CN106222187A - Transgene carrier, transgene mouse model and the construction method thereof of inducible c myc process LAN - Google Patents
Transgene carrier, transgene mouse model and the construction method thereof of inducible c myc process LAN Download PDFInfo
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Abstract
The invention discloses transgene carrier, transgenic instrument mice and the construction method thereof of a kind of inducible c myc process LAN, c myc coding region is connected acquisition pcDNA3 c myc plasmid with pcDNA3 carrier；Loxp Stop Loxp fragment is connected acquisition pcDNA3 Loxp Stop Loxp c myc plasmid with pcDNA3 c myc plasmid；EF1a promoter fragment is connected acquisition transgene carrier with pcDNA3 Loxp Stop Loxp c myc plasmid, then builds transgene mouse model based on this transgene carrier.It is derivable Leukemia Model that the present invention builds the transgene mouse model obtained, in the case of not having derivant, c myc does not express and does not fall ill, c myc process LAN in the case of derivant effect, cause leukemic generation, more conventional transgene mouse model has more preferable controllability, can need to adjust the population quantity that need to maintain according to experiment.
Description
Technical field
The invention belongs to gene engineering technology field, more particularly it relates to an can induce in hemopoietic system
Type c-myc transgene carrier and construction method thereof and in hemopoietic system inducible c-myc transgene mouse model and
Construction method.
Background technology
Leukemia is that a class betides hemopoietic organ, with the propagation of the leukocyte in blood and bone marrow and precursor thereof and
The malignant disease that abnormal development is characterized.According to the report of World Health Organization (WHO), the whole world in 2000 there are about 210,000 people and dies from white blood
Disease, wherein 90% is adult.The feature of mouse hemopoietic system is similar with the mankind, and mouse leukemia and human leukemia are in many
Aspect is very much like.The research and development of mouse leukemia model has become research human leukemia pathogenesis, biochemical immunity
Feature, pathophysiological change, Cytobiology and molecular biology characteristic and carry out the powerful of experimental therapy, significant.
Research in recent years shows, leukemic generation and the activation of cellular proto-oncogene and the disappearance of antioncogene or sudden change have
Close relationship.Wherein c myc is to study one of most commonly used oncogene at present, and it is former that this gene belongs to myc
One of Oncogene family member (c-Myc, N-myc and L-myc).Myc albumen is basic-helix-loop-helix
Zipper (bHLHZ) class transcription factor, it can form heterodimer with Max and be attached to special CAC (G/A) TG ' E-box '
Sequence, by the expression of a large amount of target genes that combine with other factors various.Myc is considered to participate in most basic cell and lives
Dynamic process, such as propagation, growth, differentiation and apoptosis etc., plays a significant role in fetal development and tumor generating process.
In hemopoietic system, each stage that c-myc gene not only generates at hematopoietic cell plays regulatory role, and its expression is different
Often and the generation of disease in the blood system, the most all types of leukemic generations are closely related.Research shows, almost all of
Male's Burkitt ' s B cell lymphoma case all can finding, the c-myc genome molding caused because of chromosome rearrangement is expressed；
In acute lymphoblastic leukemia and acute myelocytic leukemia, though not finding the transposition of c-myc gene, but still can find to lead to
Cross other chromosome rearrangements, to initiations such as phosphorylation modification abnormal for c-myc, c-myc upstream positive regulating gene abnormal activations
Process LAN on c-myc gene function；In the whole pathogenic process of chronic myelocytic leukemia, all can find because of c-myc upstream
The process LAN expressing the c-myc caused of regulation and control kinases bcr/abl, and it was found that the mRNA level in-site of c-myc is thin along with chronic grain
The leukemic progression of born of the same parents, constantly raises to acute change from chronic phase, accelerated period the phase；Recent studies have shown that c-myc's is general
Elementization regulation played a significant role in the initial of chronic myelogenous leukemia and evolution.
The research of C-myc transgenic mice shows: the c-myc gene driven by Ig heavy chain enhancer is in bone-marrow-derived lymphocyte
High expressed may result in the generation of lymphoma and in early days B cell leukemia.This transgene mouse model is as spontaneous white
The researchs such as the classical model of disorders of blood, is widely used in leukemia pathogenesis, related drugs screening.Infected at bone marrow by virus
In cell, the research of process LAN c-myc shows, medullary cell process LAN c-myc may result in the generation of acute myeloid leukaemia.This
A little evidences show that c-myc gene plays a significant role in the leukemic generation of polytype, evolution.This gene-correlation turns
The foundation of genetic model, simulates the generating process of human leukemia, promotes the understanding to c-myc gene function, accelerates
Research and development process to leukemia medicament.
