CN104419700A - Humanization method of animal organ and human-animal chimera model - Google Patents
Humanization method of animal organ and human-animal chimera model Download PDFInfo
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Abstract
The invention relates to a humanization method of an animal organ; a DTA-Cre double-transgenic animal is established by using high-efficiency DTA as a virulent gene product; human stem cells planted in the double-transgenic animal can be developed into human body cells to replace original viscera or blood in the animal partially or totally and achieve the effect that the human cell can replace a target organ or blood in the animal partially or totally; the expression of toxic DTA can be strictly controlled by regulating and controlling a tetracycline-controlled expression system. The human-animal chimera established through the above method can replace a traditional transgenic mice; the method is simply operated. Moreover, the invention further relates to a human-animal chimera model established through the humanization method of the animal organ.
Description
Technical field
The present invention relates to biomedicine field, the people-animal chimeric model especially relating to a kind of humanized method of animal organ and use the method to build.
Background technology
Hepatitis B virus (Hepatitis B virus, HBV) belongs to Hepadna Virus section, and Hepadna Virus has stronger species specificity and tissue specificity.The natural reservoir (of bird flu viruses) of such as human hepatitis B virus only has the mankind and minority non-human primate (as chimpanzee), usually only invades the liver organization of host.Laboratory animal mouse the most frequently used in medical experiment limits the use in this research field because its liver cannot infect HBV.And man and animal is fitted together to hepatic model just in time can solve this difficult problem, because this animal pattern can infect HBV in natural situation.Study except for HBV, this model accurately can also measure the usefulness of Anti-HBV activity carrier.Except the research being applied to HBV, the chimeric liver of humans and animals can also be applied to other a lot of aspects, comprises the production and artificial liver transplanting etc. of human hepatocytes.
Liver has an outstanding feature, and namely liver cell has the potentiality of propagation.After partially hepatectomized, or when the hepatocyte cell be dispersed in viral hepatitis is dead, hepatocellular propagation can make up the loss of liver cell until liver reinstatement.This physiology controlling mechanism of liver cell group has been used to manufacture man and animal and has been fitted together to liver: set up a kind of host hepatocytes slow death mechanism, thus the human hepatocytes that generation one increment signal stimulates transplanting to come in.Existing two Animal Models are on this mechanism at present, i.e. plasma urokinase-type plasminogen activator (urokinase plasminogen activator, uPA) transgene mouse model and fumarylacetoacetic acid lytic enzyme (fumaryl acetoacetate hydrolase, FAH) gene knock-out mice model.In liver cell there is low-level expression in uPA usually, and excessive expression will cause cytotoxicity thus destroy host cell.In transgenosis uPA model, the uPA gene of albumin gene promoters driven multiple copied, the uPA albumen of synthesis intoxicating dosage, result causes the necrocytosis of host's Chronic Liver.In FAH model, because knocked out a gene FAH in TYR alienation path, cause toxic metabolites urokinase-type plasminogen to build up thus caused host's Chronic Liver downright bad.Although consistent human liver cell can fill up the host's liver substituted up to 70% in above-mentioned two models, cause the frequent death of filial generation because toxic product is accumulative and make these two kinds of models be difficult to go down to posterity.
Summary of the invention
Based on this, be necessary to provide a kind of can comparatively easily go down to posterity and the humanized method of animal organ that can substitute animal organ completely and the people-animal mosaic using the method to build.
A kind of humanized method of animal organ, comprises the steps:
Build the transgenic animal containing DTA gene and the transgenic animal containing Cre gene, wherein, promotor and the DTA gene of the described transgenic animal containing DTA gene are stopped sequence separating by two LoxP and, being added by described LoxP stops sequence suppressing to express, and the transactivator that the promotor of the Cre gene of the described transgenic animal containing Cre gene is relied on by the tsiklomitsin proceeding to the startup of animal body internal specific promotor starts;
By the described transgenic animal containing DTA gene and the described transgenic animals containing Cre gene, and the double transgenic animal that genescreen obtains containing Cre gene and DTA gene is carried out to filial generation;
Human stem cell is implanted into, the obtained double transgenic animal containing human body cell to the described double transgenic animal body containing Cre gene and DTA gene;
To the double transgenic feeding animal containing human body cell or injection tsiklomitsin or tetracycline derivant, abduction delivering Cre, thus the restructuring in mediation two Loxp sites, remove stopping sequence, promotor expresses DTA gene close to also starting, kill animal body internal object cell, promote the hyperplasia of Humanized cell, obtain people-animal chimeric model.
