with sequence 4 qualifications has 90% above homology, and the protein DNA molecule of encoding and having uPA activator function.

Said promotor is following 16) or 17) or 18) dna molecular:

16) its nucleotide sequence is the dna molecular shown in the sequence 6 in the sequence table;

17) under stringent condition with 16) dna sequence dna hybridization that limits and dna molecular with identical function;

18) with sequence table in the dna sequence dna that limits of sequence 6 have 90% above homology, and have the dna molecular of identical function.

Said tsiklomitsin response factor (TRE) is following 19) or 20) or 21) dna molecular:

19) its nucleotide sequence is the dna molecular shown in the sequence 7 in the sequence table;

20) under stringent condition with 19) dna sequence dna hybridization that limits and dna molecular with identical function;

21) with sequence table in the dna sequence dna that limits of sequence 7 have 90% above homology, and have the dna molecular of identical function.

The recombinant expression vector that contains said dna fragmentation II also belongs to protection scope of the present invention.

Said recombinant expression vector can import adenovirus expression carrier with said dna fragmentation II and obtain.

Said recombinant expression vector specifically can be Adeno-TRE-uPA, and said Adeno-TRE-uPA imports the Adeno-X carrier with said dna fragmentation II and obtains.

The recombinant adenovirus that the said recombinant expression vector of cultivation obtains in mammalian cell also belongs to protection scope of the present invention.

Said mammalian cell specifically can be the HEK293 cell.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The present invention also protects a kind of making method of liver damage animal model, may further comprise the steps:

1) described dna fragmentation I is imported in the animal, obtain the transgenic founder animal;

2) said transgenic founder animal and scid/bg animal are backcrossed, obtain the animal that carries dna fragmentation I of scid/bg background;

3) with the animal that carries dna fragmentation I of said recombinant adenovirus injection scid/bg background, obtain adjustable liver injury animal.

Said animal specifically can be mouse.

After common backcross for 3 times, can obtain the mouse of the background of immunodeficient.In the actually operating, can backcross 3-5 time.

Use the liver injury model mouse that method of the present invention obtains; Overcome the shortcoming of A1b-uPA mouse; Expression of gene is activated by vibra-(verivate of tsiklomitsin), and expression intensity changes with the dosage variation of Dox, and maximum expression intensity and CMV promotor directly start quite.This systemic characteristic is very high, does not almost have nonspecific effect.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.

Description of drawings

Fig. 1 is the electrophorogram of pcr amplification uPA gene.

Fig. 2 cuts evaluation figure for the enzyme of pGEMT-uPA plasmid.

Fig. 3 cuts evaluation figure for the enzyme of PTRE-uPA.

Fig. 4 cuts evaluation figure for the enzyme of Adeno-TRE-uPA

Fig. 5 is the segmental electrophoresis result of A1b-rtTA.

Fig. 6 is the PCR qualification result of transgenic mice.

Fig. 7 is that the RT-PCR of transgenic mice identifies.

Fig. 8 is the immunohistochemical analysis result of model mice.

Fig. 9 is the RT-PCR analytical results of model mice.

Figure 10 is the HE coloration result of model mice.

Figure 11 is the evaluation that liver injury mouse model external source liver cell of the present invention is rebuild.

Embodiment

Experimental technique among the following embodiment like no specified otherwise, is ordinary method.

The structure of embodiment 1, pAlb-rtTA-polyA plasmid

One, the structure of Psk-rtTA-polyA plasmid

1, cuts pTet-On-Advanced carrier (Clontech company with restriction enzyme EcorI and 37 ℃ of enzymes of HindIII; Catalog No.:630930) spends the night; After enzyme is cut end; Enzyme is cut product and is carried out 1% agarose gel electrophoresis, reclaims the rtTA-polyA fragment that test kit reclaims about 1.2kb with ancient cooking vessel state glue.The dna fragmentation that reclaims is dissolved in the 10ul ultrapure water.The dna fragmentation that reclaims is checked order, see the sequence 1 in the sequence table, the aminoacid sequence of rtTA genes encoding is seen the sequence 2 in the sequence table.

