CN100540660C - A kind of transgenic mice hepatopathy animal model, its preparation method and application - Google Patents
A kind of transgenic mice hepatopathy animal model, its preparation method and application Download PDFInfo
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- CN100540660C CN100540660C CNB2004100540862A CN200410054086A CN100540660C CN 100540660 C CN100540660 C CN 100540660C CN B2004100540862 A CNB2004100540862 A CN B2004100540862A CN 200410054086 A CN200410054086 A CN 200410054086A CN 100540660 C CN100540660 C CN 100540660C
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Abstract
The invention discloses a kind of transgenic mice hepatopathy animal model, the genome conformity of this transgenic mice animal model has dna fragmentation LLTK, described dna fragmentation LLTK is by mouse albumin gene promotor ALB/pro and enhanser ALB/en, and downstream gene herpes simplex thymidine kinase (HSV-TK) the cDNA formation that is subjected to its regulation and control.The invention also discloses the preparation method and the application of this transgenic mice animal model, induce liver to produce the hepatitis diseases related of clinical biochemical index and pathomorphism and human toxin-induced by tail vein injection GCV.This animal model damage is easy to control, and mechanism is more clear, and the disease type is clearer and more definite, and model stability can repeat.The medicine that can be used for screening treatment hepatopathy (hepatitis) is estimated the method for the treatment of hepatopathy (hepatitis).
Description
Technical field
The invention belongs to medical care evaluation, detection technique field, specifically, the invention relates to a kind of transgenic mice hepatopathy animal model, its preparation method and application.
Background technology
Hepatic diseases is one of human modal disease, and human health in serious threat.The animal model tolerable duplicates some disease that can not directly observe on mankind and study, as the important means of medical research.The foundation of hepatopathy animal model is for pathogenesis, pathophysiological process, pathomorphism change and the medical treatment of inquiring into hepatopathy have been opened up wide prospect.But the cell of some particular type of kill animals body optionally, thus certain disease imitated, and present method is very limited.For example, by surgical operation or laser, can not be on molecular level designated cell, and the material that can point to specific cells automatically is also seldom.The means that are usually used in setting up hepatopathy model now have: by mechanical stimuluss such as surgical operation common bile duct ligation; Immunologic injury (albumin tail vein injection, heterogenous animal serum abdominal injection); Alcohol or malnutrition; Pathogenic infection; Damage due to chemical substance is poisoned; Comparatively the common chemical material has D-Gal (Gal-NH
2), concanavalin A, N-ethyl nitrosamine (DEN), thioacetamide, tetracol phenixin (CCl
4).1979, Galanos etc. adopt GalN inducing mouse toxic liver injury, hepatic injury mechanism mainly is an interfere RNA and proteinic synthetic, cause endotoxemia, produce oxyradical and lipid peroxidation and cause the film dysfunction, the lesion wire plastochondria causes the impaired grade of oxidative phosphorylation (GalanosC.et al.Proc.Natl.Acad.Sci.U.S.A.1979 Nov; 76 (11): 5939-43).The concanavalin A intravenous injection mouse of development such as Tiegs in 1992 is induced hepar damnification (Tiegs G.et al.J.Clin.Invest.1992 Jul; 90 (1): 196-203).This class model is similar to human viral hepatitis, extensively be used in the research of drug screening, yet symptom is fulminating often.Use N-ethyl nitrosamine (DEN), thioacetamide to be difficult for grasping dosage.CCl
4It is the most widely used the earliest hepatotoxin of bringing out experimental hepatic fibrosis, be used to the kinds of experiments animal, produce chronic or acute liver poisoning (Allis JW.et al.Fundamental and Applied Toxicology.15:558-570,1990 by gastrogavage, subcutaneous injection or inhalation; Pritchard DJ et al.Journal of Applied Toxicology.7:229-236,1987; Ray SD et al.Fundamental and Applied Toxicology.15:429-440,1990).Yet, because CCl
4Toxicity is bigger, and the animal surviving rate is not high, the stability of model, and repeatability is lower.
