CN102860282B - 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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- CN102860282B CN102860282B CN201110187441.3A CN201110187441A CN102860282B CN 102860282 B CN102860282 B CN 102860282B CN 201110187441 A CN201110187441 A CN 201110187441A CN 102860282 B CN102860282 B CN 102860282B
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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, 5 ' the end promoter regulation region that the size that is specifically related to utilize PCR method human cloning fetal development genes involved EDAG gene is 2.3kb 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
Mammals hematopoiesis is a complicated dynamic process.7.5 days mice embryonic phases, endothelium and hematopoietic cell that extraembryonic mesoderm derived cell produces form blood island jointly, and now hematopoietic cell is mainly pronormoblast, and core is round, cell space is large and express embryo type oxyphorase, this process is lasted of short duration, is called as original hematopoiesis.10.5 days embryonic stages, in embryo, aorta-gonad-mesonephros district, site (AGM) starts to produce hematopoietic stem cell, at 11.5 days, peaks, and this type of cell can be to a plurality of hematopoietic cell lineage differentiation.11 days embryonic stages, the blood circulation that hemopoietic stem cell starts by having set up migrates to tire liver, and continues to reach maturity, the most remarkable with erythroid differentiation.Now seedless, the synthetic adult form globin of red corpuscle, indicates the foundation of permanent hematopoiesis.The hematopoiesis of AGM district is active in and finished 13 days embryonic stages, and tire liver becomes hematopoiesis main place thereafter.While closing on birth, fetal liver hemopoietic activity is disappeared, and by marrow, is replaced, and the latter becomes lifelong hemocytopoietic organ (Dzierzak E.Ontogenic emergence of definitive hematopoietic stem cells.Curr Opin Hematol, 2003,10:229-234).
It is expression of specific gene or process reticent and acquisition particular phenotype that embryonic hematopoiesis is grown, the multiple gene relevant with hematopoietic regulation or gene family have been found, as GATA, C/EBP and AML/CBF/Runx1 etc., in the different steps of hematopoietic development, have an effect respectively.In these cores, the Harmony of hematopoiesis genes involved is expressed under a series of complicated signal network regulation and control and is completed, as Notch, Wnt, Sonic, hedgehog (Shh), Smad etc.Along with the arrival in functional genome's epoch, to comprise that the gene function playing a role of said gene and the research of the mechanism of action seem 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, but knocking out the gene relevant to fetal development usually causes mice embryonic deformity even embryo, in early days death to occur and cannot carry out follow-up Journal of Sex Research.Adopt tissue-specific gene knockout's technology of the recombination system mediation of Cre/Loxp can specificity to control specific expressed in tissue of some important gene, and then study it in the effect of different developmental phases.
EDAG gene is the new gene of hemopoietic tissue specifically expressing, and research shows, EDAG gene specific is expressed in the prematurity hematopoietic cell (CD34 in hemopoietic tissue (grow up marrow, embryonic liver)
+cell, be hemopoietic 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, a kind of and the separated of the closely related new gene of hematopoietic regulation and confirm. Acta Biochimica et Biophysica Sinica, 2001,33:641-646).But high expression level EDAG in leukemia human peripheral blood cell and leukemia cell K562, at induction K562 cells such as protoheme Hemin, dyers' grapes PMA, after red system or the differentiation of macronucleus system, EDAG expresses rapidly and lowers; Utilize antisense nucleic acid to suppress after EDAG expression, K562 cell proliferation is obstructed, and colony formation ability declines, and cell is more responsive to inductor reaction.The hematopoiesis specifically expressing EDAG transgenic mouse that research CD11a drives finds that its thymus development is abnormal, and spleen is loose; Peripheral blood lymphocyte paramophia, ratio decline, and neutrophil leucocyte ratio rises; Marrow B220
+cell obviously declines, sca-1
+c-kit
+lin
-cell increases; Spleen DC cytosis, B220
+positive cell reduces; Thymus gland T cell development is blocked in early days, DN cytosis; And Bone Marrow Cells In Vitro differentiation capability obviously declines.These results suggest EDAG high expression level may block hemopoietic stem cell normal differentiation, and its down-regulated expression is essential with growth by hematopoietic cell differentiation.These researchs show that EDAG/ specifically expressing regulates and controls closely related (Li CY in early stage hematopoietic cell/hemopoietic stem cell and with hematopoietic differentiation, 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 are in the process of transgenic mice of preparing the specific expressed Cre recombinase of hemopoietic system, and the regulating and controlling sequence that application PCR method has been cloned people EDAGEDAG 5 ' end 2.3kb is for instructing hemopoietic system specific expressed of Cre recombinase gene.