Wherein in an embodiment, the described transgenic animal containing DTA gene are start the animal stopping sequence and DTA gene containing LoxP of expressing by ROSA26 promotor, and described LoxP stops sequence between described ROSA26 promotor and described DTA gene.
Wherein in an embodiment, the described transgenic animal containing DTA gene after described ROSA26 promotor successively containing, for example lower sequence: SA splice acceptor sequence, LoxP stop sequence, eGFP sequence box, pFK-Neo sequence box, 3 SV40poly A sequences, LoxP stop sequence and DTA gene orders.
Wherein in an embodiment, described animal body internal specific promotor is animal livers specificity promoter Bai Danbai gene promoter (albumin gene promoter, Alb), described human stem cell behaviour liver stem cells.
Wherein in an embodiment, the described transgenic animal containing Cre gene are a kind of TetOn regulator control systems, the transactivator that reverse tsiklomitsin wherein expressed by TetOn regulator control system relies on is started by described promotor Alb expresses, and can be started and expresses after described Cre recombinase sequence is positioned at the hCMV promotor that the transactivator that relied on by described reverse tsiklomitsin starts by described hCMV promotor.
Wherein in an embodiment, described animal body internal specific promotor is animal hemopoietic stem cell specific gene promoter, and described human stem cell is human hematopoietic stem cell.
Wherein in an embodiment, described animal is mouse, pig or goat.
A kind of people-animal mosaic built by the humanized method of the animal organ described in above-mentioned any embodiment.
The humanized method of above-mentioned animal organ builds the people-animal chimeric model obtained and uses efficient DTA(diphtheria toxin) as toxic gene product, build DTA-Cre double transgenic animal, implant the human stem cell in this double transgenic animal body, the somatocyte of adult can be grown, partially or completely substitute former internal organ or blood in animal body, reach the effect that human body cell partially or completely substitutes animal body internal object internal organs or blood, and by using tetracycline-controlled expression system regulation and control, can the expression of strict control toxicity DTA.The people built by aforesaid method-animal mosaic replaces traditional transgenic mice, easy and simple to handle.
Accompanying drawing explanation
Fig. 1 is the gene fragment schematic diagram of the transgenic animal containing DTA gene;
Fig. 2 is the gene fragment schematic diagram of the transgenic animal containing Cre gene;
Fig. 3 is the gene fragment schematic diagram containing the double transgenic animal of Cre gene and DTA gene after Cre expresses.
Embodiment
Main by reference to the accompanying drawings to the humanized method of animal organ of invention and build by the method the people-animal mosaic obtained and be described in further detail below.
The humanized method of animal organ of one embodiment, comprises the steps:
Step one: build containing DTA(diphtheria tosin, diphtheria toxin) gene transgenic animal and containing Cre(Crerecombinase, derive from a kind of intergrase of P1 phage) transgenic animal of gene.Wherein, the promotor of the transgenic animal containing DTA gene and DTA gene are by two LoxP recombination sequences and stop sequence separating, being added by LoxP stops sequence suppressing to express, and the promotor of the Cre gene of the transgenic animal containing Cre gene is started by the transactivator proceeding to the tsiklomitsin dependence that animal body internal specific promotor starts.
Animal described in present embodiment can be the laboratory animal such as mouse, pig or goat.
In the present embodiment, should the transgenic animal containing DTA gene be start the animal of expressing DTA gene by ROSA26 promotor, LoxP stops sequence between ROSA26 promotor and DTA gene.As shown in Figure 1, specifically should transgenic animal containing DTA gene after ROSA26 promotor successively containing, for example lower sequence: SA splice acceptor sequence, LoxP stop sequence, eGFP sequence box, pFK-Neo sequence box, 3 SV40poly A sequences (three poly A is called for short tpA), LoxP stop sequence and DTA gene orders.In normal state, because LoxP stops the existence of sequence, DTA gene silencing does not express DTA.