But, these models also have its limitation: with the c-myc transgenic mice (E μ-myc) of Ig heavy chain enhancer driving
As a example by, the process LAN of c-myc starts from period of embryo, and the leukemia symptoms such as B cell volume increase i.e. be can be observed in mouse post-natal,
Hinder the understanding of myc gene function in the leukemia generating process that c-myc is caused, in this transgene mouse model,
Myc causes the average life of E μ-myc transgenic mice to only have 12 weeks from the most lasting high expressed, needs in research process
Maintain substantial amounts of population.
In sum, continue high expressed owing to the most conventional c-myc transgenic leukemia mouse model has c-myc,
The defects such as disease time is uncontrollable, in the urgent need to setting up a kind of derivable spontaneous leukemia transgenic models.
Summary of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides one and can induce in hemopoietic system
The transgene carrier of type c-myc process LAN, mouse model and construction method thereof.
In order to realize foregoing invention purpose, this invention takes techniques below scheme:
The construction method of a kind of inducible c-myc process LAN transgene carrier, comprises the following steps:
A, to comprise c-myc ORF cDNA plasmid as template, SEQ ID No:1 and SEQ ID No:2 is primer, and PCR expands
The c-myc coding region of increasing acquisition Serial No. SEQ ID No:3, XhoI and XbaI enzyme cutting c-myc coding region and pcDNA3 carrier,
Connect and obtain pcDNA3-c-myc plasmid；
B, with comprise Loxp-Stop-Loxp element plasmid as template, SEQ ID No:4 and SEQ ID No:5 is for drawing
Thing, PCR amplification obtains the Loxp-Stop-Loxp fragment of Serial No. SEQ ID No:6, EcoRI and NotI enzyme action Loxp-
Stop-Loxp fragment and pcDNA3-c-myc plasmid, connect and obtain pcDNA3-Loxp-Stop-Loxp-c-myc plasmid；
C, with pLVX-EF1a-ires-puro plasmid as template, SEQ ID No:7 and SEQ ID No:8 is primer, PCR
Amplification obtain Serial No. SEQ ID No:9 EF1a promoter fragment, BamHI and EcoRI enzyme action EF1a promoter fragment and
PcDNA3-Loxp-Stop-Loxp-c-myc plasmid, connects and obtains inducible c-myc process LAN transgene carrier.
Present invention also offers above-mentioned construction method and build the inducible c-myc process LAN transgene carrier obtained.
Present invention also offers the construction method of a kind of inducible c-myc process LAN transgene mouse model, including with
Lower step:
(1), the above-mentioned inducible c-myc process LAN transgene carrier of SmaI and PvuI enzyme action, gel reclaim comprise EF1a
The fragment of promoter-Loxp-Stop-Loxp-c-myc, after the super ovulation of the female Mus of germ cell donor, female Mus hero Mus mates, and will see bolt
Female Mus put to death to collect and be in the germ cell of one cell stage, carry out male pronucleus microinjection；It is bicelluar being subject to by normal development
Essence ovum transfer is transgenic F0 for mice to pseudo-fetus female Mus uterus, the given birth to mice of the female Mus of pseudo-fetus；
(2), by above-mentioned transgenic F0 for mice and wild-type mice copulation, pass on to build and be, it is thus achieved that F1 generation mice；Select
The F1 generation mice of the equal high expressed of bone marrow, spleen, lymph node and thymus is as follow-up experiment strain, it is thus achieved that transgenic instrument is little
Mus；
(3) the transgenic instrument mice, by step (2) obtained and Cre transgenic mice copulation, acquisition can induce c-myc
The transgenic instrument mice of process LAN.
Present invention also offers above-mentioned construction method and build the inducible c-myc process LAN transgenic mice mould obtained
Type.
Present invention also offers above-mentioned inducible c-myc process LAN transgene carrier and inducible c-myc process LAN
Transgene mouse model application in anti-leukemia medicine screens.
