CN102860282A - Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system - Google Patents
Preparation method of transgenic mouse of specificity expression Cre recombinase of hematopoietic system Download PDFInfo
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- CN102860282A CN102860282A CN2011101874413A CN201110187441A CN102860282A CN 102860282 A CN102860282 A CN 102860282A CN 2011101874413 A CN2011101874413 A CN 2011101874413A CN 201110187441 A CN201110187441 A CN 201110187441A CN 102860282 A CN102860282 A CN 102860282A
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Abstract
The invention relates to a preparation method of a transgenic mouse of specificity expression Cre recombinase of a hematopoietic system. The invention discloses a transgenic mouse animal model, a deoxyribonucleic acid (DNA) fragment pEDAG-Cre is integrated in a genome of the transgenic mouse animal model, the DNA fragment comprises a promoter at an end of a gene (EDAG) 5' which is related to human embryonic development, a Cre recombinase gene and a human growth hormone gene polyA fragment. The invention also comprises a preparation method of the transgenic mouse animal model. The method comprises the steps of cloning 2.3 kb of a control region of the promoter at the end of the EDAG gene 5' by using a polymerase chain reaction (PCR) method, constructing a transgenic expression vector pEDAG-Cre, leading 5.6 kb of transgenic fragments into a male pronucleus of the mouse by using a micro-injection method, and identifying a positive transgenic mouse by using the PCR method. The RT-PCR method and a mouse-identifying result report ROSA26LacZ show that the specificity expression Cre recombinase of the transgenic mouse can be achieved only in the hematopoietic system.
Description
Technical field
The invention relates to a kind of preparation method of transgene mouse model, be specifically related to utilize the size of PCR method human cloning embryonic development related gene EDAG gene to be 5 ' the end promoter regulation zone of 2.3kb and a kind of preparation method who utilizes this regulating and controlling sequence construction of expression vector and prepare the transgenic mice of the specific expressed Cre recombinase of hemopoietic system.
Background technology
Mammal hematopoiesis is the dynamic process of a complexity.7.5 days mice embryonic phases, endothelium and hematopoietic cell that the extraembryonic mesoderm derived cell produces form blood island jointly, and this moment, hematopoietic cell mainly was pronormoblast, and nuclear is round, cell space is large and express the embryo type haemoglobin, this process is lasted of short duration, is called as original hematopoiesis.10.5 days embryonic period, embryonic phases, aorta-gonad-mesonephros district, site (AGM) begins to produce hematopoietic stem cell in the embryo, peaks at 11.5 days, and this type of cell can be to a plurality of hematopoietic cell lineage differentiation.11 days embryonic period, embryonic phases, candidate stem cell begins to migrate to the tire liver by the blood circulation of having set up, and continues to reach maturity, and is the most remarkable with erythroid differentiation.Seedless, the synthetic adult form globin of red blood cell this moment indicates the foundation of permanent hematopoiesis.The hematopoiesis of AGM district is active in and finished 13 days embryonic period, embryonic phases, and the tire liver becomes the hematopoiesis main place thereafter.When closing on birth, the fetal liver hemopoietic activity is disappeared, and is replaced by marrow, and the latter becomes lifelong blood forming organ (Dzierzak E.Ontogenic emergence of definitive hematopoietic stem cells.Curr Opin Hematol, 2003,10:229-234).
It is expression of specific gene or reticent and obtain the process of particular phenotype that embryonic hematopoiesis is grown, the multiple gene relevant with hematopoietic regulation or gene family have been found, such as GATA, C/EBP and AML/CBF/Runx1 etc., have an effect in the different phase of hematopoietic development respectively.The harmony of hematopoiesis related gene is expressed under a series of complicated signal network regulation and control and is finished in these nuclears, such as Notch, Wnt, Sonic, hedgehog (Shh), Smad etc.Along with the arrival in functional genome's epoch, the gene function and the Research on the effect mechanism that play a role that comprises said gene seemed particularly urgent in the especially early stage hemopoietic system growth course of hemopoietic system.Traditional gene Knockout is being brought into play important effect as effective research means aspect this, usually cause the mice embryonic deformity even can't carry out follow-up Journal of Sex Research in the death of embryo's Development but knock out the gene relevant with embryonic development.Adopt tissue-specific gene knockout's technology of the recombination system mediation of Cre/Loxp then can specificity to control specific expressed in tissue of some important gene, and then study it in the effect of different developmental phases.