The transgenic animal technology is a brand new technical in the life science that grows up of recent two decades, in the medicine and pharmacology field, both can be used as " bio-reactor " and used, and also can be used as disease animal model and be used for disease mechanism and chemotherapeutics screening study.Basic skills of transgenic animal and principle all are to be based upon gene fragment exchange reorganization (plasmid reorganization), on the basis that specific gene expression and karyomit(e) outer-gene shift.But when causing particular organization's expressing protein toxin with engineered method, animal can not be controlled with these methods experimentally in the early stage death of life.As the mid-90, the hepatic diseases mouse model of development: alb-uPA transgenic mice.Sangren etc. produce hepatotoxicity (Sangren EP.et al.Cell, 1991,66:245~256) by urokinase profibr(in)olysin incitant uPA gene is inserted into the albumin promoter downstream to mediate its expression special in liver.Generalized case, most of uPA is secreted into blood, thereby is considered to the thrombolytic agent factor.At this model, some uPA still are retained in liver and cause serious inflammation.The alternate model similar to uPA mouse transplantation model is fumarylacetoacetic acid salt hydrolysis enzyme (FAH
-) knock out mice (Grompe M.et al.Genes.Dev., 1993,7 (12A): 2298~2307).This model is by the animal model of development such as Grompe as human inheritance's property tyrosinemia I type (HT1).This model mice lacks FAH, and the catabolic final step of tyrosine is hindered, and causes the gathering of fumarylacetoacetic acid salt (FAA) and precursor (MAA) substrate, and FAA and MAA are toxic to liver.These two models are liver heredopathia mouse.Yet, alb-uPA transgenic mice and FAH
-The raising of transgenic mice has certain limitation with breeding, because only under the effect of the condition of adding (medicine), mouse just can be dead, and liver function depletion does not take place yet.The normal need of its liver function is kept by medicine.
Heyman etc. proposed the notion (Heyman that TK knocks out (TK obliteration) as far back as 1989, R.A.et al.Proc.Natl.Acad.Sci.USA 86:2698-2702,1989), promptly in the transgenic mice body, knock out specific cells by the cell type specificity promotor.Herpes simplex thymidine kinase (HSV-TK) is different from mammiferous nucleoside kinase, and HSV-TK is phosphorylated nucleosides and nucleoside analog such as ganciclovir (GCV) effectively, and it is synthetic to hinder DNA, thereby causes the death of cell.Its mechanism of action is: HSV-TK genes encoding thymidine kinase, and this enzyme can be a diphoponate with nucleoside analog (NA) nontoxicity antiviral GCV metabolism, the latter produces triphosphoric acid GCV under the effect of desmo enzyme; Triphosphoric acid GCV is toxic, and it can obviously suppress cell dna polymerase, thereby arrestin matter is synthetic, killer cell.HSV-TK or GCV do not have influence substantially to body separately.This system allows the investigator by time and stage that GCV control cell knocks out, is the effective means of understanding the physiological function of particular cell types.As under cell specificity promotor control, knocking out the growth hormone secretion cell, T cell and B cell, il-22,4 express cells, dendritic cell, fibroblast (Borrelli, E.et al.Nature (London) 339:538-541,1989; Dzierzak, E.et al.Int.Immunol.5:975-984,1993; Kamogawa, Y.et al.Cell 75:985-995,1993; Minasi, L.E.et al.J.Exp.Med.177:1451-1459,1993; Salomon, B.et al.J.Immunol.152:537-548,1994; Bin Tian et al.American Journal of Pathology, Vol.163, No.2, August2003).
Summary of the invention
The transgenic mice hepatopathy animal model that provides a kind of damage to induce and to regulate and control just is provided purpose of the present invention.
Another object of the present invention is to provide the preparation method of the transgenic mice hepatopathy animal model that a kind of damage can induce and regulate and control.
Another purpose of the present invention is to provide the application of the transgenic mice hepatopathy animal model that a kind of damage can induce and regulate and control.
Because the mouse growth cycle is short, be easy to feeding and management, obtain mouse of inbreeding mating qualitatively and genetic mutant easily, comparatively economical and practical as laboratory animal, be the model animals systems close such as genome structure, ontogenetic process, histocyte structure with the mankind.