2, cut pBluscript carrier (Fermentas company) with restriction enzyme EcorI and 37 ℃ of enzymes of HindIII and spend the night, after enzyme was cut end, enzyme was cut product and is carried out 1% agarose gel electrophoresis, reclaimed the pSK carrier framework that test kit reclaims about 3kb with ancient cooking vessel state glue.The dna fragmentation that reclaims is dissolved in the 10ul ultrapure water.

3, the pSK carrier framework that rtTA-polyA fragment that step 1 is obtained and step 2 obtain is hatched 2h for 16 ℃, connects.

4, (day root biochemical technology ltd CB104-03), puts on ice and slowly thaws, and adds the connection product of 10ul step 3, mixing to get 1 pipe (100ul) TOP10 competent cell; Continue to place 30min in the ice-water bath; Heat shock 90sec is left standstill in 42 ℃ of water-baths; Be transferred to rapidly and cool off 1-2min in the ice-water bath; Add 600ul and do not contain antibiotic LB substratum, pat mixing gently, put 37 ℃ of shaking table incubation 45min.Getting the LB solid medium of two ammonia benzyl resistances, dull and stereotyped (Amp 125U/ml), respectively is coated with 100ul bacterium liquid; After room temperature left standstill and treats that bacterium liquid absorbs, 37 ℃ of incubators were inverted and are cultivated 10-15hr.

5, from the agar plate that transforms, choose single colony inoculation to the LB solid medium of ammonia benzyl resistance (Amp, 125U/ml) in 37 ℃ of cultivations.

Be extracted in the plasmid of the bacterium colony of growing on the substratum, carry out enzyme with restriction enzyme HindIII and EcorI and cut, carry out agarose gel electrophoresis then.The band that shows about 1.2kb and about 3kb on the gel.Enzyme is cut the male plasmid check order, the result shows and has obtained the purpose plasmid, with its called after Psk-rtTA-polyA.

Two, the structure of pAlb-rtTA-polyA plasmid

1, the acquisition of Alb promotor/enhanser

To be arranged in the enhanser fragment of the 1.99kb between the mouse albumin gene 5 ' upper reaches 10.4-8.5kb and the promoter fragment of 0.33kb and be fused into Alb promotor/enhanser that length is 2.3kb at pAlb-hGH; Concrete experimental technique is referring to document (Pinkert CA et al.An ALB enhancer located 10kb upstreamfunctions along with its promoter to direct efficient, liverspecificexpression in transgenic mice.Genes Dev 1987; 1:268-276.) (Peking University).Alb promotor/the enhancer sequence that obtains is seen the sequence 3 in the sequence table.

2, cut pAlb-Hgh 3-4 hour with restriction enzyme BamH I enzyme; Add the T4 archaeal dna polymerase then, smooth end; Add restriction enzyme Sac I then and carry out enzyme and cut, obtain Alb promotor/enhanser fragment of 2.3kb.

3, cut Psk-rtTA-polyA 3-4 hour with restriction enzyme EcorI enzyme; Add the T4 archaeal dna polymerase then, smooth end; Add restriction enzyme Sac I then and carry out enzyme and cut, obtain the carrier segments of 4.2kb.

4, the Alb promotor/enhanser fragment of step 2 preparation and the carrier segments mixing of step 3 preparation are hatched, connect.

5, with connect product transform the TOP10 competent cell (day root biochemical technology ltd, CB104-03).Getting the LB solid medium of ammonia benzyl resistance, dull and stereotyped (Amp 125U/ml), is coated with 100ul bacterium liquid; After room temperature left standstill and treats that bacterium liquid absorbs, 37 ℃ of incubators were inverted and are cultivated 10-15hr.

6, from the agar plate that transforms, choose single colony inoculation to the LB solid medium of ammonia benzyl resistance (Amp, 125U/ml) in 37 ℃ of cultivations.

Be extracted in the plasmid of the bacterium colony of growing on the substratum, carry out enzyme with restriction enzyme SacI and KpnI and cut, carry out agarose gel electrophoresis then.Show the band of about 3.5kb and about 3kb on the gel, show to have obtained the purpose plasmid, its called after pAlb-rtTA-polyA.