Animal body internal specific promotor can be animal livers specificity promoter Alb or animal hemopoietic stem cell specific gene promoter etc., and accordingly, the human stem cell that subsequent step is implanted can be people's liver stem cells or hemopoietic stem cell etc.Be appreciated that this animal body internal specific promotor can also be the specificity promoter of other internal organs of animal, the human body cell that subsequent step is implanted can be corresponding other internal organs cells of human body.
Specifically in the present embodiment, as shown in Figure 2, should the transgenic animal containing Cre gene be a kind of TetOn regulator control systems, the transactivator (reverse tetracycline-responsive transactivator, rtTA) that reverse tsiklomitsin wherein expressed by TetOn regulator control system relies on is started by the special specificity promoter Alb of animal livers expresses.Can be started by hCMV promotor and express after Cre recombinase sequence is positioned at the hCMV promotor that the transactivator that relied on by reverse tsiklomitsin starts.The startup of hCMV depends on the expression of the TetO gene order that rtTA expresses.Therefore, when after this feeding animal or injection tsiklomitsin or tetracycline derivant, TetO gene order is expressed, thus starts hCMV promotor, starts the expression Cre recombinase of Cre recombinase sequence further.
Step 2: by the transgenic animal containing DTA gene with containing the transgenic animals of Cre gene, and the double transgenic animal that genescreen obtains containing Cre gene and DTA gene is carried out to filial generation.
Step 3: be implanted into human stem cell to the double transgenic animal body containing Cre gene and DTA gene, the obtained double transgenic animal containing human body cell.
Step 4: to the double transgenic feeding animal containing human body cell or injection tsiklomitsin or tetracycline derivant, obtain people-animal chimeric model.
When after this double transgenic feeding animal or injection tsiklomitsin or tetracycline derivant (as doxycycline DOX etc.), Cre genetic expression Cre recombinase, this Cre recombinase can cause the gene order between two LoxP sites to recombinate, LoxP stops sequence being removed, as shown in Figure 3, thus express DTA and cause the death of host's target cell.By regulation and control feeding or the tsiklomitsin of injection or the amount of tetracycline derivant, partially or completely can remove the target cell in host, and allow the human body cell implanted obtain procreation and alternate host body internal object cell completely.
The humanized method of this animal organ builds the people-animal chimeric model obtained and uses efficient DTA(diphtheria toxin) substitute traditional uPA and FHA as toxic gene product, animal body internal object internal organs or blood cell can be removed completely, reach the effect that human body cell substitutes animal body internal object internal organs or blood completely, and by using tetracycline-controlled expression system regulation and control, can the expression of strict control toxicity DTA.The people built by aforesaid method-animal mosaic replaces traditional transgenic mice, easy and simple to handle.
The people obtained by the method-animal chimeric model can carry out morbidity and the Therapy study of HBV as the research model of HBV, in addition, this people-animal mosaic can also be applied in other various fields, as:
(1) human hepatocytes source: Human primary liver cell is difficult to cultivate and go down to posterity in vitro, applies above-mentioned people-animal chimeric model and just constantly can generate human hepatocytes in the body of animal.If with the animal analogue formation that build is larger, the donor source that the liver transplantation of the mankind provides sufficient can be thought.
(2) Other diseases model: with induced multi-potent stem cells (the induced pleuripotent stemcell of patient self, iPS) cord blood stem cell is replaced to originate as Humanized cell, patient-specific hepatic diseases animal model can be made, such as A type and haemophilia B, hypercholesterolemia, thus greatly promote the gene to these diseases and cell therapy research.
(3) pharmacokinetics research.Liver is metabolic organ main in human body, endogenous and the exogenous compounds of the overwhelming majority all carry out metabolism in liver, and metabolizing enzyme systems in human hepatocytes and animal is obviously different, so the chimeric hepatic model of this people-animal can provide an animal model easily for the pharmacokinetics research of the mankind.
(4) people-animal follow board of other organ.Utilize the principle making chimeric hepatic model, the follow board of other organ can be made, such as chimeric Hemic and immune system, chimeric cardiovascular and neural system; The transgenic animal having two chimeric organs can also be made, such as chimeric liver and the two chimeric organ animal model of blood, this pair of chimeric organ animal model will be conducive to the interaction studying human immune system and much virus (such as HBV and HCV).