Albumin gene promotor ALB/pro high conservative during evolution, and the liver cell specificity regulatory mechanism of height becomes ALB/pro to be suitable for the promotor of liver cell specifically expressing study on regulation.
We regulate and control herpes simplex thymidine kinases (HSV-TK) at the adult hepatic specifically expressing with albumin gene enhanser ALB/en and ALB/pro, utilize mouse to make the liver injury experimental animal model.Inartificial giving and GCV do not have influence substantially to body if this kind model only has the expression of HSV-TK, so be easy to captive breeding.
Key of the present invention is to utilize the GCV drug susceptibility of HSV-TK and the liver specificity of mouse albumin gene promotor and enhanser to regulate and control, produce transgenic mice, injection by GCV produces hepar damnification, and realizes the adjustable of hepar damnification by GCV time of administration and dosage.
The preparation method of this transgenic mice comprises the following steps:
One, the clone of mouse albumin gene promotor ALB/pro;
Two, the clone of mouse albumin gene enhanser ALB/en;
Three, pcr amplification obtains HSV-TK cDNA;
Four, be goal gene with HSV-TK cDNA, ALB/pro and ALB/en are controlling element, make up liver specificity and kill and wound carrier pLLTK;
Five, digestion with restriction enzyme linearizing pLLTK reclaims the also fragment of the about 4130bp of purifying;
Six, microinjection mouse male pronucleus obtains positive transgenic mice.
The Mouse Liver disease model damage that present method produces is easy to control, and mechanism is more clear, and the disease type is clearer and more definite, and model stability can repeat.Because goal gene tissue specific expression under the control of liver specificity controlling element, the damage high specificity is compared with the transgenic animal model that general toxin protein is expressed, and is easy to captive breeding.Can be used for screening the medicine of treatment hepatitis, estimate the method for treatment hepatitis.
Description of drawings
Fig. 1 is the structure schema of plasmid pLLTK.
Fig. 2 cuts the evaluation electrophoretogram for the enzyme of pLLTK, wherein M:1kb marker; 1:AgeI+XhoI:1.9kb+6.5kb; 2:KpnI:2.1kb+6.3kb.
Fig. 3 is F0 and the F1 PCR detected result for transgenic mice, wherein 1: positive control; 2: negative control; 3: blank; 4: positive transgenic mice F0 generation; M:marker 100bp; 5~13:F1 is for the transgenic mice offspring.
Fig. 4 is the PCR detected result of F2 for transgenic mice, and wherein 1~10:F2 is for transgenic mice; 11: positive transgenic mice F1 generation; 12: blank; 13: negative control: 14: positive control; M:marker 100bp.
Fig. 5 is the PCR detected result of F3 for transgenic mice, and wherein 1~11:F3 is for transgenic mice; 12: blank; 13: negative control; 14: positive control; 15: positive transgenic mice F2 generation; M:marker 100bp.
Fig. 6 is the agarose gel electrophoresis figure that the HSV-TK of transgenic mice expresses, wherein M:marker100bp; 1: blank; 2: negative control; 3: positive control; 4: the heart; 5: liver; 6: spleen; 7: kidney; 8: brain; 9: pancreas; 10: intestines; 11: blood; 12: skin; 13: testis.
Fig. 7 is an immunohistochemical analysis HSV-TK protein expression situation synoptic diagram, and wherein A is the TK mouse liver; C is the TK mouse kidney; B is the non-transgenic mouse liver; D is a non-transgenic mouse liver kidney.
Fig. 8 handles the variation synoptic diagram of front and back biochemical values for GCV.
Fig. 9 is the painted result schematic diagram of liver organization HE, and A, C are that GCV handles back TK mouse liver; B is the TK transgenic mice liver that is untreated; D is that GCV handles back ALB transgenic mice liver.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.Should be understood that these embodiment only are used to the present invention is described but not are used to limit scope of the present invention.