Embodiment 2, making TRE-PminCMV-uPA adenovirus

One, amplification uPA activator (uPA) gene

The a pair of primer of uPA mRNA sequence (NM_008873) design according to mouse among the Gene bank is following:

Upstream primer (29bp): 5 '-GGC GCTAGCCACC ATGAAAGTCTGGCTGGCGAG-3 ';

Downstream primer (29bp): 5 '-TCT GGTACCGAGAGGACGGTCAGCATGGG-3 '.

In the above primer, what underscore marked is restriction enzyme site, and tiltedly runic is expressed for the Kozak sequence is used for strengthening.Upstream primer is positioned at the initial ATG of uPA mRNA, contains the NheI restriction enzyme site.Downstream primer is positioned at 76bp place, terminator codon downstream, contains Kpn I restriction enzyme site.

1, extracting CD-1 mouse (available from dimension tonneau China) kidney RNA and reverse transcription and become cDNA, is template with cDNA, adopts above-mentioned primer, uses the Pyrobest DNA Polymerase of TaKaRa to carry out the PCR reaction.

2, the PCR product is after gel electrophoresis; Application of DNA fast purifying/recovery test kit (Beijing ancient cooking vessel state biotech firm, cat.no A014-1) reclaims, and ultraviolet spectrophotometer is measured A260/280 OD; Estimate the concentration and the purity of PCR product, the DNA of recovery be stored in-20 ℃ subsequent use.The electrophorogram of PCR product is seen Fig. 1.

3, the PCR product that reclaims through external add the A reaction after, be connected into the pGEM-T carrier (available from Promega, catalog number: A3600).

1. add the A reaction

In 200ul PCR pipe, add following solution (TV 10ul):

PCR product 7.2ul

10×PCR?buffer 1ul

dNTP(2.5mM) 0.8ul

Taq archaeal dna polymerase (Takara article No. DRR001A) 0.2ul

Reaction is 30 minutes under 70 ℃ of conditions.

2) the PCR product cloning is gone into the pGEM-T carrier

1. ligation adds following solution to final volume 10ul in the 1.5ml centrifuge tube:

2×T4?DNA?Ligase?buffer 5ul；

pGEM-T(50g/ul) 0.5ul；

Add the PCR product 3.5ul behind the A;

T4DNA ligase enzyme (Promega M1801) 1ul;

After the incubated at room 1 hour, be used for transformation experiment.

2. transform

Getting 5ul connection product joins in the 100ul Top10 competence; Ice bath 30 minutes was hatched 90 seconds for 42 ℃, and ice bath is after 2 minutes again; It is transferred in the 0.5ml LB nutrient solution; Cultivated 45 minutes under 37 ℃, 100rpm condition, draw 50ul then and evenly coat on the 10cm LB flat board of handling through IPTG and X-gal, cultivation is 18 hours under 37 ℃ of conditions.

3. recombinate the selecting and identifying of positive bacterium colony

Select 8～10 single bacterium colonies of finely disseminated white and go into respectively in the 5ml liquid LB substratum, cultivated 12 hours under 37 ℃, 250rpm condition, when treating that substratum becomes muddiness, obtain 1ul bacterium liquid and be used for PCR and identify reorganization, preserve under 4 ℃ of conditions of all the other bacterium liquid.

The Wizard Plus SV Minipreps DNA Purification System test kit of Using P romega company extracts the plasmid that PCR identifies positive strain, identifies with restriction enzyme MluI and NheI.The positive plasmid of plasmid that 3kb and two DNA bands of 1.3kb occur.The result sees Fig. 2.Among Fig. 2, M:marker; 1: the plasmid of positive strain; 2:pGEM-T carrier (negative control).With positive plasmid called after pGEMT-uPA.

Send Beijing three biological Ltd of rich polygala roots to check order the pGEMT-uPA plasmid, sequencing result shows and has obtained correct uPA.

Two, preparation Adeno-TRE-uPA adenovirus

System for use in carrying is the Adeno-X Tet-on expression system of Clontech company, article No. 631050.

1, makes up pTRE-uPA

With NheI and KpnI the uPA fragment is scaled off from the pGEMT-uPA plasmid, be connected on the skeleton of the pTRE-Shuttle2 carrier (Clontech, Adeno-X Tet-on expression system, article No. 631050) that digested with NheI and KpnI.Conversion reaction is the same.The bacterium colony of that resistance of picking card is cultivated, and enzyme is cut the screening positive clone (see figure 3), is pTRE-uPA.

