CN101955963A

CN101955963A - Expression vector, recombinant plasmid and use thereof

Info

Publication number: CN101955963A
Application number: CN2010102445938A
Authority: CN
Inventors: 陈其奎; 于涛
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-08-04
Filing date: 2010-08-04
Publication date: 2011-01-26
Anticipated expiration: 2030-08-04
Also published as: CN101955963B

Abstract

The invention discloses an expression vector, which comprises a mouse Musashi1 gene promoter and a report gene expressed under the control of the mouse Musashi1 gene promoter. The invention also discloses a recombinant plasmid containing the expression vector and the use of the expression vector and the recombinant plasmid in cytobiology. In the invention, after the expression vector transfects a cell population, the mouse Musashi1 gene promoter controls the expression of the report gene, and Musashi1 positive expression cells can be sorted out conveniently without influencing the activity of the cells according to an expression signal of the report gene.

Description

Expression vector, recombinant plasmid and their application

Technical field

The present invention relates to a kind of the contain expression vector of mouse Musashi1 gene promoter and reporter gene, the recombinant plasmid that contains this expression vector and their application in cytobiology.

Background technology

Musashi albumen is a kind of rna binding protein of high conservative, finds in the fruit bat body, and is proved to be one of sign of fruit bat neural stem cell, and great expression is in neural system.Existing known Musashi is the crucial regulon of the asymmetric splitted of fruit bat neural stem cell, after it impels stem cell division, a cell keeps the self ability, another cell has further differentiation capability, thereby is considered to play an important role in the keeping of stem cell state, differentiation of stem cells and tumour form.The Musashi1 gene is its isoformgene mouse, at the neural precursor high expression level, can detect Musashi1 genetic expression equally in embryo stage Musashi1 selectivity in the adult neural stem cell.Studies show that subsequently Musashi1 the dryness of neural stem cell keep and differentiation in keying action is arranged, so Musashi1 is considered to one of sign of mouse neural stem cell.

Small intestinal mucosa is the tissue of another high expression level Musashi1 except that central nervous system, has pointed out the in close relations of Musashi1 gene and intestinal epithelial cells.The intestinal mucosa epithelium is made up of countless simple columnar epitheliums, the lifelong constantly quick self of intestinal mucosa epithelial cell, average 4-6 of replacement cycle days.Because the fast updating of intestinal mucosa epithelium, the someone infers the existence of gut epithelium stem cell very early.Think that at present the small intestine epithelium stem cell is positioned at the basis pontis of little intestinal crypts, apart from the position of 2-7 the cell in crypts bottom, the nearly 4-6 of each crypts; The colonic epithelium stem cell is positioned at the basis pontis of colon body of gland, the nearly 1-4 of each crypts.Gut epithelial stem cells continues propagation, differentiation, and to top of villi migration (paneth's cell moves downwards), replace the outer differentiation of end eventually mucosal epithelium cell gradually, the okioplast apoptosis of moving to top of villi comes off and falls into enteric cavity, and the death of okioplast comes off and the division of stem cell between keep certain balance.

Though the existence of gut epithelial stem cells obtains investigator's common recognition, the research of the sign of gut epithelial stem cells but makes little progress.2003, the colon crypts Musashi1 positive expression zone of the normal people's colon of discoveries such as Nishimura sample was positioned at the basis pontis of crypts, with the position consistency of gut epithelial stem cells.At the little intestinal crypts of adult mice, Musashi1 is expressed on the paneth's cell and is close to several cells in paneth's cell, the Musashi1 positive cell the enteric epithelium crypts generally express and suitable number hints that all the Musashi1 positive cell is small intestinal mucosa epithelial stem cell or progenitor cell.Gut epithelial stem cells is under the Musashi1 gene action, a cell is kept stem cell character, stay in the position of gut epithelial stem cells, another cell that division produces is as the precursor cell-of short duration amplifying cells of enteric epithelium, these of short duration amplifying cells continue propagation and differentiation gradually, produce paneth's cell, absorptive epithelium cell, neuroendocrine cell and goblet cell.By research to signal path, the investigator finds as a kind of transcription inhibition factor, Musashi1 regulates the Notch signal, final Hairy and enhancer of split 1 (Hes1) expression of gene of regulating, this adjusting finally has influence on the formation of goblet cell, endocrine cell, paneth's cell, and the coexpression cell of hint Musashi1 and Hes1 may be exactly a gut epithelial stem cells.

