CN106222187B - Transgene carrier, transgene mouse model and its construction method that inducible c-myc is overexpressed - Google Patents
Transgene carrier, transgene mouse model and its construction method that inducible c-myc is overexpressed Download PDFInfo
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Abstract
The invention discloses a kind of transgene carrier, transgenosis tool mouse and its construction method that inducible c-myc is overexpressed, the code area c-myc is connect with pcDNA3 carrier obtains pcDNA3-c-myc plasmid；Loxp-Stop-Loxp segment is connect with pcDNA3-c-myc plasmid obtains pcDNA3-Loxp-Stop-Loxp-c-myc plasmid；EF1a promoter fragment is connect with pcDNA3-Loxp-Stop-Loxp-c-myc plasmid obtains transgene carrier, then constructs transgene mouse model based on the transgene carrier.The transgene mouse model that the present invention constructs is derivable Leukemia Model, c-myc, which is not expressed, in the case where no inducer does not fall ill, c-myc is overexpressed in the case where inducer effect, cause the generation of leukaemia, more conventional transgene mouse model has better controllability, can be needed to adjust the population quantity that need to be maintained according to experiment.
Description
Technical field
The invention belongs to gene engineering technology fields, more particularly it relates to which one kind can induce in hemopoietic system
Type c-myc transgene carrier and its construction method and in hemopoietic system inducible c-myc transgene mouse model and its
Construction method.
Background technique
Leukaemia is that one kind betides blood forming organ, with the proliferation of leucocyte and its precursor in blood and marrow and
The malignant disease that dysplasia is characterized.According to the report of the World Health Organization, there are about 210,000 people to die of white blood for the whole world in 2000
Disease, wherein 90% is adult.The characteristics 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 the powerful for carrying out experimental therapy, are of great significance.
Research in recent years shows that the missing or mutation of the generation of leukaemia and the activation of cellular proto-oncogene and tumor suppressor gene have
Close relationship.Wherein c myc is to study one of most commonly used oncogene at present, which belongs to myc original
One of Oncogene family member (c-Myc, N-myc and L-myc).Myc albumen is basic-helix-loop-helix
Zipper (bHLHZ) class transcription factor can form heterodimer with Max and be integrated to special CAC (G/A) TG ' E-box '
Sequence, by the expression for combining a large amount of target genes with various other factors.Myc is considered participating in most basic cell work
Dynamic process, is such as proliferated, growth, and differentiation and apoptosis etc. play a significant role in embryonic development and tumour generating process.
In hemopoietic system, c-myc gene is not only played regulatory role in each stage that hematopoietic cell generates, and is expressed different
Often with the generation of disease in the blood system, the generation of especially all types of leukaemia is closely related.Studies have shown that almost all
C-myc gene constitutive expression caused by being found because of chromosomal rearrangement in male's Burkitt ' s B cell lymphoma case；
In acute lymphoblastic leukemia and acute myelocytic leukemia, though not finding the transposition of c-myc gene, but still it can find to lead to
Cross other chromosomal rearrangements, to initiations such as phosphorylation modification, the upstream the c-myc positive regulating gene abnormal activations of c-myc exception
Overexpression on c-myc gene function；In the entire pathogenic process of chronic myelocytic leukemia, can it find because of the upstream c-myc
Regulate and control the overexpression of the c-myc of the expression initiation of kinases bcr/abl, and it was found that the mRNA level in-site of c-myc is thin with chronic grain
The progression of born of the same parents' leukaemia is constantly increased from chronic phase, accelerated period to acute change's phase；Recent studies have shown that c-myc's is general
Elementization adjusting plays a significant role in the starting and development process of chronic myelogenous leukemia.
C-myc transgenic mice research shows that: by Ig heavy chain enhancer drive c-myc gene in bone-marrow-derived lymphocyte
High expression can lead to the generation of lymthoma and early stage B cell leukemia.The transgene mouse model has been used as spontaneous white
The classical model of blood disease is widely used in leukaemia pathogenesis, the research such as related drugs screening.By virus infection in marrow
C-myc's studies have shown that bone marrow cell, which is overexpressed c-myc, can lead to the generation of acute myeloid leukaemia is overexpressed in cell.This
A little evidences show that c-myc gene plays a significant role in the generation, development process of multiple types leukaemia.The gene-correlation turns
The foundation of genetic model simulates the generating process of human leukemia, promotes the understanding to c-myc gene function, accelerates
To the research and development process of leukemia medicament.