The EDAG gene is the new gene of hematopoietic tissue specifically expressing, studies show that, the EDAG gene specific is expressed in the prematurity hematopoietic cell (CD34 in the hematopoietic tissue (grow up marrow, embryonic liver)
+Cell, be candidate stem cell), in the mature cells such as the heart, liver, spleen, lung, kidney, brain, muscle and peripheral blood lymphocytes, do not express (gate of a village army, Deng .EDAG-1, the separating and confirm of a kind of and the closely related new gene of hematopoietic regulation. Acta Biochimica et Biophysica Sinica, 2001,33:641-646).But high expressed EDAG in leukemia human peripheral blood cell and leukaemia K562 induces the K562 cell after red system or the differentiation of macronucleus system at ferroheme Hemin, dyers' grapes PMA etc., and EDAG expresses rapidly downward modulation; After utilizing antisensenucleic acids to suppress the EDAG expression, K562 cell proliferation is obstructed, and colony formation ability descends, and cell is more responsive to the derivant reaction.The hematopoiesis specifically expressing EDAG trangenic mice that research CD11a drives finds that its thymus development is unusual, and spleen is loose; Peripheral blood lymphocyte paramophia, ratio descend, and the neutrophil leucocyte ratio rises; Marrow B220
+Cell obviously descends, sca-1
+C-kit
+Lin
-Cell increases; Spleen DC cytosis, B220
+Positive cell reduces; Thymus gland T cell development is blocked in early days, the DN cytosis; And the Bone Marrow Cells In Vitro differentiation capability obviously descends.These results suggest EDAG high expressed may block the candidate stem cell normal differentiation, and its down-regulated expression is essential with growth by the hematopoietic cell differentiation.These studies show that the EDAG/ specifically expressing in early stage hematopoietic cell/candidate stem cell and with closely related (the Li CY of hematopoietic differentiation regulation and control, et al.EDAG regulates the proliferation and differentiation of hematopoietic cells and resists cell apoptosis through the activation of nuclear factor-kappa B.Cell Death Differ, 2004,11:1299-1308; Li CY, et al.Overexpression of a hematopoietic transcriptional regulator EDAG induces myelopoiesis and suppresses lymphopoiesis in transgenic mice.Leukemia, 2007,21:2277-2286; Yang LV, et al.Hemogen is a novel nuclear factor specifically expressed in mouse hematopoietic development and its human homologue EDAG maps to chromosome 9q22, a region containing breakpoints of hematological neoplasms.Mech Dev, 2001,104:105-111).Therefore we use PCR method and have cloned hemopoietic system specific expressed that the regulating and controlling sequence of people EDAGEDAG 5 ' end 2.3kb is used for instructing the Cre recombinase gene in the process of the transgenic mice of the specific expressed Cre recombinase of preparation hemopoietic system.
The acquisition of this transgenic mice is studied the development related gene biological function in the different developmental phases and the domestic strong research tool of expression pattern body thereof in hemopoietic system for realize series of genes reorganization operation, preparation tissue specific expression animal model in hemopoietic system by the Cre/Loxp recombination system.
Summary of the invention
The object of the present invention is to provide transgenic mice animal model and the preparation method of the specific expressed Cre recombinase of a kind of hemopoietic system.
Key of the present invention be to utilize the genetic recombination of Cre recombinase and knock out specificity and the early stage hematopoiesis of EDAG gene specific expressed, produce transgenic mice, realize the specific expressed regulation and control in hemopoietic system of Cre recombinase by the EDAG promotor.
The preparation method of this transgenic mice may further comprise the steps:
One, the clone of people's embryonic development related gene EDAG 5 ' end promotor;
Two, utilize EDAG 5 ' the end promoter sequence that obtains to make up the specific expressed Cre recombinase expression vector pEDAG-Cre of hematopoietic cell;
Three, digestion with restriction enzyme linearisation pEDAG-Cre expression vector, recovery and purifying be the linearisation fragment of 5.6Kb approximately;
Four, the linearisation expression vector is carried out microinjection mouse male pronucleus, successfully prepared the transgenic mice that is integrated with this expression vector in the genome.
Description of drawings
Fig. 1 is the structural representation of transgene expression vector pEDAG-Cre.
Fig. 2 is that the enzyme of expression vector pEDAG-Cre is cut the evaluation electrophoresis pattern.Nucleic acid molecular weight standard: DL2000marker wherein; Right side nucleic acid molecular weight standard: DL15000Marker.
Fig. 3 is the electrophoresis qualification result that PCR identifies the genetically modified head of the EDAG-Cre person of building (F0 generation) mouse.M:DL2000DNA molecular weight standard wherein; 1: positive control; 2～10: build mouse children musculus cdna group DNA PCR result through the head that obtains behind the pronuclear microinjection.