Compared with prior art, the method have the advantages that
1, the present invention builds the transgene mouse model obtained is derivable Leukemia Model, is not having the feelings of derivant
Under condition, c-myc does not express and does not fall ill, c-myc process LAN in the case of derivant effect, causes leukemic generation, more conventional
Transgene mouse model there is more preferable controllability, can need to adjust the population quantity that need to maintain according to experiment；
2, the leukemia transgene mouse model that the present invention sets up gives the time of derivant by control, controls c-myc
Process LAN opportunity, thus control leukemic disease time, research c-myc is in the function of leukemia morbidity different times；With
Set up the leukemia animal model of animal integral level based on this model, carry for the leukemic medicine of different times for screening
Supply to support；The Leukemia Model that leukemia mouse model is spontaneous type that the present invention sets up, relatively tumor cell transplantation model etc.
More can onset state in analogue body；
3, the present invention set up medicaments sifting model, with c-myc proto-oncogene as target spot, this gene with polytype in vain
The generation of disorders of blood, develop closely related, provide new selection for leukemia medicament screening.
Accompanying drawing explanation
Fig. 1 is the structure of the transgene carrier of the inducible c-myc gene overexpression built in the embodiment of the present invention 1
Figure；
Fig. 2 is the function of the transgene mouse model of the inducible c-myc gene overexpression that the embodiment of the present invention 2 builds
Figure.
Detailed description of the invention
The present invention, in following example if no special instructions, institute is described in detail below in conjunction with the drawings and specific embodiments
Raw material is used to derive from commercially available, the conventional practices that institute's employing method is well known to those skilled in the art.
The structure of the transgene carrier of embodiment 1 inducible c-myc gene overexpression
The construction method of the transgene carrier of this embodiment, including step in detail below:
A, design are for the forward of c-myc coding region and reverse primer, and primer adds XhoI and XbaI enzyme cutting site respectively,
The c-myc coding region of about 1.4kb is obtained, by the way of enzyme action from comprising amplification c-myc ORF cDNA plasmid by PCR
It is connected into the pcDNA3 carrier through XhoI and XbaI enzyme cutting and obtains pcDNA3-c-myc plasmid；
B, design are for the forward of Loxp-Stop-Loxp element and reverse primer, and primer adds EcoRI and NotI respectively
Site, expands the Loxp-Stop-Loxp sheet obtaining about 1.3kb from the plasmid comprising Loxp-Stop-Loxp element by PCR
Section, is connected into the pcDNA3-c-myc carrier through EcoRI and NotI enzyme action by the way of enzyme action and obtains pcDNA3-Loxp-Stop-
Loxp-c-myc plasmid；
C, design are for the forward of EF1alpha promoter and reverse primer, and primer adds BamHI and EcoRI enzyme action respectively
Site, is expanded the EF1a promoter fragment obtaining about 1.3kb from pLVX-EF1a-ires-puro plasmid, passes through enzyme by PCR
The pcDNA3-Loxp-Stop-Loxp-c-myc carrier that the mode cut is connected into through BglII and EcoRI enzyme action obtains pcDNA3-
EF1a promoter-Loxp-Stop-Loxp-c-myc plasmid, is called for short: EF1a-Stop-myc, plasmid construct is as shown in Figure 1.
The structure of the transgenic instrument mice of embodiment 2 inducible c-myc gene overexpression
The construction method of the transgenic mice of the inducible c-myc gene overexpression of this embodiment, including in detail below
Step:
1, the structure of EF1a-Stop-myc transgenic founder Mus
A, the EF1a-Stop-myc plasmid obtaining embodiment 1 are taken out in carrying out；
B, by SmaI and PvuI, EF1a-Stop-myc plasmid being carried out enzyme action, gel reclaims about 5.8kb and comprises EF1a
The fragment of promoter-Loxp-Stop-Loxp-c-myc；
Prepared by C, the super ovulation of the female Mus of germ cell donor and the female Mus of pseudo-fetus: the female Mus that 4-6 is all is injected the pregnant mare serum of about 5IU
Promoting sexual gland hormone (PMSG), PMSG injects the human chorionic gonadotropin (hCG) of about 5IU, injects hCG after injecting 48 hours
After, female Mus hero Mus mates；Meanwhile, male with ligation for the female Mus being used for replace-conceive Mus is mated copulation to be used for producing the female Mus of pseudo-fetus；
D, mate rear morning female Mus inspection bolt, will see that the female Mus of bolt puts to death to collect and be in the germ cell of one cell stage,
Carry out male pronucleus microinjection；
After E, microinjection second day, be that bicelluar zygote transplation is to pseudo-fetus female Mus uterus, pseudo-fetus by normal development
The given birth to mice of female Mus is EF1a-Stop-myc transgenic F0 for mice；
F, transgenic F0 cut tail for mice after being born 2 weeks, extract genome, use the method for PCR to transgenic mice
Identifying, PCR is accredited as the mice of the positive and is EF1a-Stop-myc transgenic founder Mus.
