CN104419700B - The method of animal organ's humanization and people-animal chimeric model - Google Patents
The method of animal organ's humanization and people-animal chimeric model Download PDFInfo
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Abstract
The present invention relates to a kind of methods of animal organ's humanization, it is by using efficient DTA as toxic gene product, build DTA Cre double transgenic animals, the human stem cell being implanted into the double transgenic animal body, the body cell of adult can be developed, former internal organ or blood in animal body is partially or completely substituted, achievees the effect that human body cell partially or completely substitutes target internal organs or blood in animal body, and is regulated and controled by using tetracycline-controlled expression system, can tightly control the expression of toxicity DTA.Traditional transgenic mice is replaced by people's animal chimera that the above method is built, it is easy to operate.Moreover, it relates to people's animal chimeric model that a kind of method using above-mentioned animal organ's humanization is built.
Description
Technical field
The present invention relates to biomedicine fields, a kind of method more particularly, to animal organ's humanization and use this method
The people of structure-animal chimeric model.
Background technology
Hepatitis type B virus（Hepatitis B virus, HBV）Belong to Hepadna Virus section, Hepadna Virus has stronger
Species specificity and tissue specificity.Such as the natural reservoir (of bird flu viruses) of human hepatitis B virus only has the mankind and a small number of non-human primates
（Such as chimpanzee）, usually only invade the liver organization of host.In medical experiment most common experimental animal mouse because its liver without
Method infects HBV and limits the use in this research field.And people's hepatic model chimeric with animal can just solve this
Problem, because this animal pattern can infect HBV in natural conditions.Except being studied for HBV, this model can also be accurate
Ground measures the efficiency of Anti-HBV activity carrier.Except the research applied to HBV, the chimeric liver of humans and animals can also be applied to other very
Various aspects include production and the artificial liver transfer operation etc. of human hepatocytes.
There are one outstanding features, i.e. liver cell to have the potentiality of proliferation for liver.After partially hepatectomized, or viral
When the hepatocyte cell death being dispersed in hepatitis, the proliferation of liver cell can make up the loss of liver cell until liver returns
Restore shape.This physiology controlling mechanism of liver cell group is already used to manufacturer's liver chimeric with animal：Establish a kind of place
Main liver cell slow death mechanism rises in value signal come the human hepatocytes for stimulating transplanting to come in generate one.Have at present
Two Animal Models are on this mechanism, i.e. plasma urokinase-type plasminogen activator（urokinase plasminogen
Activator, uPA）Transgene mouse model and fumaroyl acetoacetate hydrolase（fumaryl acetoacetate
Hydrolase, FAH）Gene knock-out mice model.In liver cell usually there is low-level expression in uPA, and excessive expression will be led
It is cytotoxicity caused to destroy host cell.In transgenosis uPA models, albumin gene promoter drives the uPA of multicopy
Gene synthesizes the uPA albumen of intoxicating dosage, as a result leads to the chronic hepatocyte death of host.In FAH models, because knocking out
A gene FAH in tyrosine alienation access, causes toxic metabolites urokinase-type plasminogen to build up so as to cause place
Main chronic hepatonecrosis.Although in above-mentioned two model consistent human liver cell can fill up be substituted up to 70% host's liver,
Cause that filial generation is frequently dead since toxic product is accumulative and so that both models are difficult to pass on.
Invention content
Based on this, it is necessary to provide a kind of being relatively easy to passage and animal device that animal organ can be substituted completely
The method of official's humanization and the people-animal chimera built using this method.