Fig. 4 is the electrophoresis qualification result collection of illustrative plates that the RT-PCR method is identified the tissue specific expression of transgenic mice Cre recombinase.Wherein, M:DL2000DNA standard molecular weight; 1: positive control; 2: marrow; 3: thymus gland; 4: peripheral blood; 5: spleen; 6: liver; 7: kidney; 8: lung; 9: muscle; 10: the tire liver; 11: the tire brain; 12: negative control.
Fig. 5 EDAG-Cre transgenic mice and ROSA26 mouse mating situation.
Fig. 6 is the expression activity of Cre recombinase in ROSA26 mouse bone marrow cells and thymus gland.The expression activity of LacZ in the A. bone marrow cell wherein, the expression activity of LacZ in the B. spleen cell (
*P＜0.05,
* *P＜0.001).
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.Should understand these case study on implementation only for explanation the present invention but not for limiting scope of the present invention.
Materials and methods
1. material
Recombinase Cre expression plasmid A1.0-Cre is so kind as to give by professor Yang Xiao of BIO ENGINEERING INST MILITARY.The plasmid pEDAG-GFP that includes the EDAG promotor is made up and is preserved by this chamber.Bacterial isolates DH5a, JM109 are preserved by this chamber preparation.Restriction enzyme and T4 ligase etc. are available from NEB company.The C57B/6 mouse is provided by the court's Experimental Animal Center.ROSA26 reporter gene mouse is provided by model animal research institute of Nanjing University.After successfully constructing, preserves in Military Medical Science Institute's Experimental Animal Center the EDAG-Cre transgenic mice.
2.EDAG-Cre targeting vector makes up
The plasmid DNA of EDAG promoter region-2214 to+96 fragments take pEDAG-GFP is template, through behind the pcr amplification, behind the KpnI/SalI restriction enzymes double zyme cutting, be cloned into and make up the Cre recombinase targeting vector EDAG-Cre that EDAG drives in the A1.0-Cre carrier, confirm that through order-checking sequence is correct.
Cre PCR primer sequence is as follows:
Forward primer: CGG GGT ACC TAC AGT CCC TCC ACA CTC TGC TTC
Reverse primer: CGG GTC GAC CTT CCT GCT ATT TTG GTC TGA CTT CC
3.EDAG-Cre the preparation of transgenic mice
The EDAG-Cre plasmid is cut through KpnI/SacII linearisation enzyme, and dna fragmentation reclaims, and carries out routinely fertilized egg pronuclear microinjection and oviduct embryo transplantation, is finished by Institute of Experimental Animals, Chinese Academy of Medical Sciences.Laboratory animal is used the C57Bl/6 mouse.
4.EDAG-Cre first evaluation of building mouse
The about 0.3-0.5cm of 10-14 days young mouse tail point adds 55ul and organizes lysate after the clip birth, after 55 ℃ of overnight incubation, 100 ℃ of deactivations 5 minutes, add 100ul distilled water after, the centrifuging and taking supernatant is as the Genomic PCR template.Cre recombinase universal primer is as follows, amplification purpose sheet segment length 354bp:
Forward primer sequence: GGA CAT GTT CAG GGA TCG CCA GGC G
Reverse primer sequence: CCA TGA GTG AAC GAA CCT GG
PCR condition: 94 ℃ of denaturations 3 minutes; 94 ℃ of sex change 30s, 58 ℃ of renaturation 30s, 72 ℃ are extended 30s,
Carry out 42 circulations; Last 72 ℃ were extended 5 minutes.
5.EDAG-Cre the expression of recombinase gene in the mouse
Put to death the positive transgenic mice of EDAG-Cre in 6 ages in week, get the various tissues that comprise liver, spleen, kidney, lung, thymus gland, marrow, peripheral blood, muscle; In addition with the male mouse of Cre transgenic positive and wild female mouse mating, determine the mating date by checking female mouse vaginal plug, collect E13.5 in age days mice embryonic of embryo, identify the positive embryo of Cre with yolk sac genomic DNA PCR, get tire liver and the brain of positive mice embryonic, use the Trizol of Invitrogen company
Kit extracts total RNA of above-mentioned tissue, prepares cDNA with Takara RNA PCR reverse transcription kit, and carry out PCR with Takara rTaq and above-mentioned Cre primer and identify, take GAPDH as the internal reference gene, purpose sheet segment length 150bp:
Forward primer: TGC CCC CAT GTT TGT GAT G
Reverse primer: TGT GGT CAT GAG CCC CTT TCC
6.EDAG-Cre the tissue distribution of recombinase active
EDAG-Cre mouse and ROSA26 mouse post-coitum are got two positive (EDAG-Cre in 8 ages in week
+/ LacZ
+) mouse and LacZ
+Mouse bone marrow cells and spleen are separated into unicellularly, get 1X10
6Cell is used the beta galactosidase kit and is detected galactosidase activity (LacZ dyeing), in 37 ℃ hatch 1～2h after, the absorbance under microplate reader detection 405nm wavelength.