Embodiment 1The clone of mouse albumin promoter ALB/pro
The tail vein is taked Kunming white mouse peripheral blood 200 μ l, extracts genomic dna according to conventional phenol chloroform method, is template with the genomic dna, and is by nest-type PRC amplification Kunming white mouse albumin gene promotor ALB/pro, specific as follows:
1, primer:
pro?1?kpn?I:5′-cttaggtacctccatgccaaggcccaca-3′
pro2?Age?I:5′-cgtataccggtagtggggttgataggaaagg-3′
2, reaction system:
Masterplate: 1.0 μ l
10×buffer 2.5μl
dNTP(25mM) 2.5μl
pro3(10mM) 1.0μl
pro4(10mM) 1.0μl
TagE(5U/μl) 0.25μl
ddH
2
O 16.75μl
25.0μl
3, loop parameter condition:
94 ℃ 5 minutes;
95 ℃ 30 seconds-1 minute, 50 ℃-55 ℃ 30 seconds-1 minute, 72 ℃ 30 seconds-1 minute, altogether 28-35 does not follow bad;
72 ℃ 10 minutes.
Obtain the Kunming white mouse albumin gene promotor ALB/pro shown in the SEQ ID No.1 by above amplification, total length 335bp is positioned at transcripting start point downstream 310bp to upstream 25bp.
Embodiment 2The clone of mouse albumin gene enhanser ALB/en
With the Kunming white mouse gene group DNA is template, by pcr amplification clone mouse albumin gene enhanser ALB/en, specific as follows:
1, primer:
en?1:5′-acgag?tctaga?gtggagctta?cttctttgatttga-3′
en2?kpnI:5′-aaacggtaccatgccatgtaaatcaact-3′
2, reaction system:
DNA(20-100ng): 1.0μl
10×buffer 2.5μl
dNTP(25mM) 2.5μl
en1(10mM) 1.0μl
en2(10mM) 1.0μl
TagE(5U/μl) 0.25μl
ddH
2
O 16.25μl
25.0μl
3, loop parameter condition:
94 ℃ 5 minutes;
95 ℃ 30 seconds-1 minute, 50 ℃-55 ℃ 30 seconds-2 minutes, 72 ℃ 30 seconds-2 minutes, altogether 28-35 is followed bad;
72 ℃ 10 minutes.
Obtain the Kunming white mouse albumin gene enhanser ALB/en shown in the SEQ ID No.2 by above amplification, the about 2059bp of total length is positioned at transcripting start point upstream-9192bp and arrives-11250bp.
Embodiment 3Pcr amplification obtains herpes simplex thymidine kinase (HSV-TK) cDNA
With plasmid pTK-neo (novagen company) is template, by pcr amplification HSV-TK cDNA, specific as follows:
1, primer:
tk?1?ageI?5‘-gtataccggtggcaccatggcttcgtaccccggc-3’
tk25‘-ccg?cgt?cga?cgg?aaa?agc?gcc?tcc?cct?ac-3’
2, reaction system:
DNA(20-100ng): 1.0μl
10×buffer 2.5μl
dNTP(25mM) 2.5μl
tk1(10mM) 1.0μl
tk2(10mM) 1.0μl
TagE(5U/μl) 0.25μl
ddH
2
O 16.25μl
25.0μl
3, loop parameter condition:
94 ℃ 5 minutes;
95 ℃ 30 seconds-1 minute, 45 ℃-55 ℃ 1 minute-2 minutes, 72 ℃ 1 minute-2 minutes, altogether 28-35 is followed bad;
72 ℃ 10 minutes.
Obtain HSV-TK cDNA (TK) by above amplification, its sequence is shown in SEQ ID No.3.
Embodiment 4Liver specificity kills and wounds the structure of carrier
As shown in Figure 1, with HSV-TK cDNA is goal gene, with ALB/pro and ALB/en is controlling element, (Invitrogen company) is framework with pCDNA3.1 (+)/Zeo plasmid, restriction enzyme site by the DNA two ends is put down, cohesive end connects, establishing target carrier-liver specificity kills and wounds carrier pLLTK, and is specific as follows:
1, the linearizing of frame carrier
Cut carrier for expression of eukaryon pCDNA3.1 (+)/Zeo with restriction enzyme NruI and NheI enzyme and obtain the terminal linear fragment flat, cohesive end that is.