2, building of EF1a-Stop-myc transgenic mice is
A, by the EF1a-Stop-myc founder Mus of above-mentioned acquisition and wild-type mice copulation, founder Mus is carried out
Pass on to build and be, it is thus achieved that F1 generation mice；
B, the F1 generation mice from different F0 generations is taken bone marrow, spleen, lymph node and thymus extracting RNA, pass through
The expression of Stop-myc mRNA is compared by the method for Realtime-PCR, select bone marrow, spleen, lymph node and
The strain of the equal high expressed of thymus is as follow-up experiment strain, and the mice obtained is transgenic instrument mice.
In this transgenic instrument mice, c-myc gene expression frame is by a composition type expression promoter EF1a (human
Elongation factor 1alpha) driving, this promoter all has transcriptional activity in almost all of cell type.?
Inserting a Loxp-Stop-Loxp element between promoter and c-myc expression cassette, in this element, the existence of Stop sequence is led to
Cross the mode adding termination codon, stop the translation of follow-up c-myc expression cassette；Two Loxp in Loxp-Stop-Loxp element
Site can be identified by recombinase Cre, and in the presence of Cre enzyme, the Stop sequence between two Loxp sites is removed,
Follow-up c-myc gene can normally be translated, and structure is as shown in Figure 2.
3, hemopoietic system inducible c-myc gene overexpression transgenic mouse lines is obtained
Step 2 obtain EF1a-Stop-myc transgenic mice in, although c-myc encoder block can by normal transcription,
But because above having a translation termination expression cassette (Stop), c-myc cannot be translated, the most not function；Only when
After Cre enzyme effect, translation termination expression cassette (Stop) is deleted, and c-myc can normally translate, function.In this step,
By by EF1a-Stop-myc transgenic instrument mice, (strain is Mx1-Cre transgenic mice, and this is little with Cre transgenic mice
In Mus, Cre is expressed as Mx1 (myxovirus influenza virus resistance 1) promoter and is driven) copulation
Mode obtains the double positive mice (being abbreviated as myc:cre) of EF1a-Stop-myc Yu Cre, is in hemopoietic system and can induce c-
The transgenic instrument mice that myc expresses.
The transgenic instrument mice of the induced c-myc process LAN that the present embodiment obtains, Mx1 promoter is in normal condition
Under, do not express；In the presence of having interferon or interferon inducer (poly dI:dC), various at hemopoietic system
Can great expression in the short time in cell type.Therefore can be made by the way induction of mouse peritoneal injection poly dI:dC
The expression of Mx1 promoters driven Cre in blood system, removes the Stop sequence in EF1a-Stop-myc structure, and c-myc is carried out
Normal translation, function.Therefore it may only be necessary to controlled the time of injection poly dI:dC by mouse peritoneal, control Cre and play
The time of effect, and then control the c-myc process LAN time in hemopoietic system.
The phenotype of leukemia analysis that test example 1 c-myc gene overexpression causes
Comprise the following steps:
1. induction c-myc expresses: the double positive mice of myc:cre transgenic (inducible c-myc gene overexpression turn base
Because of instrument mice) 3 weeks pneumoretroperitoneums of being born inject 250 μ g poly dI:dC, take bone marrow after injecting one week, and extract proteins is to c-myc
Expression carry out western checking；
2. the leukemia Development process that detection c-myc process LAN causes: pass through eye week about after poly dI:dC injection
The mode of socket of the eye blood sampling takes blood and carries out routine blood test detection, is monitored the number change situation of various types of cells, and then speculates white blood
Sick Development process；
The type of leukemia the most clearly fallen ill: combine routine analysis of blood, passes through blood smear, stream to the leukemia mouse of morbidity
The type of leukemia of the methods such as formula cell instrument detects, pathological section clearly sent out c-myc induction
4. the leukemic sickness rate of monitoring c-myc induction, mouse survival curve, enter the stability of this Leukemia Model
Row is evaluated.