However, these models also have its limitation: the c-myc transgenic mice (E μ-myc) driven with Ig heavy chain enhancer
For, the overexpression of c-myc starts from embryonic period, embryonic phase, the leukaemia symptoms such as B cell volume increase can be observed in mouse post-natal,
The understanding for myc gene function in leukaemia generating process caused by c-myc is hindered, in the transgene mouse model,
Myc causes the average life span of E μ-myc transgenic mice there was only 12 weeks from early stage lasting high expression, needs in the course of the research
Maintain a large amount of population.
In conclusion since there is currently used c-myc transgenosis leukemia mouse model c-myc to continue high expression,
The defects of disease time is uncontrollable, there is an urgent need to establish a kind of derivable spontaneous leukaemia transgenic models.
Summary of the invention
Based on this, in order to overcome the defects of the prior art described above, the present invention provides one kind can induce in hemopoietic system
Transgene carrier, mouse model and its construction method that type c-myc is overexpressed.
In order to achieve the above-mentioned object of the invention, this invention takes following technical schemes:
A kind of inducible c-myc is overexpressed the construction method of transgene carrier, comprising the following steps:
A, using comprising c-myc ORF cDNA plasmid, as template, SEQ ID No:1 and SEQ ID No:2 is primer, PCR expands
Increase the code area c-myc for obtaining Serial No. SEQ ID No:3, XhoI and the code area XbaI enzyme cutting c-myc and pcDNA3 carrier,
Connection obtains pcDNA3-c-myc plasmid；
B, using the plasmid comprising Loxp-Stop-Loxp element as template, SEQ ID No:4 and SEQ ID No:5 is to draw
Object, PCR amplification obtain the Loxp-Stop-Loxp segment of Serial No. SEQ ID No:6, EcoRI and NotI digestion Loxp-
Stop-Loxp segment and pcDNA3-c-myc plasmid, connection obtain pcDNA3-Loxp-Stop-Loxp-c-myc plasmid；
C, using 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 digestion EF1a promoter fragment and
PcDNA3-Loxp-Stop-Loxp-c-myc plasmid, connection obtain inducible c-myc and are overexpressed transgene carrier.
The present invention also provides the inducible c-myc that above-mentioned construction method constructs to be overexpressed transgene carrier.
The present invention also provides the construction methods that a kind of inducible c-myc is overexpressed transgene mouse model, including with
Lower step:
(1), the above-mentioned inducible c-myc of SmaI and PvuI digestion is overexpressed transgene carrier, and gel recycling includes EF1a
The segment of promoter-Loxp-Stop-Loxp-c-myc, female mice hero mouse mates after the super ovulation of fertilized eggs donor female mice, will see bolt
Female mice put to death collect be in one cell stage fertilized eggs, carry out male pronucleus microinjection；By normal development be it is bicelluar by
To false pregnancy female mice uterus, the given birth to mouse of false pregnancy female mice is transgenosis F0 for mouse for smart ovum transfer；
(2), above-mentioned transgenosis F0 is mated for mouse with wild-type mice, passage, which is built, is, obtains F1 generation mouse；Selection exists
It is small to obtain transgenosis tool as subsequent experiment strain for the highly expressed F1 generation mouse of marrow, spleen, lymph node and thymus gland
Mouse；
(3), the transgenosis tool mouse that step (2) obtain is mated with Cre transgenic mice, obtaining can induce c-myc
The transgenosis tool mouse of overexpression.
The present invention also provides the inducible c-myc that above-mentioned construction method constructs to be overexpressed transgenic mice mould
Type.
Transgene carrier is overexpressed the present invention also provides above-mentioned inducible c-myc and inducible c-myc is overexpressed
Application of the transgene mouse model in anti-leukemia medicine screening.