Though some researchs recently make the investigator to the sign statement into question of Musashi1 gene as gut epithelial stem cells, and leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) have been reported, Acheate-scute like 2 (ASCL-2), the marker gene of Doublecortin and CaM kinase-like-1 candidate's gut epithelial stem cells such as (DCAMK-1), but no matter be Lgr5 or ASCL-2, not only to limit to be expressed in the intestinal mucosa epithelium, discovery is all arranged in other tissue, and these new candidate genes do not obtain the support of gene knockout experiment yet.As the marker gene of candidate's gut epithelial stem cells, Musashi1 remains one of candidate gene of gut epithelial stem cells or progenitor cell as early.The emerging position of Musashi1 positive cell is to be positioned between paneth's cell, and be long column shape more, these cells itself are exactly the positive position of other emerging candidate's gut epithelial stem cells marker gene (for example Lgr5), and also have the investigator to think that gut epithelial stem cells is the long column shape cell between paneth's cell.Therefore, the Musashi1 gene still can be used as gut epithelial stem cells or is candidate's marker gene of progenitor cell at least.

Gut epithelial stem cells is in the microenvironment " niche "." niche " is made up of mesenchymal cell around the gut epithelial stem cells and matrix, provides to keep gut epithelial stem cells propagation and self desired signal.Gut epithelial stem cells has hyperplasia, Selfstabilizing characteristic (being that quantity keeps relative stability), can produce a large amount of intestinal mucosa functioning cells again, energy Regeneration and Repair intestinal mucosa after the damage of intestines wall, therefore, gut epithelial stem cells is the first-selected target cell of intestinal tract disease gene therapy theoretically.The existing gut epithelial stem cells that studies confirm that external survival growth can be by the transfection of alien gene institute, so the gut epithelium stem cell can become the ideal basis of gene engineering treatment intestinal tract disease because of carrier cell.And for the heavy losses of gut epithelial stem cells, stem cell transplantation may be unique method that can be used in treatment.The transplantation treatment first-selection that realizes gut epithelial stem cells is to obtain enough gut epithelial stem cells, and can't obtain gut epithelial stem cells in a large number from intestinal mucosa at present.

Research for the common utilization of stem cell in the intestinal function reparation of bone marrow stem cell and embryonic stem cell two classes also deepens continuously.Wherein, bone marrow stem cell is easy to obtain, but, limited the utilization of hemopoietic stem cell (Allo-HSCT) in intestinal function is repaired popularizing greatly along with autologous peripheral blood hematopoietic stem cell transplantation (Auto-PBSCT) because there are complication such as immunological rejection, infection, GVHD.And because the aspects such as validity of Allo-HSCT mechanism that effective constituent, the state of an illness are recovered in the intestinal function repair process and treatment all can not be definite fully.Therefore, bone marrow stem cell can't damage the desirable derived cell of transplantation treatment and gut epithelial stem cells research at present as gut epithelial stem cells.For embryonic stem cell, it can break up the clone of various histoorgans, for transplantation medicine has been established theoretical basis as a kind of totipotency stem cell in ontogenetic process.The separation of embryonic stem cell successfully reaches external evoked differentiation achievement in research and has shown that it has application promise in clinical practice in stem cell transplantation.After the gut epithelial stem cells that studies show that vitro culture is transplanted to small intestine, still can continue survival, hyperplasia and differentiation, transplanting for gut epithelial stem cells provides experimental basis.But if isolate gut epithelial stem cells from embryonic stem cell deutero-cell, an important ring is to find the gut epithelial stem cells sign.Candidate's gut epithelial stem cells sign has at present: Musashi1, Lgr5, ASCL-2 etc.And wherein study history at most, the most deep candidate's gut epithelial stem cells marker gene is Musashi1, obtain enough Musashi1 positive cells and can promote the research of gut epithelial stem cells, for the cellular replacement therapy or the gene therapy of enteron aisle refractory disease provides important research basis and cell to originate.Simultaneously because the propagation of Musashi1 and kinds of tumors is relevant, obtains the Musashi1 positive cell and help research, thereby understand the biological characteristics of tumour better the Musashi1 gene.But Musashi1 albumen is present in nucleus and tenuigenin, do not express at surface of cell membrane, can't adopt the method for flow cytometer (FACS) or magnetic bead cell sorting (MACS) to carry out the sorting of Musashi1 protein positive cells according to Musashi1 protein expression situation.And existing method needs pair cell to carry out chemical fixation or cytolemma punching, could use the intracellular Musashi1 albumen of Musashi1 antibodies, and then the mark or the sorting of realization Musashi1 cell, but the cell through fixing or punching is all dead, lose cell activity, be difficult to carry out the research of follow-up cell function.