2, make up Adeno-TRE-uPA

With restriction enzyme PI-Sce I and I-Ceu I the TRE-PminCMV-uPA fragment of 3kb is scaled off from pTRE-uPA; Be cloned into adenovirus carrier Adeno-X (Adeno-X Tet-on expressionsystem; Article No. 631050) in, obtains Adeno-TRE-uPA.Connect before the product conversion, connect product with swaI digestion and eliminate background.The bacterium colony of picking Amp resistance is cultivated, the PCR screening positive clone, and positive colony is cut the affirmation (see figure 4) with enzyme.

3, packing is produced the Adeno-TRE-uPA adenovirus

1) transfection is 12-24 hour before, inoculation (1-2) * 10 in the 6cm petridish ⁶HEK293 cell (Adeno-XTet-on expression system, article No. 631050) is in order to raise the efficiency transfection when cell 50%-70%.

2) cell is at 37 ℃, 5%CO ₂Condition is cultivated.

3) each 6cm petridish is with the Adeno-TRE-uPA of calcium phosphate or liposome transfection 10ug Pac I digestion.

4) after one day, detect the cytopathy reaction every day.

5) after the week, transitional cell is to 15ml centrifuge tube (making sure to keep in mind not use trysinization), and the cell that pastes closely can scrape off transfer gently.

6) room temperature 1500g * 5min centrifugal enrichment cell.

7) supernatant discarded, resuspended being deposited among the aseptic PBS of 500ul.

8) three cracking cells of multigelation: in dry ice/ethanol bath, freeze, dissolve in 37 ℃ of water-baths, but do not reach 37 ℃, each back vortex concussion cell that melts.

9) after the freeze thawing three times, frozen in-20 ℃, or be used for for the 10th step.

10) in the petridish of a 6cm, add the 250ul cell lysate, directly be added to lysate in the substratum, a Zhou Yihou should see the cytopathy reaction.

11) if the cell more than 50% comes off from petridish, the cell lysate of-70 ℃ of frozen fronts if do not see the cytopathy reaction, explains that virus titer is too low.

4, amplification recombinant adenovirus

1) infect preceding 24 hours inoculation HEK293 cells to the T75 culturing bottle, cell infects when growing to the 50-70% of culturing bottle.

2) cell is at 37 ℃, 5%CO ₂Condition is cultivated.

3) second day, change liquid with the substratum that contains adenovirus, the virus of the corresponding 1-5pfu of each cell.

4) 37 ℃, 5%CO ₂Condition was cultivated 90 minutes.

5) change the fresh growth medium of 10ml.

6) 37 ℃, 5%CO ₂Condition was cultivated 3-4 days.

7) detect the cytopathy reaction, when 50% cell detachment, the transitional cell suspension centrifuge tube of falling 15ml makes sure to keep in mind not use trysinization, and the cell that pastes closely can scrape off transfer gently.

8) freeze-thaw method results virus.

9) (Clontech Cat.No.631028) measures virus titer with Adeno-X Rapid Titer Kit.

The preparation of embodiment 3, liver injury mouse

One, the preparation of Alb-rtTA mouse

(1) the segmental enzyme of Alb-rtTA cut, recovery and purifying

1, with restriction enzyme SacI and and KpnI37 ℃ of enzyme cut the pAlb-rtTA-polyA plasmid and spend the night.

2, enzyme is cut product and is carried out 1% agarose gel electrophoresis.Electrophoresis result is seen Fig. 5.Downcut the band of 3.5kb size, reclaim test kit (ancient cooking vessel state) with DNA glue and reclaim DNA.

3, be further purified dna fragmentation with Promega Wizard DNA clear-up kit (Cat:A7280).

4, TE (7mM Tris-HCl, 0.15mM EDTA, pH7.5) DNA behind the dissolving purifying.

(2) preparation supplies the DNA sample of procaryotic injection

Measure the OD value of DNA sample, DNA is diluted to 2-5ng/ul with TE; 12000g * 10min (4 ℃) is centrifugal; The careful sucking-off of last 1/3 part solution with after centrifugal is sub-packed in some centrifuge tubes, and is frozen in-20 ℃ of refrigerators.