Compared with prior art, the invention has the following advantages:
1, the transgene mouse model that present invention building obtains is derivable Leukemia Model, in the feelings of not inducer
C-myc is not expressed and is not fallen ill under condition, and c-myc is overexpressed in the case where inducer effect, causes the generation of leukaemia, more routinely
Transgene mouse model there is better controllability, can be needed to adjust the population quantity that need to maintain according to experiment；
2, the leukaemia transgene mouse model that the present invention establishes gives the time of inducer by control, controls c-myc
Overexpression opportunity, to control the disease time of leukaemia, function of the research c-myc in leukaemia morbidity different times；With
The leukaemia animal model of animal integral level is established based on the model, the drug for screening for different times leukaemia mentions
It has supplied to support；The leukemia mouse model that the present invention establishes is the Leukemia Model of spontaneous type, compared with tumor cell transplantation model etc.
Intracorporal onset state can more be simulated；
3, the medicaments sifting model established of the present invention, using c-myc proto-oncogene as target spot, the gene with it is a plurality of types of white
Generation, the development of blood disease are closely related, provide new selection for leukemia medicament screening.
Detailed description of the invention
Fig. 1 is the structure of the transgene carrier of the inducible c-myc gene overexpression constructed in the embodiment of the present invention 1
Figure；
Fig. 2 is the function of the transgene mouse model for the inducible c-myc gene overexpression that the embodiment of the present invention 2 constructs
Figure.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be described in detail, in following embodiment unless otherwise specified, institute
The conventional practices commercially available, used method is well known to those skilled in the art are derived from using raw material.
The building of the transgene carrier of the inducible c-myc gene overexpression of embodiment 1
The construction method of the transgene carrier of the embodiment, comprising the following specific steps
A, design is directed to the forward and reverse primer of the code area c-myc, and primer adds XhoI and XbaI enzyme cutting site respectively,
By PCR from the code area c-myc for obtaining about 1.4kb comprising amplification on c-myc ORF cDNA plasmid, by way of digestion
It is connected into and obtains pcDNA3-c-myc plasmid through the pcDNA3 carrier of XhoI and XbaI enzyme cutting；
B, design is directed to the forward and reverse primer of Loxp-Stop-Loxp element, and primer adds EcoRI and NotI respectively
Site, by PCR, amplification obtains the Loxp-Stop-Loxp piece of about 1.3kb from the plasmid comprising Loxp-Stop-Loxp element
Section is connected into the pcDNA3-c-myc carrier through EcoRI and NotI digestion by way of digestion and obtains pcDNA3-Loxp-Stop-
Loxp-c-myc plasmid；
C, design is directed to the forward and reverse primer of EF1alpha promoter, and primer adds BamHI and EcoRI digestion respectively
Site is expanded the EF1a promoter fragment for obtaining about 1.3kb from pLVX-EF1a-ires-puro plasmid by PCR, passes through enzyme
The mode cut is connected into the pcDNA3-Loxp-Stop-Loxp-c-myc carrier through BglII and EcoRI digestion and obtains pcDNA3-
EF1a promoter-Loxp-Stop-Loxp-c-myc plasmid, referred to as: EF1a-Stop-myc, plasmid construct are as shown in Figure 1.
The building of the transgenosis tool mouse of the inducible c-myc gene overexpression of embodiment 2
The construction method of the transgenic mice of the inducible c-myc gene overexpression of the embodiment, including in detail below
Step:
1, the building of EF1a-Stop-myc transgenosis founder mouse
A, it is taken out in the EF1a-Stop-myc plasmid progress obtained to embodiment 1；
B, digestion is carried out to EF1a-Stop-myc plasmid by SmaI and PvuI, it includes EF1a that gel, which recycles about 5.8kb,
The segment of promoter-Loxp-Stop-Loxp-c-myc；
C, the super ovulation of fertilized eggs donor female mice and the preparation of false pregnancy female mice: to the pregnant mare serum of 4-6 weeks female mice injection about 5IU
Promoting sexual gland hormone (PMSG), PMSG inject the human chorionic gonadotrophin (hCG) of about 5IU after injecting 48 hours, inject hCG
Afterwards, female mice hero mouse mates；Meanwhile the female mice for being used for replace-conceive being mated to mate with the male mouse of ligation and is used to generate false pregnancy female mice；
D, rear morning female mice inspection bolt is mated, the female mice for seeing bolt is put to death and collects the fertilized eggs for being in one cell stage,
Carry out male pronucleus microinjection；
It E, is bicelluar zygote transplation to false pregnancy female mice uterus, false pregnancy by normal development second day after microinjection
The given birth to mouse of female mice is EF1a-Stop-myc transgenosis F0 for mouse；
F, transgenosis F0 cuts tail after being born 2 weeks for mouse, genome is extracted, using the method for PCR to transgenic mice
It is identified, it is EF1a-Stop-myc transgenosis founder mouse that PCR, which is accredited as positive mouse,.