2, the enzyme of junction fragment is cut
A) cut ALB/pro with KpnI and AgeI enzyme and obtain the linear fragment that two ends are cohesive end;
B) cut ALB/en with the KpnI enzyme and obtain the linear fragment that two ends are respectively flush end and cohesive end;
C) cut TK with the AgeI enzyme and obtain the linear fragment that two ends are respectively flush end and cohesive end.
3, connect
4, the clone makes up and carries out according to molecular cloning test guide (J. Sa nurse Brooker), obtains carrier pLLTK.
5, enzyme is cut evaluation
Carrier pLLTK is carried out digestion with restriction enzyme, reacts as follows:
(1) select AgeI (10u/ μ l, buffer L, promega), XhoI (10u/ μ l, buffer H, TakaRo),
(2) (10u/ μ l, buffer 4, NEB) to select KpnI
(3) enzyme is cut product and is carried out agarose gel electrophoresis and identify, its electrophoretogram as shown in Figure 2, institute's fragment length that obtains is consistent with expected results.
Embodiment 5Produce the transgenic mice family
The plasmid that embodiment 4 is obtained carries out plasmid purification (carrying out according to QIAGEN company, Protocol for EndoFree Plasmid Maxi Kit) with no intracellular toxin plasmid large-scale purification test kit; With restriction enzyme Hand III linearization for enzyme restriction, agarose gel electrophoresis reclaims the dna fragmentation LLTK of about 4130bp, carries out purifying (carrying out according to QIAGEN company, QIAquick Gel Extraction Kit Protocol) then; Use Schleicher ﹠amp at last; The S ﹠amp of Schuell company; S ELUTIP Minicolumns purifying (carries out Schleicher ﹠amp according to Basic Protocol for DNA for DNA Purification from 50bp-50Kb; Schuell company); DNA is dissolved in 10mMTris, 0.1mMEDTA, PH7.4 carries out the microinjection of mouse male pronucleus according to 4ng/ μ l; Step is according to conventional transgenic mice preparation process.
1, the PCR initial analysis proves former generation and the genetically modified integration of offspring mouse;
Transgenic mice detects its offspring's positive rate after passing for 3 generations, it is as follows that PCR detects design of primers:
stk1:5’-gtataccggtatgcccacgctactgcgg-3’
SH552:5‘-gatggcggtcgaagatgagg-3’
Product length: 390bp
Detected result is shown in Fig. 3-5:
As seen from Figure 3, F0 and F1 for the PCR detected result of transgenic mice are:
6,8,10,11,13:F1 is for positive transgenic mice offspring; 5,7,9,12:F1 is for negative transgenic mice offspring, positive rate is 5/9=56%;
As seen from Figure 4, F2 for the PCR detected result of transgenic mice is:
1,2,3,5,7:F2 is for positive transgenic mice; 4,6,8,9,10:F2 is for negative transgenic mice, positive rate is 5/10=50%.
As seen from Figure 5, F3 for the PCR detected result of transgenic mice is:
1,4,5,7,8:F2 is for positive transgenic mice; 2,3,6,9,10,11:F2 is for negative transgenic mice, positive rate is 5/11=45%.
The above results shows that after passing for 3 generations, offspring's positive rate is 45%~56%, illustrate transgenosis stable integration in the genome of transgenic mice, and by reproductive tract genetic stability between the generation.
Embodiment 6The tissue specific expression of HSV-TK
Being object with the heart, liver, spleen, kidney, brain, pancreas, intestines, blood, skin and testis respectively, is internal reference with 260bp β-actin, analyzes the tissue specific expression of HSV-TK by RT-PCR, its result as shown in Figure 6:
Wherein primer is as follows:
β-actin primer:
actin?1:5‘-ctacaatgagctgcgtgtggc-3’
actin?2:5‘-caggtccagacgcaggatggc-3’
The tk primer is the same.
As shown in Figure 6,390bp HSV-TK purpose band only occurs expressing at liver and testis.
Embodiment 7Immunohistochemical methods
The immunohistochemical methods step is according to conventional steps.