(3) the protokaryon microinjection is made the Alb-rtTA transgenic mice

To use the protokaryon microinjection by the pAlb-rtTA linearizing dna fragmentation that as above the step purifying is good and make the pAlb-rtTA transgenic mice, concrete experimental procedure is following:

1, choose female ICR mouse in 4～5 ages in week (available from dimension tonneau China), the vaginal tract sealing, as donor, about afternoon 3:00, every mouse peritoneal is injected 10 IU pregnant mare serums (PMSG);

2, after 47～48 hours, every mouse peritoneal is injected people's trophoblast hormone HCG of 10 IU, and mates with normal public mouse; The only of the right age female mouse of peek (more than 2 monthly ages) is chosen the female mouse of oestrusing of vaginal tract flush as acceptor in addition, mates with the public mouse of ligation;

3, observe donor, acceptor before the next morning 9:00, have smart bolt person to take out subsequent use.The acceptor cage is taken out and is performed quarantine measures;

4, about 10:30, disconnected neck is put to death donor, and whole uterine tube is taken out in operation, puts into and contains 0.05% Unidasa M2 liquid, and microscopically is torn ampulla of uterine tube with tweezers, and zygote is promptly together flowed out in company with granulosa cell;

5, examine the zygote that is placed in the Unidasa M2 liquid, when the granulosa cell around the zygote breaks away from,, put into M2 liquid and wash, be placed at last in the M16 liquid and put into 5%CO the zygote sucking-off ₂, 37 ℃ of incubators are cultivated;

6, examine under a microscope, it is full to select cell, and zona pellucida is clear, and the apparent zygote of male pronucleus is for use;

7, install and to hold ovum pin and entry needle, make its end be parallel to Stage microscope, splash into a M2 liquid, cover Yellow Protopet 2A, move into zygote to be injected in the central authorities of depression slide.The bubble of DNA in entry needle should be walked by formerly whole bullets.

8, under high power lens, entry needle is touched ovum holding tube, DNA is slowly flowed out and control its flow; Pressure-vaccum zygote makes it be in the optimum position repeatedly, and entry needle is thrust the male pronucleus of zygote, promptly withdraws from until seeing that protokaryon expands.The zygote of injecting He do not inject is placed apart up and down, be unlikely to mix and stir, after injection finishes, put into 5%CO ₂, 37 ℃ of incubators are cultivated.

9, with acceptor anesthesia, injection measurement is 1% vetanarcol 0.01ml/g, abdominal injection.Operation is taken out ovary and is connected uterine tube, fixes with fatty tweezer, finds the uterine tube opening at microscopically.Draw injection after cultivate into zygote alive, the absorption method is to inhale one section long M2 earlier, inhales a bubble, draws zygote then, closely arranges as far as possible, inhales one section liquid again, inhales a bubble, inhales one section liquid again, three bubbles of totally four sections liquid.Remove long that section liquid, remaining liquid is roughly about 1cm, about bubble 0.2cm.To transplant the mouth of pipe and insert the uterine tube mouth, gently the liquid in the grafts will be blown into, and see that ampulla of uterine tube expands and clearly see three bubbles, i.e. successful transplantation.Ovary is put back to the abdominal cavity together with uterine tube, suture muscles and skin.

10, acceptor is whenever weighed once at a distance from a week, when increasing than weighing for the first time for the second time, can tentatively judge pregnancy.The newborn mouse childbirth in 19～21 days of operation back is transgenic (Founder) mouse of establishing.

(4) evaluation of male mouse

1, PCR identifies

Positive mouse tail genomic dna is carried out pcr analysis, identify whether it is the transgenic positive mouse.

Adopt Alb-rtTA to stride gene primer and carry out the PCR detection.Upstream primer is based on Alb promoter sequence (Alb/p), and downstream primer (primer II) is based on the rtTA gene order, and target fragment is 700bp.Primer sequence is following:

Upstream primer (primer I): 5 '-tgg ctg aaa gtt act gtg gga-3 ';

Downstream primer (primer II): 5 '-aga tgg tgc gct cct gga cgt-3 '.