2, building for EF1a-Stop-myc transgenic mice is
A, the EF1a-Stop-myc founder mouse of above-mentioned acquisition is mated with wild-type mice, founder mouse is carried out
Passage, which is built, is, obtains F1 generation mouse；
B, marrow, spleen, lymph node and thymus gland extract RNA are taken to the F1 generation mouse from different F0 generations, passed through
The method of Realtime-PCR is compared the expression quantity of Stop-myc mRNA, selection marrow, spleen, lymph node and
For the highly expressed strain of thymus gland as subsequent experiment strain, mouse obtained is transgenosis tool mouse.
In the transgenosis tool mouse, c-myc gene expression frame is by a composition type expression promoter EF1a (human
Elongation factor 1alpha) driving, which has transcriptional activity in almost all of cell type.?
A Loxp-Stop-Loxp element is inserted between promoter and c-myc expression cassette, the presence of Stop sequence is logical in the element
The mode for crossing addition terminator codon, prevents the translation of subsequent 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 sites Loxp is removed,
Subsequent c-myc gene can be translated normally, and structure is as shown in Figure 2.
3, the inducible c-myc gene overexpression transgenic mouse lines of hemopoietic system are obtained
Step 2 obtain EF1a-Stop-myc transgenic mice in, although c-myc encoder block can by normal transcription,
But because there is a translation termination expression cassette (Stop) in front, c-myc can not be translated, therefore also not functioned；Only when
After Cre enzyme effect, translation termination expression cassette (Stop) is deleted, and c-myc can be translated normally, be functioned.In this step,
By by EF1a-Stop-myc transgenosis tool mouse, (strain is Mx1-Cre transgenic mice, this is small with Cre transgenic mice
Cre is expressed as Mx1 (myxovirus influenza virus resistance 1) promoter and is driven in mouse) mating
Mode obtains the mouse (being abbreviated as myc:cre) of EF1a-Stop-myc Yu the bis- positives of Cre, can induce c- as in hemopoietic system
The transgenosis tool mouse of myc expression.
The transgenosis tool mouse that the inducible c-myc that the present embodiment obtains is overexpressed, Mx1 promoter is in normal condition
Under, it does not express；In the presence of having interferon or interferon inducer (poly dI:dC), in the various of hemopoietic system
It can be with great expression in the short time in cell type.Therefore the method induction that poly dI:dC can be injected by mouse peritoneal is made
The expression of Mx1 promoter driving Cre, the Stop sequence in EF1a-Stop-myc structure is removed, c-myc is carried out in blood system
Normal translation, functions.Therefore it may only be necessary to control the time of injection poly dI:dC by mouse peritoneal, control Cre is played
The time of effect, and then control overexpression time of the c-myc in hemopoietic system.
The phenotype of leukemia analysis that 1 c-myc gene overexpression of test example causes
The following steps are included:
1. induction c-myc expression: (inducible c-myc gene overexpression turns base for myc:cre transgenosis double positive mices
Because of tool mouse) 250 μ g poly dI:dC are injected intraperitoneally after 3 weeks in birth, and injection takes marrow after a week, and extract proteins are to c-myc
Expression carry out western verifying；
2. detection c-myc is overexpressed the leukaemia Development process caused: passing through eye every other week after poly dI:dC injection
The mode of socket of the eye blood sampling takes blood to carry out blood routine detection, is monitored to the quantity situation of change of various types of cells, and then speculates white blood
The Development process of disease；
3. the type of leukemia clearly fallen ill: in conjunction with routine analysis of blood, passing through blood film, stream to the leukemia mouse of morbidity
The detection of formula cell instrument, the type of leukemia that clear the sent out c-myc of the methods of pathological section induces
4. monitor the disease incidence for the leukaemia that c-myc induces, mouse survival curve, to the stability of the Leukemia Model into
Row evaluation.