A kind of method of animal organ's humanization, includes the following steps：
Build the transgenic animals containing DTA gene and the transgenic animals containing Cre genes, wherein described to contain
The promoter of the transgenic animals of DTA gene stops sequence by two LoxP and one with DTA gene and separates, and is added by the LoxP
Upper stopping sequence inhibiting expression, and the promoter of the Cre genes of the transgenic animals containing Cre genes is by being transferred in animal body
The transactivator that the tetracycline that specificity promoter starts relies on starts；
By the transgenic animals containing DTA gene and the transgenic animals containing Cre genes, and to miscellaneous
It hands over offspring to carry out genescreen and obtains the double transgenic animal containing Cre genes and DTA gene；
To the double transgenic animal et al. Ke human stem cell containing Cre genes and DTA gene, it is made and contains someone
The double transgenic animal of body cell；
To the double transgenic feeding animal containing human body cell or injection tetracycline or tetracycline derivant, induced expression
Cre is removed to mediate the recombination in two sites Loxp and is stopped sequence, and promoter approaches and start expression DTA gene, is killed
Target cell in animal body promotes the hyperplasia of Humanized cell, obtains people-animal chimeric model.
The transgenic animals containing DTA gene are to start table by ROSA26 promoters in one of the embodiments,
What is reached stops the animal of sequence and DTA gene containing LoxP, and the LoxP stops sequence and is located at the ROSA26 promoters and institute
It states between DTA gene.
The transgenic animals containing DTA gene are after the ROSA26 promoters in one of the embodiments,
Contain following sequence successively：SA splice acceptor sequences, LoxP stop sequence, eGFP sequence boxes, pFK-Neo sequence boxes, 3
SV40poly A sequences, LoxP stop sequence and DTA gene sequence.
The animal body internal specific promoter is the white denier of animal's liver specificity promoter in one of the embodiments,
White gene promoter（Albumin gene promoter, Alb）, the human stem cell behaviour liver stem cells.
The transgenic animals containing Cre genes are a kind of TetOn regulator control systems in one of the embodiments,
The transactivator that reversed tetracycline expressed by middle TetOn regulator control systems relies on is started by the promoter Alb expresses, described
Cre recombination enzyme sequences are located at after the hCMV promoters that the transactivator relied on by the reversed tetracycline starts and can be by institutes
It states hCMV promoters and starts expression.
The animal body internal specific promoter is animal candidate stem cell specific gene in one of the embodiments,
Promoter, the human stem cell are human hematopoietic stem cell.
The animal is mouse, pig or goat in one of the embodiments,.
People-animal chimera that a kind of method by animal organ's humanization described in any of the above-described embodiment is built.
The people that the method for above-mentioned animal organ's humanization is built-animal chimeric model uses efficient DTA（Diphtheria
Toxin）As toxic gene product, DTA-Cre double transgenic animals are built, the human body being implanted into the double transgenic animal body is dry
Cell can develop the body cell of adult, partially or completely substitute former internal organ or blood in animal body, reach human body cell part
Or the effect of target internal organs or blood in animal body is substituted completely, and regulated and controled by using tetracycline-controlled expression system, Ke Yiyan
The expression of close control toxicity DTA.Traditional transgenic mice, operation are replaced by people-animal chimera that the above method is built
It is easy.
Description of the drawings
Fig. 1 is the genetic fragment schematic diagram of the transgenic animals containing DTA gene；
Fig. 2 is the genetic fragment schematic diagram of the transgenic animals containing Cre genes；
Fig. 3 is the genetic fragment schematic diagram of the double transgenic animal containing Cre genes and DTA gene after Cre expression.
Specific implementation mode
The people-built below mainly in combination with attached drawing to the method for animal organ's humanization of invention and by this method is dynamic
Object chimera is described in further detail.
The method of animal organ's humanization of one embodiment, includes the following steps：
Step 1：Structure contains DTA（Diphtheria tosin, diphtheria toxin）The transgenic animals of gene and contain
Cre（Crerecombinase derives from a kind of integrase of P1 bacteriophages）The transgenic animals of gene.Wherein, containing DTA bases
The promoter of the transgenic animals of cause is separated with DTA gene by two LoxP recombination sequences and stopping sequence, is added and is stopped by LoxP
Only sequence inhibits expression, the promoter of the Cre genes of the transgenic animals containing Cre genes to be opened by being transferred to animal body internal specific
The transactivator that the tetracycline that mover starts relies on starts.
Animal described in present embodiment can be the experimental animals such as mouse, pig or goat.