In 32 positive mouse, have 4 PCR and be accredited as the positive, be transgenic mice.The PCR qualification result of 4 transgenic mices is seen Fig. 6.Among Fig. 6, PC: plasmid pAlb-rtTA-polyA (positive control); 1-5,2-9,2-10, a 4-6:4 transgenic mice; NC:ICR mouse (negative control).

2, RT-PCR identifies

The procaryotic injection transgenic method; The purpose fragment is a random integration; The situation of transgene silencing and express spectra mistake takes place sometimes; For the rtTA that confirms to obtain transgenic mice whether express and express spectra whether correct, the method that adopts RT-PCR tissue to the heart, liver, lung, kidney and the pancreas of mouse on the mRNA level detects.

4 transgenic mices are all expressed rtTA, and express spectra is correct, and are strong special at the liver expression goal gene, except liver, have only kidney that faint expression is arranged.Wherein the qualification result of a transgenic mice is seen Fig. 7.

Two, backcross and obtain the Alb-rtTA transgenic mice of immunodeficient background

In order to accept the external source liver cell, need the mouse background of this model conversion, and have only the mouse of immunodeficient could be by the adenovirus repeated infection to immunodeficient.Scid/bg is a kind of immunodeficient mouse, no sophisticated T cell and B cell in the body, NK cell disappearance.This mouse can be accepted foreign cell and tissue, thus we with the Alb-rtTA mouse through repeatedly backcrossing, convert the Scid/bg background into.

(1) transfers to the SPF environment to the Alb-rtTA transgenic mice under the conventional environment through the c-section purification

1, selects the Alb-rtTA transgenic mice of the mating purification object of cutting open the belly not in 6～8week age.Post-coitum is observed cloudy bolt after second day continuous a couple of days, and writes down cloudy bolt and the date occurs, and as becoming pregnant the date, 19 ± 1d is as the expected date of childbirth for gestation, i.e. the c-section date.

2,, after disconnected cervical vertebra is put to death, soak 2～3s with 2% Peracetic Acid immediately to the pregnant mouse of the expected date of childbirth; Promptly on operating table, fix, cut open the belly, clamp nearly anus virgin uterine neck, cut off the uterus with aseptic scissors from anus one side of mosquito forceps again with aseptic mosquito forceps; After taking out whole uterus, put 2% Peracetic Acid liquid and soak 1～2s, in the aqueduct liquid (thimerosal) of operation shield retaining, the uterus is transferred in the aseptic operation shield retaining rapidly; Cut the uterus, take out the young baby, little young whole body is wiped clean with sterile gauze; Scarlet to little young whole body again, till freely breathing.

3, the young baby alive of c-section gained; (tie up the aseptic ICR mouse that tonneau China Experimental Animal Center is raised, mating shifts to an earlier date in 3～4d) boxes than the Alb-rtTA mouse of cutting open the belly, and every nurse is for about 8 of breasts to put into the nurse mouse; And produce after little son fully contacts with nurse, put to death nurse and produce little son.

4, be transferred to aseptic shield retaining from the operation shield retaining and raise, 21d is from breast.After each item microorganism detection is qualified, is transferred in the SPF barrier system and raises.

5, changed the Alb-rtTA mouse of raising in the SPF barrier system over to, per season censorship is (5～10) once, make each item microorganism detection.

(2) the Alb-rtTA transgenic mice of acquisition scid/bg background

Transgenic mice that purifies and scid/bg mouse (available from dimension tonneau China) are backcrossed under the SPF environment, and mouse and scid/bg mouse that the back method through PCR of at every turn backcrossing detects transgenic positive are backcrossed next time.After backcrossing for 3 times, the scid gene test of mouse is found that transgenic mice has carried pure and mild scid site, IgG is detected discovery content far below normal mouse, explain that mouse has obtained the background of immunodeficient.In order further to reduce the immunizing power of mouse, mouse is continued to backcross.The Alb-rtTA mouse of the scid/bg background that obtains after backcrossing for 5 times carries out the test of step 3.