Test example 2 c-myc functional study in leukemia generation, evolution
1. c-myc expresses the correlation analysis with proliferation of bone marrow cells ability
Its clonality is compared by medullary cell in vitro that separate inducibility expression c-myc different time
Analyze；
2. c-myc is in the target gene comparative analysis of leukemia morbidity different times regulation and control
It has been demonstrated that, c-myc has different expressions at leukemia morbidity different times, and it may regulate and control not
With target gene, the different time expressed at induction c-myc in this test example, extracting bone marrow, spleen and lymph node mRNA and
Albumen, to downstream target gene, is analyzed as the expression of CyclinD1 etc. carries out detection.
The screening study of the test example 3 anti-leukemia medicine with c-myc gene as target spot
1, the screening of the anti-leukemia medicine with c-myc gene as target spot of cellular level
1. the leukemia medicament screening with Bone Marrow Cells In Vitro clonality as index
Extract the medullary cell of c-myc abduction delivering different time sections respectively, to clinical Common Chemotherapy medicine such as arabinose born of the same parents
Glycosides, the anti-clonality of methotrexate etc. is tested and is compared；
2. to kill the ability leukemia medicament screening as index for B cell, T cell
By the method for immuno magnetic cell separation or selected by flow cytometry apoptosis obtain specific cell type (such as: B cell or
Person's T cell), the cytotoxicity for the leukemic medicine of lymphocyte or T cell that clinic is conventional is carried out screening and compares.
2, the screening of the anti-leukemia medicine with c-myc gene as target spot of animal integral level
1. clinic is commonly used leukemia medicament and compares analysis for morbidity different times leukemia treating effect；
2. the curative effect of potential leukemia compound and Chinese herbal medicine is detected.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not 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, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. the construction method of the transgene carrier of an inducible c-myc process LAN, it is characterised in that comprise the following steps:
A, to comprise c-myc ORF cDNA plasmid as template, SEQ ID No:1 and SEQ ID No:2 is primer, and PCR amplification obtains
Obtain the c-myc coding region of Serial No. SEQ ID No:3, XhoI and XbaI enzyme cutting c-myc coding region and pcDNA3 carrier, connect
Obtain pcDNA3-c-myc plasmid；
B, with comprise Loxp-Stop-Loxp element plasmid as template, SEQ ID No:4 and SEQ ID No:5 is primer, PCR
Amplification obtains the Loxp-Stop-Loxp fragment of Serial No. SEQ ID No:6, EcoRI and NotI enzyme action Loxp-Stop-Loxp
Fragment and pcDNA3-c-myc plasmid, connect and obtain pcDNA3-Loxp-Stop-Loxp-c-myc plasmid；
C, with pLVX-EF1a-ires-puro plasmid as template, SEQ ID No:7 and SEQ ID No:8 is primer, PCR expand
Obtain Serial No. SEQ ID No:9 EF1a promoter fragment, BamHI and EcoRI enzyme action EF1a promoter fragment and
PcDNA3-Loxp-Stop-Loxp-c-myc plasmid, connects and obtains inducible c-myc process LAN transgene carrier.
2. construction method described in claim 1 builds the transgene carrier of the inducible c-myc process LAN obtained.
3. the construction method of the transgene mouse model of an inducible c-myc process LAN, it is characterised in that include following step
Rapid:
(1) transgene carrier of the inducible c-myc process LAN, described in SmaI and PvuI enzyme action claim 5, gel reclaims
Comprising the fragment of EF1a promoter-Loxp-Stop-Loxp-c-myc, after the super ovulation of the female Mus of germ cell donor, female Mus hero Mus is closed
Cage, puts to death the female Mus seeing bolt and collects the germ cell being in one cell stage, carry out male pronucleus microinjection；It is two by normal development
The zygote transplation of cell is transgenic F0 for mice to pseudo-fetus female Mus uterus, the given birth to mice of the female Mus of pseudo-fetus；
(2), by above-mentioned transgenic F0 for mice and wild-type mice copulation, pass on to build and be, it is thus achieved that F1 generation mice；Select at bone
The F1 generation mice of the equal high expressed of marrow, spleen, lymph node and thymus is as follow-up experiment strain, it is thus achieved that transgenic instrument is little
Mus；
(3) the transgenic instrument mice, by step (2) obtained and Cre transgenic mice copulation, acquisition can induce c-myc to cross table
The transgene mouse model reached.
4. the construction method described in claim 3 builds the transgene mouse model of the inducible c-myc process LAN obtained.
5. the transgene carrier of the inducible c-myc process LAN described in claim 2 or inducing described in claim 4
The application in anti-leukemia medicine screens of the transgene mouse model of type c-myc process LAN.
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