One is anti-: the HSV-TK rabbit polyclonal antibody;
Two is anti-: biotin labeled goat-anti rabbit;
Three is anti-: avidin coupling peroxidase.
Its result as shown in Figure 7, visible HSV-TK albumen is at liver expression, and do not express at kidney.
1, the biochemical indicator inspection of blood
Tail vein injection GCV induces the damage of transgenic mice.Control mice is the transgenic mice that mammary gland is expressed ALB.Tail vein blood, continuous three weeks are measured the biochemical values of blood weekly, its result as shown in Figure 8, as seen from the figure, injected for the first time back 21 days at GCV, with compare before the injection, the detected value of gpt ALT, glutamic-oxal(o)acetic transaminase AST, total bilirubin TBIL increases (P<0.05) in transgenic mice group significance, and control mice does not have significant difference.The detected value of liver acid anhydride CREA there are no significant difference (figure is row).The biochemical analysis result of blood shows the damage of liver function, renal function normal.
2, pathomorphism inspection
After putting to death mouse, get liver organization and carry out the pathomorphism analysis, method is according to conventional H E staining procedure, the result as shown in Figure 9, after the result showed that GCV handles the TK transgenic mice, the liver lobule focal necrosis appearred in liver organization, indivedual apoptotic bodies, pathological manifestations such as lymphocytic infiltration (A, C), and the TK transgenic mice liver (B) that is untreated, ALB transgenic mice liver the morphologic change of histopathology (D) do not occur substantially after GCV handles.
In sum, transgenic mice hepatopathy animal model damage of the present invention is easy to control, mechanism is more clear, and the disease type is clearer and more definite, and model stability can repeat, and owing to goal gene tissue specific expression under the control of liver specificity controlling element, the damage high specificity is compared with the transgenic animal model that general toxin protein is expressed, and is easy to captive breeding, therefore can be used for screening the medicine of treatment hepatitis, estimate the method for treatment hepatitis.
Sequence table
<110〉The Children's Hospital Attached to Shanghai Jiaotong Univ.
<120〉a kind of transgenic mice hepatopathy animal model, its preparation method and application
<130>041317C
<160>3
<170>PatentIn?version?3.1
<210>1
<211>335
<212>DNA
<213>rat
<220>
<221>promoter
<222>(25)..(308)
<223>
<400>1
ggtacctcca?tgccaaggcc?cacactgaaa?tgctcaaatg?ggagacaaag?agattaagct 60
cttatgtaaa?atttgctgtt?ttacataact?ttaatgaatg?gacaaagtct?tgtgcatggg 120
ggtgggggtg?gggttagagg?ggaacagctc?cagatggcaa?acatacgcaa?gggatttagt 180
caaacaactt?tttggcaaag?atggtatgat?tttgtaatgg?taggaaccaa?tgaaatgcga 240