Three, the acquisition of liver injury model mouse

The Alb-rtTA transgenic mice of 10 immunodeficient backgrounds is divided into two groups, 5 every group (first group and second group).10 ICR mouse are divided into two groups, 5 every group (the 3rd group and the 4th group).First group and the 3rd group of mouse in the following whole test stage with the Dox aqueous solution (1mg/ml) as tap water.Second group and the 4th group of mouse in the following whole experimental phase with ortho-water as tap water.Each is organized mouse and injects adenovirus Adeno-TRE-uPA 5 * 10 respectively ⁹/ only, dissect mouse after three days and get liver organization, carry out RT-PCR and immunohistochemical analysis.

For the injection adenovirus, DOX inductive Alb-rtTA transgenic mice was opened the abdominal cavity discovery in 1 day behind virus injection, and the mouse liver outward appearance is pale, and this hepar damnification characteristic with the Alb-uPA mouse of report is consistent.The pcr analysis result shows that respectively organizing mouse is all infected by adenovirus uPA.Immunohistochemical analysis result sees Fig. 8.Among Fig. 8, A) wild-type mice; B) Dox inductive wild-type mice; C) Alb-rtTA transgenic positive mouse; D) Dox inductive Alb-rtTA transgenic mice.Have only DOX inductive Alb-rtTA mouse can detect the expression of uPA, the liver cell of statistical result showed more than 80% all expressed uPA.And in its excess-three kind mouse, all do not detect uPA.The RT-PCR analytical results is seen Fig. 9.Among Fig. 9, WT) wild-type mice; WT+DOX) Dox inductive wild-type mice; Alb-rtTA) Alb-rtTA transgenic positive mouse; Alb-rtTA+Dox) Dox inductive Alb-rtTA transgenic mice.The result of RT-PCR conforms to the immunohistochemical methods result, promptly has only two elements to exist simultaneously and just can start expression with uPA under the DOX inductive situation.The HE coloration result is seen Figure 10.Among Figure 10, A) DOX inductive wild-type mice; B) DOX inductive Alb-rtTA transgenic mice.The wild-type mice cellular form is normal, the damage characteristic of the stem cell formation of vacuoles appearance of Alb-rtTA transgenic mice.

Four, the hepatocellular reconstruction of external source

1, the acquisition of external source liver cell

With tribromoethyl alcohol with GFP transgenic mice (available from dimension tonneau China) anesthesia after, open the abdominal cavity, the separation gate vein, the scalp acupuncture that will have flexible pipe thrusts portal vein, bulldog clamp is fixed.Flexible pipe connects syringe, pours into PBE50ml and PBC50ml successively.Cut liver then, WB washes liver.Tear the coating of liver with tweezers, sieve filtration cell with cell.800rpm * 1min is centrifugal, carefully sops up supernatant, uses the WBD re-suspended cell, and is centrifugal; Use DF12 (GIBICO) washed twice again, trypan blue dyeing detects the liver cell survival rate.

Perfusion damping fluid (PB) prescription (PH7.4): NaCl 27g, KCl 1.26g, NaHCO ₃6.3g, Glucose2.4g, Hepes 14.34g, H ₂O 3000ml.

The prescription of PBE (PH7.4): EDTA 0.37g, PB 500ml.

The prescription of PBC (PH7.4): Collagenase 4.2g, CaCl ₂-2H ₂O 0.275g, PB 500ml.

The prescription of WB (PH7.4): NaCl 17.5g, KCl 1.15g, CaCl ₂-2H ₂O 0.325g, Hepes 5.95g, BSA 2.5g, H ₂O 2500ml.

The prescription of WBD (PH7.4): MgCl ₂-6H ₂O 0.15g, MgSO ₄-7H ₂O 0.15g, DNase 0.15g, WB 1500ml.

2, the hepatocellular reconstruction of external source

The whole test stage as tap water, is respectively injected adenovirus Adeno-TRE-uPA 5 * 10 with the Alb-rtTA transgenic mice of 10 immunodeficient backgrounds with the Dox aqueous solution (1mg/ml) ⁹/ only, obtain the liver injury mouse model.

At second day (testing the 4th day) liver transplantation cell of injection adenovirus, every mouse transplants 5 * 10 ⁵Individual.Transplant per 7 days every the injected in mice 5 * 10 in back ⁹Adenovirus promotes the propagation of foreign cell.The per injection adenovirus detects the effect of transplanting after 7 days.