Summary of the invention

The purpose of this invention is to provide a kind of expression vector, after this expression vector is transfected into cell mass, mouse Musashi1 gene promoter regulating and expressing reporter gene according to the expression signal of reporter gene, is convenient to sub-elect Musashi1 positive expression cell under the situation that does not influence cytoactive.

On the one hand, the invention provides a kind of expression vector, this expression vector comprises mouse Musashi1 gene promoter and by the reporter gene of this mouse Musashi1 gene promoter regulating and expressing.

Preferably, described mouse Musashi1 gene promoter is for comprising the dna sequence dna of mouse Musashi1 gene translation starting point (ATG).

Preferred implementation as expression vector of the present invention, mouse Musashi1 gene promoter is the 823bp sequence that comprises mouse Musashi1 gene translation starting point (ATG) upstream 5 ' non-translational region, and the 316bp gene fragment in translation starting point (ATG) downstream.

As the preferred implementation of expression vector of the present invention, mouse Musashi1 gene promoter has the sequence of SEQID NO:1.

Preferably, reporter gene is green fluorescence (GTP) reporter gene.

On the other hand, the present invention also provides a kind of recombinant plasmid, the expression vector that this recombinant plasmid comprises in the technique scheme to be disclosed.

Preferably, described recombinant plasmid also comprises polyclone restriction enzyme site and kan/neo resistant gene.

Preferably, the polyclone restriction enzyme site is arranged in described mouse Musashi1 gene promoter and the mouse Musashi1 gene promoter insertion zone at this recombinant plasmid at least.Further, described polyclone restriction enzyme site is Ecl136II and BamHI double enzyme site.

The present invention also provides a kind of host cell, and it comprises expression vector or the recombinant plasmid that is disclosed in the right technique scheme.This host cell is preferably intestinal bacteria.

Expression vector and the application of reorganization matter in preparation Musashi1 positive cell and research Musashi1 positive cell biological function that the present invention also provides the present invention to disclose.

Expression vector of the present invention, after being transfected into cell mass, because the Musashi1 promoter sequence is consistent with the structure of endogenous Musashi1 promotor in the carrier, also start the exogenous Musashi1 promotor that is transfected into cell when making conjugated protein startup endogenous Musashi1 promotor, mouse Musashi1 gene promoter regulating and expressing reporter gene, according to the expression signal of reporter gene, be convenient under the situation that does not influence cytoactive, sub-elect Musashi1 positive expression cell.In a kind of preferred implementation of the present invention, based on the pAcGFP1-1 plasmid, the dna sequence dna that will comprise mouse Musashi1 gene translation starting point (ATG) is inserted into the upstream of reporter gene GFP encoding sequence, the expression of regulation and control GFP, construction recombination plasmid pMsi1-GFP is behind the transfectional cell, express the GFP that produces and send green fluorescence, according to these signal representation characteristics, use flow cytometer and can directly sub-elect Musashi1 positive expression cell, do not influence cell activity.In addition, because the Musashi1 positive cell after separating still has proliferation activity, and brings into play the ability of its multidirectional differentiation, can be divided into neurocyte and intestinal epithelial cells.

Description of drawings

Fig. 1 is a plasmid pMsi1-GFP structure: (A) structure iron of empty carrier pAcGFP1-1; (B) the Musashi1 promoter fragment synoptic diagram that is inserted.

Fig. 2 (A) is the luciferase expression synoptic diagram of mESCs different times noble cells transfection pMsi1-GFP and pPAcGFP1-1.Difference transfection mESCs, the 7th day differentiation cell, the 10th day differentiation cell.With Hochest transfect cell nuclear, transfection was observed under fluorescent microscope after 24 hours.Bars＝25μm。(B, C) be with pMsi1-GFP (go up group) and pAcGFP1-1 (group down) transfection mESCs, the 7th day differentiation cell, broke up cell on the 10th day.Transfection detected GFP positive cell rate after the transfection with flow cytometer after 24 hours.