The acquisition of this transgenic mice for realizing series of genes reorganization operation by Cre/Loxp recombination system, preparing tissue specific expression animal model and study the development related gene biological function in different developmental phases and the domestic strong research tool of expression pattern body thereof in hemopoietic system in hemopoietic 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 gene recombination of Cre recombinase and knock out specificity and the early stage hematopoiesis of EDAG gene specific expressed, produce transgenic mice, by EDAG promotor, realize the specific expressed regulation and control in hemopoietic system of Cre recombinase.
The preparation method of this transgenic mice comprises the following steps:
One, the clone of people's fetal development genes involved EDAG 5 ' end promotor;
Two, utilize EDAG 5 ' the end promoter sequence obtaining to build the specific expressed Cre recombinase expression vector pEDAG-Cre of hematopoietic cell;
Three, digestion with restriction enzyme linearizing pEDAG-Cre expression vector, reclaims the also linearizing fragment of the about 5.6Kb of purifying;
Four, linearizing expression vector is carried out to microinjection mouse male pronucleus, successfully prepared the transgenic mice that is integrated with this expression vector in genome.
Accompanying drawing explanation
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 evaluation electrophoretogram.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: the head obtaining after pronuclear microinjection builds mouse children musculus cdna group DNA PCR result.
Fig. 4 is the electrophoresis qualification result collection of illustrative plates that 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: tire liver; 11: 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 A. medullary cell wherein, the expression activity of LacZ in 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 the present invention is described 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 EDAG promotor is built and is preserved by this chamber.Bacterial isolates DH5a, JM109 are preserved by this chamber preparation.Restriction enzyme and T4 ligase enzyme etc. are purchased from NEB company.C57B/6 mouse is provided by the court's Experimental Animal Center.ROSA26 reporter gene mouse is provided by model animal institute of Nanjing University.EDAG-Cre transgenic mice successfully constructs Hou Military Medical Science Institute Experimental Animal Center and preserves.
2.EDAG-Cre targeting vector builds
It is template that the plasmid DNA of pEDAG-GFP be take to+96 fragments in EDAG promoter region-2214, after pcr amplification, after KpnI/SalI restriction enzymes double zyme cutting, be cloned in A1.0-Cre carrier and build the Cre recombinase targeting vector EDAG-Cre that EDAG drives, through order-checking, confirm that 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
The preparation of 3.EDAG-Cre transgenic mice
EDAG-Cre plasmid is cut through KpnI/SacII linearizing enzyme, and DNA fragmentation reclaims, and carries out routinely zygote pronuclear microinjection and uterine tube embryo transfer, by Institute of Experimental Animals, Chinese Academy of Medical Sciences, is completed.Laboratory animal is used C57Bl/6 mouse.
4.EDAG-Cre head builds the evaluation of mouse
After clip birth, the about 0.3-0.5cm of young mouse tail point of 10-14 days, adds 55ul to organize lysate, and after 55 ℃ of overnight incubation, 100 ℃ of deactivations 5 minutes, add after 100ul distilled water, and centrifuging and taking supernatant is as Genomic PCR template.Cre recombinase universal primer is as follows, amplification object 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 ℃ are extended 5 minutes.