Hepatocellular implantation method is following: with tribromoethyl alcohol model mice is anaesthetized, side position is on experiment table.Spleen position, left side skin is picked hair, cut off skin and muscle layer, the about 1cm of openings of sizes.Isolate spleen with tweezers, from the spleen end liver cell is injected spleen slowly with syringe.After having annotated, have an acupuncture treatment the position, hole in case hemorrhage with toe-in.Then with the spleen replaced, suture muscles layer and skin layer successively.Mouse after the transplanting is put on the insulation tire and is incubated to reviving.

The result sees Figure 11.Among Figure 11, P1-P5 promotes once to five times for transplanting back adenovirus Adeno-TRE-uPA.For the injection adenovirus, and DOX inductive Alb-rtTA transgenic mice, behind virus injection, can observe liver 4 days the time has point-like regeneration, and the regeneration zone of joint knot shape appears in liver in the time of 7 days, and whole liver recovers fully after 10 days.The GFP liver cell is rebuild the mouse liver of uPA damage.After promoting for the first time, can detect the cell mass of 4-5 cell in the liver of mouse; After promoting for the second time, these cell masses are further bred; Promote for three times the back cell mass to reach more than 100 cell, the reconstruction degree reaches 20%; Promote for four times the back cell mass to begin to link in flakes, the reconstruction rate reaches 50%; Continue to promote that with adenovirus uPA foreign cell reconstruction rate is near 80%.

Sequence table

< 110>Peking University

< 120>making method of adjustable liver damage animal model and special DNA fragment thereof

<130> CGGNARY81956

<160> 7

<210> 1

<211> 1228

<212> DNA

< 213>artificial sequence

<220>

<223>

<400> 1

<210> 2

<211> 248

<212> PRT

< 213>artificial sequence

<220>

<223>

<400> 2

<210> 3

<211> 2372

<212> DNA

< 213>artificial sequence

<220>

<223>

<400> 3

<210> 4

<211> 1414

<212> DNA

< 213>Mus mouse kind (Mus musculus)

<400> 4

<210> 5

<211> 433

<212> PRT

< 213>Mus mouse kind (Mus musculus)

<400> 5

<210> 6

<211> 129

<212> DNA

< 213>artificial sequence

<220>

<223>

<400> 6

<210> 7

<211> 312

<212> DNA

< 213>artificial sequence

<220>

<223>

<400> 7

Claims (8)

1. a recombinant expression vector imports adenovirus expression carrier with the dna fragmentation II and obtains;

Said dna fragmentation II is for being connected in sequence by tsiklomitsin response factor, promotor and the uPA activator encoding sox of the Tet-off/Tet-on gene expression system order by the upper reaches to downstream.

2. recombinant expression vector as claimed in claim 1 is characterized in that: said recombinant expression vector is Adeno-TRE-uPA, and said Adeno-TRE-uPA imports the Adeno-X carrier with said dna fragmentation II and obtains.

3. according to claim 1 or claim 2 recombinant expression vector is characterized in that: the protein that said uPA activator is made up of the aminoacid sequence shown in the sequence in the sequence table 5.

4. according to claim 1 or claim 2 recombinant expression vector, it is characterized in that: the encoding sox of said uPA activator is following 1) or 2) dna molecular:

1) in the sequence table sequence 4 from the dna molecular shown in the deoxyribonucleotide of the terminal 44-1346 of 5 ˊ position;

2) its nucleotide sequence is the dna molecular shown in the sequence 4 in the sequence table.

5. recombinant expression vector as claimed in claim 4 is characterized in that: said promotor is that nucleotide sequence is the dna molecular shown in the sequence 6 in the sequence table.

6. recombinant expression vector as claimed in claim 5 is characterized in that: said tsiklomitsin response factor is that nucleotide sequence is the dna molecular shown in the sequence 7 in the sequence table.

7. in mammalian cell, cultivate the recombinant adenovirus that arbitrary said recombinant expression vector obtains among the claim 1-6.

8. recombinant adenovirus as claimed in claim 7 is characterized in that: said mammalian cell is the HEK293 cell.

Images