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. the humanized method of animal organ, is characterized in that, comprise the steps:
Build the transgenic animal containing DTA gene and the transgenic animal containing Cre gene, wherein, promotor and the DTA gene of the described transgenic animal containing DTA gene are stopped sequence separating by two LoxP and, being added by described LoxP stops sequence suppressing to express, and the transactivator that the promotor of the Cre gene of the described transgenic animal containing Cre gene is relied on by the tsiklomitsin proceeding to the startup of animal body internal specific promotor starts;
By the described transgenic animal containing DTA gene and the described transgenic animals containing Cre gene, and the double transgenic animal that genescreen obtains containing Cre gene and DTA gene is carried out to filial generation;
Human stem cell is implanted into, the obtained double transgenic animal containing human body cell to the described double transgenic animal body containing Cre gene and DTA gene;
To the double transgenic feeding animal containing human body cell or injection tsiklomitsin or tetracycline derivant, abduction delivering Cre, thus the restructuring in mediation two Loxp sites, remove stopping sequence, promotor expresses DTA gene close to also starting, kill animal body internal object cell, promote the hyperplasia of Humanized cell, obtain people-animal chimeric model.
2. the humanized method of animal organ as claimed in claim 1, it is characterized in that, the described transgenic animal containing DTA gene are start the animal stopping sequence and DTA gene containing LoxP of expressing by ROSA26 promotor, and described LoxP stops sequence between described ROSA26 promotor and described DTA gene.
3. the humanized method of animal organ as claimed in claim 2, it is characterized in that, the described transgenic animal containing DTA gene after described ROSA26 promotor successively containing, for example lower sequence: SA splice acceptor sequence, LoxP stop sequence, eGFP sequence box, pFK-Neo sequence box, 3 SV40polyA sequences, LoxP stop sequence and DTA gene orders.
4. the humanized method of animal organ as claimed in claim 1, it is characterized in that, described animal body internal specific promotor is animal livers specificity promoter Bai Danbai gene promoter, described human stem cell behaviour liver stem cells.
5. the humanized method of animal organ as claimed in claim 4, it is characterized in that, the described transgenic animal containing Cre gene are a kind of TetOn regulator control systems, the transactivator that reverse tsiklomitsin wherein expressed by TetOn regulator control system relies on is started by described promotor Alb expresses, and can be started and expresses after described Cre recombinase sequence is positioned at the hCMV promotor that the transactivator that relied on by described reverse tsiklomitsin starts by described hCMV promotor.
6. the humanized method of animal organ as claimed in claim 1, it is characterized in that, described animal body internal specific promotor is animal hemopoietic stem cell specific gene promoter, and described human stem cell is human hematopoietic stem cell.
7. the humanized method of the animal organ according to any one of claim 1-6, is characterized in that, described animal is mouse, pig or goat.
8. people-animal the chimeric model of the humanized method structure of the animal organ according to any one of claim 1-7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108310368A (en) * | 2017-01-15 | 2018-07-24 | 上海市公共卫生临床中心 | A method of structure liver humanized mouse model |
| CN109735485A (en) * | 2019-01-29 | 2019-05-10 | 深圳市拓普生物科技有限公司 | Humanization liver animal model and its construction method and application |
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| CN102653764A (en) * | 2012-04-26 | 2012-09-05 | 天津农学院 | Method for introducing frame-shift mutation in MSTN (myostatin) genes of cattle |
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| WO2009114400A1 (en) * | 2008-03-07 | 2009-09-17 | Regeneron Pharmaceuticals, Inc. | Es cell-derived mice from diploid host embryo injection |
| CN102653764A (en) * | 2012-04-26 | 2012-09-05 | 天津农学院 | Method for introducing frame-shift mutation in MSTN (myostatin) genes of cattle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108310368A (en) * | 2017-01-15 | 2018-07-24 | 上海市公共卫生临床中心 | A method of structure liver humanized mouse model |
| CN109735485A (en) * | 2019-01-29 | 2019-05-10 | 深圳市拓普生物科技有限公司 | Humanization liver animal model and its construction method and application |
| CN109735485B (en) * | 2019-01-29 | 2021-04-16 | 深圳市拓普生物科技有限公司 | Humanized liver animal model and construction method and application thereof |
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