Functional study of 2 c-myc of test example in leukaemia generation, development process
1. the correlation analysis of c-myc expression and proliferation of bone marrow cells ability
The bone marrow cell of separation inducibility expression c-myc different time is in vitro compared its clonality
Analysis；
2. c-myc is in the target gene comparative analysis of leukaemia morbidity different times regulation
It has been demonstrated that c-myc has different expression quantity in leukaemia morbidity different times, may regulate and control not
With target gene, in the different time of induction c-myc expression in this test example, extracting marrow, spleen and lymph node mRNA and
Albumen tests and analyzes downstream target gene, such as the expression of CyclinD1.
Test example 3 is using c-myc gene as the screening study of the anti-leukemia medicine of target spot
1, cellular level using c-myc gene as the screening of the anti-leukemia medicine of target spot
1. being screened by the leukemia medicament of index of Bone Marrow Cells In Vitro clonality
C-myc inducing expression bone marrow cell in different time periods is extracted respectively, to clinical Common Chemotherapy drug such as arabinose born of the same parents
Glycosides, the anti-clonality of methotrexate etc., which is tested, to be compared；
2. to be screened for B cell, T cell killing ability as the leukemia medicament of index
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), screening comparison is carried out to the clinically used cytotoxicity for lymphocyte or the drug of T cell leukaemia.
2, animal integral level using c-myc gene as the screening of the anti-leukemia medicine of target spot
1. being compared analysis for morbidity different times leukemia treating effect to the common leukemia medicament of clinic；
2. being detected to the curative effect of potential anti-leukemia compound and Chinese herbal medicine.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot 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 guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (4)
1. a kind of construction method for the transgene carrier that inducible c-myc is overexpressed, which comprises the following steps:
A, using comprising c-myc ORF cDNA plasmid, as template, SEQ ID No:1 and SEQ ID No:2 is primer, PCR amplification is obtained
Obtain the code area c-myc of Serial No. SEQ ID No:3, XhoI and the code area XbaI enzyme cutting c-myc and pcDNA3 carrier, connection
Obtain pcDNA3-c-myc plasmid；
B, using the plasmid comprising Loxp-Stop-Loxp element as template, SEQ ID No:4 and SEQ ID No:5 is primer, PCR
Amplification obtains the Loxp-Stop-Loxp segment of Serial No. SEQ ID No:6, EcoRI and NotI digestion Loxp-Stop-Loxp
Segment and pcDNA3-c-myc plasmid, connection obtain pcDNA3-Loxp-Stop-Loxp-c-myc plasmid；
C, using 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 digestion EF1a promoter fragment and
PcDNA3-Loxp-Stop-Loxp-c-myc plasmid, connection obtain inducible c-myc and are overexpressed transgene carrier.
2. the transgene carrier that the inducible c-myc that construction method described in claim 1 constructs is overexpressed.
3. a kind of construction method for the transgene mouse model that inducible c-myc is overexpressed, which is characterized in that including following step
It is rapid:
(1), the transgene carrier that SmaI and PvuI digestion inducible c-myc as claimed in claim 2 is overexpressed, gel recycling
Segment comprising EF1a promoter-Loxp-Stop-Loxp-c-myc, female mice hero mouse is closed after the super ovulation of fertilized eggs donor female mice
The female mice for seeing bolt is put to death and collects the fertilized eggs for being in one cell stage, carries out male pronucleus microinjection by cage；It is two by normal development
For the zygote transplation of cell to false pregnancy female mice uterus, the given birth to mouse of false pregnancy female mice is transgenosis F0 for mouse；
(2), above-mentioned transgenosis F0 is mated for mouse with wild-type mice, passage, which is built, is, obtains F1 generation mouse；Selection is in bone
It is small to obtain transgenosis tool as subsequent experiment strain for the highly expressed F1 generation mouse of marrow, spleen, lymph node and thymus gland
Mouse；
(3), the transgenosis tool mouse that step (2) obtain is mated with Cre transgenic mice, obtaining, which can induce c-myc, crosses table
The transgene mouse model reached.
4. transgene carrier or building side as claimed in claim 3 that inducible c-myc as claimed in claim 2 is overexpressed
The transgene mouse model that the inducible c-myc that method constructs is overexpressed is in preparing anti-leukemia medicine screening system
Using.
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