Fig. 3 is that pMsi1-GFP is transfected into mESCs differentiation in the 10th day cell GFP positive cell that selected by flow cytometry apoptosis goes out after 24 hours.

Fig. 4 is that pMsi1-GFP is transfected into mESCs the 10th day differentiation cell selected by flow cytometry apoptosis figure after 24 hours.

Fig. 5 is that pMsi1-GFP is transfected into mESCs differentiation in the 10th day cell, and the proteic western blot of Musashi1 detects in the GFP positive cell that selected by flow cytometry apoptosis goes out after 24 hours, the negative cells group.

Fig. 6 is the cell amplification curve during the external adherent culture of sorting cells, Musashi1 positive cell and negative cells not.

Fig. 7 is that graft form to be analyzed: (A) not sorting cells, the Musashi1 positive and negative cells to be injected into mouse back subcutaneous.The Musashi1 positive cell is injected in back (arrowhead), left side, and the Musashi1 negative cells is injected in back, right side (black arrow); (B) sorting cells group graft not, big or small 2.0x1.2x0.8cm3; (C) Musashi1 negative cells group graft, big or small 2.0x1.2x1.0cm3.(D) Musashi1 positive cell group graft, big or small 1.2x0.5x0.5cm3.Not sorting cells group and visible pseudocartilage tissue of Musashi1 negative cells group graft surface (black arrow) and pigment sample tissue (arrowhead).

Fig. 8 is the staining analysis of graft.HE staining analysis: (A) sorting cells group graft not, visible big pseudocartilage tissue (black arrow); (B) Musashi1 positive cell group graft, more crack (arrowhead) or cystic structures (black arrow); (C) Musashi1 negative cells group graft, visible more pseudocartilage tissue (black arrow) (Bars=25 μ m).

Fig. 9 is the immunohistochemical methods result of Nestin in the graft: (D) expression of Nestin albumen in sorting cells group graft not; (E) expression of Nestin albumen in Musashi1 positive cell group graft; (F) expression (Bars=25 μ m) of Nestin albumen in Musashi1 negative cells group graft.

Figure 10 is the proteic immunohistochemical methods result of Tubulin β III in the graft: (G) expression of Tubulin β III albumen in sorting cells group graft not; (H) expression of Tubulin β III albumen in Musashi1 positive cell group graft; (I) expression of Tubulin β III albumen in Musashi1 negative cells group graft; The agglomerating appearance of most of Tubulin β III, part is around crack shape or nido tissue distribution (black arrow).(Bars＝25μm)。

Figure 11 is the immunohistochemical methods result of Villin in the graft: (J) expression of Villin albumen in sorting cells group graft not; (K) expression of Nestin albumen in Musashi1 positive cell group graft; (L) expression of Villin albumen in Musashi1 negative cells group graft; Most of Villin positive cell forms cystic structures (arrowhead), and part is undefined structure (black arrow) (Bars=25 μ m).

Figure 12 is the immunohistochemical methods result of FABP2 in the graft: (M) expression of FABP2 albumen in sorting cells group graft not, and most of FABP2 positive cell forms cystic structures (black arrow); (N) expression of FABP2 albumen in Musashi1 positive cell group graft; (O) expression (Bars=25 μ m) of FABP2 albumen in Musashi1 negative cells group graft.

Embodiment

For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.

The experimental technique of unreceipted actual conditions among the following embodiment, usually according to normal condition, for example the Sambrook equimolecular is cloned the condition described in the enforcement manual.

In the following embodiment of the present invention, with the pAcGFP1-1 plasmid that contains green fluorescent protein (GFP) reporter gene is carrier is carrier, insert mouse Musashi1 gene promoter in the upstream of GFP encoding sequence, make up the GFP reporter gene recombinant plasmid pMsi1-GFP that expresses by mouse Musashi1 gene specific promoter regulation.Example with this kind embodiment expression vector of the present invention as an illustration and recombinant plasmid illustrates implementation of the present invention.