In the present embodiment, it is to start expression DTA bases by ROSA26 promoters to be somebody's turn to do the transgenic animals containing DTA gene
The animal of cause, LoxP stop sequence between ROSA26 promoters and DTA gene.As shown in Figure 1, should specifically contain DTA bases
The transgenic animals of cause contain following sequence successively after ROSA26 promoters：SA splice acceptor sequences, LoxP stop sequence,
EGFP sequence boxes, pFK-Neo sequence boxes, 3 SV40poly A sequences（Three poly A, abbreviation tpA）, LoxP stop sequence
And DTA gene sequence.In normal state, since LoxP stops the presence of sequence, DTA gene silence does not express DTA.
Animal body internal specific promoter can be that animal's liver specificity promoter Alb or animal candidate stem cell are special
Property gene promoter etc., accordingly, the human stem cell of subsequent step implantation can be people's liver stem cells or candidate stem cell
Deng.It is appreciated that the animal body internal specific promoter can also be the specificity promoter of other internal organs of animal, subsequently
The human body cell of step implantation can be other internal organs cells of corresponding human body.
Specifically in the present embodiment, as shown in Fig. 2, it is a kind of TetOn regulation and control to be somebody's turn to do the transgenic animals containing Cre genes
System, the transactivator that the reversed tetracycline wherein expressed by TetOn regulator control systems relies on（reverse
Tetracycline-responsive transactivator, rtTA）Table is started by animal's liver spy's specificity promoter Alb
It reaches.Cre recombination enzyme sequences are located at after the hCMV promoters that the transactivator relied on by reversed tetracycline starts and can be by
HCMV promoters start expression.Expression of the startup of hCMV dependent on the TetO gene orders of rtTA expression.Therefore, when dynamic to this
After object feeding or injection tetracycline or tetracycline derivant, the expression of TetO gene orders, to start hCMV promoters, further
Start the expression Cre recombinases of Cre recombination enzyme sequences.
Step 2：By the transgenic animals containing DTA gene and the transgenic animals containing Cre genes, and to miscellaneous
It hands over offspring to carry out genescreen and obtains the double transgenic animal containing Cre genes and DTA gene.
Step 3：To the double transgenic animal et al. Ke human stem cell containing Cre genes and DTA gene, it is made and contains
There is the double transgenic animal of human body cell.
Step 4：To the double transgenic feeding animal containing human body cell or injection tetracycline or tetracycline derivant, obtain
To people-animal chimeric model.
When to the double transgenic feeding animal or injection tetracycline or tetracycline derivant（Such as doxycycline DOX）Afterwards,
Cre gene expression Cre recombinases, the Cre recombinases can cause the gene order between two sites LoxP to recombinate,
LoxP stops sequence and is removed, as shown in figure 3, to express the death that DTA causes host's target cell.By regulate and control feeding or
The tetracycline of injection or the amount of tetracycline derivant can partially or completely remove the target cell in host, and allow implantation
Human body cell obtain target cell in procreation and completely substituted host body.
The people that the method for animal organ's humanization is built-animal chimeric model uses efficient DTA（Diphtheria poison
Element）Traditional uPA and FHA are substituted as toxic gene product, can completely remove target internal organs or blood cell in animal body,
Achieve the effect that human body cell substitutes target internal organs or blood in animal body completely, and by using tetracycline-controlled expression system tune
Control, can tightly control the expression of toxicity DTA.Traditional transgenosis is replaced by people-animal chimera that the above method is built
Mouse, it is easy to operate.
By this method obtain people-animal chimeric model can be used as HBV research model carry out HBV morbidity with
Therapy study, in addition, the people-animal chimera can be applied in other various fields, such as：
（1）Human hepatocytes source：Human primary liver cell is difficult to cultivate and pass in vitro, using above-mentioned people-animal
Chimeric model can constantly generate human hepatocytes in vivo in animal.If making mould with the larger animal of build
Type, you can sufficient donor source is provided with the liver transplant for the mankind.