ggtaagtatg?gttaatgatc?tacagttatt?ggttaaagaa?gtatattaga?gcgagtcttt 300
ctgcacacag?atcacctttc?ctatcaaccc?cacta 335
<210>2
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<220>
<221>enhancer
<222>(1)..(2059)
<223>
<400>2
gaaattaccc?atcttcctaa?gcttctgctt?ctctcagttt?tctgcttgct?cattccactt 60
ttccagctga?ccctgccccc?taccaacatt?gctccacaag?cacaaattca?tccagagaaa 120
ataaattcta?agttttatag?ttgtttggat?cgcataggta?gctaaagagg?tggcaaccca 180
cacatcctta?ggcatgagct?tgattttttt?tgatttagaa?ccttcccctc?tctgttccta 240
gactacacta?cacattctgc?aagcatagca?cagagcaatg?ttctacttta?attactttca 300
ttttcttgta?tcctcacagc?ctagaaaata?acctgcgtta?cagcatccac?tcagtatccc 360
ttgagcatga?ggtgacacta?cttaacatag?ggacgagatg?gtactttgtg?tctcctgctc 420
tgtcagcagg?gcactgtact?tgctgatacc?agggaatgtt?tgttcttaaa?taccatcatt 480
ccggacgtgt?ttgccttggc?cagttttcca?tgtacatgca?gaaagaagtt?tggactgatc 540
aatacagtcc?tctgccttta?aagcaatagg?aaaaggccaa?cttgtctacg?tttagtatgt 600
ggctgtagaa?agggtataga?tataaaaatt?aaaactaatg?aaatggcagt?cttacacatt 660
tttggcagct?tatttaaagt?cttggtgtta?agtacgctgg?agctgtcaca?gctaccaatc 720
aggcatgtct?gggaatgagt?acacggggac?cataagttac?tgacattcgt?ttcccattcc 780
atttgaatac?acacttttgt?catggtattg?cttgctgaaa?ttgttttgca?aaaaaaaccc 840
cttcaaattc?atatatatta?ttttaataaa?tgaattttaa?tttatctcaa?tgttataaaa 900
aagtcaattt?taataattag?gtacttatat?acccaataat?atctaacaat?catttttaaa 960
catttgttta?ttgagcttat?tatggatgaa?tctatctcta?tatactctat?atactctaaa 1020
aaagaagaaa?gaccatagac?aatcatctat?ttgatatgtg?taaagtttac?atgtgagtag 1080
acatcagatg?ctccatttct?cactgtaata?ccatttatag?ttacttgcaa?aactaactgg 1140
aattctagga?cttaaatatt?ttaagtttta?gctgggtgac?tggttggaaa?attttaggta 1200
agtactgaaa?ccaagagatt?ataaaacaat?aaattctaaa?gttttagaag?tgatcataat 1260
caaatattac?cctctaatga?aaatattcca?aagttgagct?acagaaattt?caacataaga 1320
taattttagc?tgtaacaatg?taatttgttg?tctatttctt?ttgagataca?gttttttctg 1380
tctagctttg?gctgtcctgg?accttgctct?gtagaccagg?ttggtcttga?actcagagat 1440
ctgcttgcct?ctgccttgca?agtgctagga?ttaaaagcat?gtgccaccac?tgcctggcta 1500
caatctatgt?tttataagag?attataaagc?tctggctttg?tgacattaat?ctttcagata 1560
ataagtcttt?tggattgtgt?ctggagaaca?tacagactgt?gagcagatgt?tcagaggtat 1620
atttgcttag?gggtgaattc?aatctgcagc?aataattatg?agcagaatta?ctgacacttc 1680
cattttatac?attctacttg?ctgatctatg?aaacatagat?aagcatgcag?gcattcatca 1740
tagttttctt?tatctggaaa?aacattaaat?atgaaagaag?cactttatta?atacagttta 1800
gatgtgtttt?gccatctttt?aatttcttaa?gaaatactaa?gctgatgcag?agtgaagagt 1860
gtgtgaaaag?cagtggtgca?gcttggcttg?aactcgttct?ccagcttggg?atccttaacc 1920
ccatgtatcc?tctcctacct?ttgaggacac?cttctcatgc?cgtaagactg?tcacacgcca 1980
cacgggaagc?acacaatcgc?agcatttcaa?ggaggaagga?ccccagggat?aattttatcc 2040
cagttgattt?acatggcat 2059
<210>3
<211>1795
<212>DNA
<213>rat
<220>
<221>gene
<222>(1)..(1795)
<223>
<400>3
gccaccatgg?cttcgtaccc?cggccatcag?cacgcgtctg?cgttcgacca?ggctgcgcgt 60
tctcgcggcc?atagcaaccg?acgtacggcg?ttgcgccctc?gccggcagca?agaagccacg 120
gaagtccgcc?cggagcagaa?aatgcccacg?ctactgcggg?tttatataga?cggtccccac 180