The result
1.EDAG-Cre targeting vector construction expression
EDAG transgenosis targeting vector is comprised of EDAG gene promoter area (2.3kb), Cre recombinase (1.2Kb) and human growth hormone (HGH) regulating and controlling sequence (hGH, 2.1kb), and total length is 5.6kb approximately; This targeting vector becomes linear DNA fragment after cutting through restriction enzyme KpnI/SacII enzyme, is used for the micro-procaryotic injection of fertilized egg subsequently.
2. transgenosis head builds the evaluation of mouse
EDAG-Cre transgenosis targeting vector being limited property restriction endonuclease KpnI and SacII linearization for enzyme restriction behind the fertilized egg pronuclear microinjection, are born young mouse after mouse coda gene group DNA PCR identifies, obtain altogether 6 positive Cre transgenosis head and build mouse.
3.Cre the hemopoietic system tissue specific expression of recombinase gene
Positive head build mouse build through going down to posterity be after, Fig. 3 is seen in the expression of Cre recombinase gene in adult mice and various each tissue of embryo.RT-PCR result's demonstration, in the body of adult mice, the Cre recombinase gene is only expressed in the hemopoietic systems such as marrow, thymus gland, peripheral blood, spleen, and wherein the expression in the tissue such as marrow and thymus gland is higher than peripheral blood and spleen; In embryonic tissue, undertaking in the tire liver of embryo's early metaphase hematopoiesis has obvious expression, then has no expression in the tire brain.
4.Cre the tissue expression of recombinase is active
Be to detect the recombinase expression activity of EDAG-Cre transgenic mice, we have carried out mating with EDAG-Cre mouse and ROSA26 mouse, get 8 all ages EDAG-Cre
+/ LacZ
+The marrow of two positive mouse and spleen tissue are with LacZ
+Mouse is contrast, carries out betagalactosidase activity and detects.The result shows that the spleen of experimental mice and marrow galactosidase activity all are higher than control group, and difference is (P＜0.05) obviously, shows that recombinase has expression activity in these two kinds of tissues.
Claims (6)
1. recombinase transgene mouse model, it is characterized in that, the genome conformity of described transgenic mice animal model has dna fragmentation, include people EDAG (Embryonic development associated gene 1, embryonic development related gene-1, be called for short EDAG) gene 5 ' end promoter region, and the Cre recombinase gene fragment that derives from phage.
2. transgene mouse model as claimed in claim 1 is characterized in that, described mouse EDAG 5 ' end regulation and control zone comprises the sequence of this regulating and controlling sequence, Cre recombinase gene, human growth hormone (HGH) poly A.
3. such as claim 1 and 2 described transgenic mice animal models, it is characterized in that, described people's embryonic development related gene (EDAG) 5 ' end promotor has the sequence shown in the SEQ ID No.1.
4. such as claim 1 and 2 described transgenic mice animal models, it is characterized in that, described Cre recombinase gene has the sequence shown in the SEQ ID No.2.
5. the preparation method of the animal model of a recombinase transgenic mice is characterized in that may further comprise the steps:
A. people's embryonic development related gene EDAG 5 ' holds the clone of promotor;
B. utilize EDAG 5 ' the end promoter sequence that obtains to make up the specific expressed Cre recombinase expression vector pEDAG-Cre of hematopoietic cell;
C. digestion with restriction enzyme linearisation pEDAG-Cre expression vector reclaims and the about linearisation fragment of 5.6Kb of purifying;
D. the linearisation expression vector is carried out microinjection mouse male pronucleus, successfully prepared the transgenic mice that is integrated with this expression vector in the genome.
6. the transgene mouse model of claim 1 is characterized in that, the Cre recombinase gene of integrating in the transgenic mice genome only has expression in hemopoietic system comprises marrow, spleen and the peripheral blood of adult mice.
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| CN111676199B (en) * | 2020-06-24 | 2022-04-19 | 武汉波睿达生物科技有限公司 | CAR-T technology for presenting and activating HSV-1 type oncolytic virus and application thereof |
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