（2）Other diseases model：With induced multi-potent stem cell (the induced pleuripotent stem of patient itself
Cell, iPS) replace cord blood stem cell as Humanized cell source, patient-specific liver diseases animal mould can be made
Type, such as A types and haemophilia B, hypercholesterolemia, gene and cell therapy to greatly promote to these diseases are ground
Study carefully.
（3）Pharmacokinetics research.Liver is main metabolic organ in human body, most endogenous and outer
Endogenous compound is all metabolized in liver, and the metabolizing enzyme systems and animal in human hepatocytes is significantly different, so should
The hepatic model of people-animal chimera can provide a convenient animal model for the Pharmacokinetics research of the mankind.
（4）The people of other organs-animal follow board.Using the principle for being fitted into hepatic model is made, can make other
The follow board of organ, such as chimeric Hemic and immune system, chimeric cardiovascular and nervous system；It can also make there are two gathering around
The transgenic animals of organ are fitted into, such as double chimeric organ animal models of chimeric liver and blood, this pair of chimeric organ move
Object model is beneficial to research human immune system and much viruses（Such as HBV and HCV）Interaction.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (7)
1. a kind of method of animal organ's humanization, which is characterized in that include the following steps：
Build the transgenic animals containing DTA gene and the transgenic animals containing Cre genes, wherein described to contain DTA bases
The promoter of the transgenic animals of cause stops sequence by two LoxP and one with DTA gene and separates, and institute is added by the LoxP
It states and stops sequence inhibition expression, the promoter of the Cre genes of the transgenic animals containing Cre genes is by being transferred in animal body
The transactivator that the tetracycline that specificity promoter starts relies on starts；
By the transgenic animals containing DTA gene and the transgenic animals containing Cre genes, and to hybridization after
In generation, carries out genescreen and obtains the double transgenic animal containing Cre genes and DTA gene；
To the double transgenic animal et al. Ke human stem cell containing Cre genes and DTA gene, it is made thin containing human body
The double transgenic animal of born of the same parents；
To the double transgenic feeding animal containing human body cell or tetracycline or tetracycline derivant are injected, induced expression Cre, from
And the recombination in two sites Loxp is mediated, the stopping sequence being removed, the promoter of DTA gene approaches and starts expression DTA bases
Cause kills target cell in animal body, promotes the hyperplasia of Humanized cell, obtain people-animal chimeric model.
2. the method for animal organ's humanization as described in claim 1, which is characterized in that described to turn base containing DTA gene
Because animal is the animal for stopping sequence and DTA gene containing LoxP for starting expression by ROSA26 promoters, the LoxP stops
Sequence is between the ROSA26 promoters and the DTA gene.
3. the method for animal organ's humanization as claimed in claim 2, which is characterized in that described to turn base containing DTA gene
Because animal contains following sequence successively after the ROSA26 promoters：SA splice acceptor sequences, LoxP stop sequence, eGFP
Sequence box, pFK-Neo sequence boxes, 3 SV40poly A sequences, LoxP stop sequence and DTA gene sequence.
4. the method for animal organ's humanization as described in claim 1, which is characterized in that the animal body internal specific starts
Son is animal's liver specificity promoter albumin gene promoter, the human stem cell behaviour liver stem cells.
5. the method for animal organ's humanization as claimed in claim 4, which is characterized in that described to turn base containing Cre genes
Because animal is a kind of TetOn regulator control systems, the transactivator of the reversed tetracycline dependence wherein expressed by TetOn regulator control systems
Started by the promoter Alb and expressed, the Cre recombinations enzyme sequence is located at the transactivator relied on by the reversed tetracycline
After the hCMV promoters of startup and can expression be started by the hCMV promoters.
6. the method for animal organ's humanization as described in claim 1, which is characterized in that the animal body internal specific starts
Son is animal candidate stem cell specific gene promoter, and the human stem cell is human hematopoietic stem cell.
7. the method for animal organ's humanization as described in any one of claim 1-6, which is characterized in that the animal is small
Mouse, pig or goat.
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| 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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| 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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| 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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