gggatgggga?aaaccaccac?cacgcaactg?ctggtggccc?tgggttcgcg?cgacgatatc 240
gtctacgtac?ccgagccgat?gacttactgg?cgggtgctgg?gggcttccga?gacaatcgcg 300
aacatctaca?ccacacaaca?ccgccttgac?cagggtgaga?tatcggccgg?ggacgcggcg 360
gtggtaatga?caagcgccca?gataacaatg?ggcatgcctt?ataccgtgac?cgacgccgtt 420
ctggctcctc?atatcggggg?ggaggctggg?agctcacatg?ccccgccccc?ggccctcacc 480
ctcatcttcg?accgccatcc?catcgccgcc?ctcctgtgct?acccggccgc?gcgatacctt 540
atgggcagca?tgacccccca?ggccgtgctg?gcgttcgtgg?ccctcatccc?gccgaccttg 600
cccggcacaa?acatcgtgtt?gggggccctt?ccggaggaca?gacacatcga?ccgcctggcc 660
aaacgccagc?gccccggcga?gcggcttgac?ctggctatgc?tgaccgcgat?tcgccgcgtt 720
tacgggctgc?ttgccaatac?ggtgcggtat?ctgcagggcg?gcgggtcgtg?gcgggaggat 780
tggggacagc?tttcggggac?ggccgtgccg?ccccagggtg?ccgagcccca?gagcaacgcg 840
ggcccacgac?cccatatcgg?ggacacgtta?tttaccctgt?ttcgggcccc?cgagttgctg 900
gcccccaacg?gcgacctgta?caacgtgttt?gcctgggcct?tggacgtctt?ggccaaacgc 960
ctccgtccca?tgcacgtctt?tatcctggat?tacgaccaat?cgcccgccgg?ctgccgggac 1020
gccctgctgc?aacttacctc?cgggatgatc?cagacccacg?tcaccacccc?aggctccata 1080
ccgacgatct?gcgacctggc?gcgcacgttt?gcacgggaga?tgggggaggc?taactgaaac 1140
acggaaggag?acaataccgg?aaggaacccg?cgctatgtcg?gcaataaaaa?gacagaataa 1200
aacgcacggg?tgttgggtcg?tttgttcata?aacgcggggt?tcggtcccag?ggctggcact 1260
ctgtcgatac?cccaccgaga?ccccattggg?gccaatacgc?ccgcgtttct?tccttttccc 1320
caccccaacc?cccaagttcg?ggtgaaggcc?cagggctcgc?agccaacgtc?ggggcggcaa 1380
gcccgccata?gccacgggcc?ccgtgggtta?gggacggggt?cccccatggg?gaatggttta 1440
tggttcgtgg?gggttattct?tttgggcgtt?gcgtggggtc?aggtccacga?ctggactgag 1500
cagacagacc?catggttttt?ggatggcctg?ggcatggacc?gcatgtactg?gcgcgacacg 1560
aacaccgggc?gtctgtggct?gccaaacacc?cccgaccccc?aaaaaccacc?gcgcggattt 1620
ctggcgccgc?cggacgaact?aaacctgact?acggcatctc?tgccccttct?tcgctggtac 1680
gaggagcgct?tttgttttgt?attggtcacc?acggccgagt?ttccgcggga?ccccggccag 1740
atcaagctta?tcgataccgt?cgaggaattc?taccgggtag?gggaggcgct?tttcc 1795
Claims (1)
1, a kind of preparation method of transgenic mice hepatopathy animal model is characterized in that may further comprise the steps:
The clone of A, mouse albumin gene promotor ALB/pro;
The clone of B, mouse albumin gene enhanser ALB/en;
C, pcr amplification obtain herpes simplex thymidine kinase cDNA;
D, be goal gene with herpes simplex thymidine kinase cDNA, ALB/pro and ALB/en are controlling element, make up liver specificity and kill and wound carrier pLLTK;
E, digestion with restriction enzyme linearizing pLLTK reclaim the also fragment LLTK of the about 4130bp of purifying;
F, microinjection mouse male pronucleus obtain positive transgenic mice.
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人肝癌特异性HSV-TK/GCV重组腺病毒相关病毒质粒的构建与研究. 李志辉等.中国普外基础与临床杂志,第11卷第3期. 2004 * |
小鼠白蛋白增强子在肝癌细胞系中无增强功能. 贾帅争等.军事医学科学院院刊,第27卷第6期. 2003 * |
小鼠肝脏特异性表达载体ALB-ATP7B的构建. 徐琳等.中山大学学报(医学科学版),第25卷第1期. 2004 * |
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