The expression of recombinase gene in 5.EDAG-Cre mouse
Put to death the positive transgenic mice of EDAG-Cre in 6 week age, get the various tissues that comprise liver, spleen, kidney, lung, thymus gland, marrow, peripheral blood, muscle; In addition by the male mouse of Cre transgenic positive and wild female mouse mating, by checking female mouse vaginal suppository, determine the mating date, collect the embryo mice embryonic of E13.5 in age days, with yolk sac genomic dna PCR, identify the positive embryo of Cre, get tire liver and the brain of positive mice embryonic, the Trizol of application Invitrogen company
test kit extracts total RNA of above-mentioned tissue, with Takara RNA PCR reverse transcription test kit, prepares cDNA, with Takara rTaq and above-mentioned Cre primer, carries out PCR evaluation, take GAPDH as internal reference gene, object sheet segment length 150bp:
Forward primer: TGC CCC CAT GTT TGT GAT G
Reverse primer: TGT GGT CAT GAG CCC CTT TCC
The tissue distribution of 6.EDAG-Cre recombinase active
EDAG-Cre mouse and ROSA26 mouse post-coitum, get two positive (EDAG-Cre in 8 week age
+/ LacZ
+) mouse and LacZ
+mouse bone marrow cells and spleen, be separated into unicellularly, gets 1X10
6cell, application beta-galactosidase enzymes test kit detects galactosidase activity (LacZ dyeing), hatches after 1～2h the absorbance under microplate reader detection 405nm wavelength in 37 ℃.
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 regulating and controlling sequence (hGH, 2.1kb), the about 5.6kb of total length; This targeting vector, after restriction enzyme KpnI/SacII enzyme is cut, becomes linear DNA fragment, for the micro-procaryotic injection of zygote 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, after zygote pronuclear microinjection, be born young mouse after mouse coda gene group DNA PCR identifies, obtains altogether 6 positive Cre transgenosis head and build mouse.
The hemopoietic system tissue specific expression of 3.Cre recombinase gene
Positive head builds after mouse builds through going down to posterity and be, Fig. 3 is shown in the expression of Cre recombinase gene in adult mice and various each tissues of embryo.The demonstration of RT-PCR result, in the body of adult mice, 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, undertake in the tire liver of embryo's early metaphase hematopoiesis and have obvious expression, in tire brain, have no expression.
The tissue expression of 4.Cre recombinase is active
For detecting the recombinase expression activity of EDAG-Cre transgenic mice, we have carried out mating by EDAG-Cre mouse and ROSA26 mouse, get EDAG-Cre in 8 week age
+/ LacZ
+the marrow of two positive mouse and spleen tissue, with LacZ
+mouse, for contrast, carries out betagalactosidase activity detection.Result shows that the spleen of experimental mice and marrow galactosidase activity are all higher than control group, and obvious difference (P < 0.05), shows that recombinase has expression activity in these two kinds of tissues.
Claims (6)
1. a recombinase transgene mouse model, it is characterized in that, the genome conformity of described recombinase transgene mouse model has DNA fragmentation, includes people's fetal development genes involved EDAG5 ' end promoter region, and the Cre recombinase gene fragment that derives from phage.
2. recombinase transgene mouse model as claimed in claim 1, is characterized in that, described people's fetal development genes involved EDAG5 ' end promotor has the sequence shown in SEQ ID No.1.
3. recombinase transgene mouse model as claimed in claim 1, is characterized in that, described Cre recombinase gene has the sequence shown in SEQ ID No.2.
4. recombinase transgene mouse model as claimed in claim 1, is characterized in that, the Cre recombinase gene of integrating in described recombinase transgenic mice genome only has expression in hemopoietic system.
5. recombinase transgene mouse model as claimed in claim 4, is characterized in that, the marrow that described hemopoietic system is adult mice, thymus gland, spleen and peripheral blood.
6. a preparation method for recombinase transgene mouse model described in claim 1-5 any one, is characterized in that comprising the following steps:
A. people's fetal development genes involved EDAG5 ' holds the clone of promotor;
B. utilize people's fetal development genes involved EDAG5 ' end promoter sequence obtaining to build the specific expressed Cre recombinase expression vector pEDAG-Cre of hematopoietic cell;
C. digestion with restriction enzyme linearizing pEDAG-Cre expression vector, reclaims the also linearizing fragment of the about 5.6Kb of purifying;
D. linearizing expression vector is carried out to microinjection mouse male pronucleus, successfully prepared the transgenic mice that is integrated with this expression vector in genome.
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| CN106222187B (en) * | 2016-08-03 | 2019-10-08 | 赣南医学院第一附属医院 | Transgene carrier, transgene mouse model and its construction method that inducible c-myc is overexpressed |
| CN106689030A (en) * | 2016-11-04 | 2017-05-24 | 南方医科大学 | Application of gene knockout animal as skin barrier function disorder animal model |
| 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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