In this embodiment, adopt the method for PCR, with the mouse gene group DNA is masterplate, after successfully cloning the Musashi1 promoter sequence, purified, order-checking is identified, be basic plasmid skeleton then with reporter gene vector plasmid pAcGFP1-1, gene recombination means such as the utilization enzyme is cut, connection, insert mouse Musashi1 gene promoter in the upstream of GFP encoding sequence, make up the GFP reporter gene recombinant plasmid pMsi1-GFP (recombinant plasmid pMsi1-GFP structure as shown in Figure 1) that expresses by mouse Musashi1 gene specific promoter regulation.Then pMsi1-GFP is transformed into bacillus coli DH 5 alpha competent cell amplification, prepare the recombinant plasmid that contains Musashi1 gene promoter and GFP reporter gene.The recombinant plasmid that structure is obtained is transfected into the cell mass (expressing Musashi1) that mouse embryo stem cell differentiates, and it can be expressed in cell, expresses the positive cell of GFP reporter gene then with selected by flow cytometry apoptosis.Can the Musashi1 positive cell that obtain further detects the differentiation capability under its ability of cell proliferation, Musashi1 albumen and mRNA expression level, culturing in vivo and two kinds of conditions of external transplanting, separate thereby estimate the activity of utilizing the constructed pMsi1-GFP of aforesaid method be used for the Musashi1 positive cell.

The mouse embryo stem cell that the present invention relates to, bacillus coli DH 5 alpha competent cell, the pAcGFP1-1 plasmid vector that contains the GFP reporter gene and other test kits and biochemical material all can be obtained by commercial, and in the following example all there is explanation in the experiment material source of adopting in the experiment.

The structure of embodiment 1 recombinant plasmid pMsi1-GFP and obtaining

1, the amplification of mouse Musashi1 promotor, purifying:

Utilize the Universal Genomic DNA Extraction Kit of Takara company from mouse embryo stem cell, to extract DNA.

According to mouse Musashi1 gene structure, analyze its possible promoter sequence position, design two ends primer:

Primer1：5’-CTT?CTT?CGA?GAG?TGC?TCT?GCT?GAC?TTA-3’

Primer2：5’-GCA?CTC?TCG?AGG?AAG?GAG?ATG?ACT?ACA?TGA-3’

Utilize the Premix PrimeSTAR of Takara company ^TMThe HS test kit carries out pcr amplification, reaction conditions: 95 ℃, and pre-sex change 5min, 94 ℃, 40s; 60 ℃, 1min; 72 ℃, 1min; Carry out 30 circulations on the PCR instrument, 72 ℃ are extended 7min.Get the PCR product, behind the agarose gel electrophoresis, cut glue, utilize the Agarose Gel DNA Purification Kit of Omega company to reclaim the PCR product.

2, the double digestion of Musashi1 gene promoter and pAcGFP1-1 plasmid vector and enzyme connect:

Product that is recovered to and pAcGFP1-1 plasmid vector reclaim by agarose gel electrophoresis after using Ecl136II and BamHI double digestion, utilize the DNA Ligation Kit LONG of Takara company that pAcGFP1-1 plasmid vector and the Musashi1 gene promoter sequence that enzyme cuts back to close carried out even reaction of enzyme then.

3, recombinant plasmid transformed competent escherichia coli cell and amplification:

(1) bacterial classification of the freezing preservation of intestinal bacteria DH-5 α, picking one ring, streak inoculation on LB solid medium flat board (activated spawn), 37 ℃ of overnight incubation (about 16 hours).

(2) single bacterium colony of picking from the long good flat board, switching in the test tube that contains 3ml LB liquid nutrient medium, 37 ℃ of shaking culture spend the night (about 16 hours).

(3) get in the 250ml triangular flask that 0.3ml bacterium liquid is inoculated in 20ml LB liquid nutrient medium, 37 ℃ of shaking culture 2～3 hours are treated OD ₆₀₀Value reaches at 0.3～0.4 o'clock, takes off triangular flask, puts ice bath immediately 10～15 minutes.

(4) bacterium is transferred in the 50ml centrifuge tube of a sterilization, centrifugal 10 minutes of 4 ℃ of 3000r/min, abandoning supernatant (as far as possible all supernatant liquors being gone only) is collected thalline.

(5) add the ice-cold 0.1mol/L CaCl of 20ml ₂Solution, the thalline that suspends again is uniformly dispersed thalline, puts in the ice bath 30 minutes.

Centrifugal 10 minutes of (6) 4 ℃ of 3000r/min, abandoning supernatant (as far as possible all supernatant liquors being gone only).

(7) add the ice-cold 0.1mol/L CaCl of 2ml again ₂Solution, the thalline that carefully suspends again (it is light that operation is wanted).In 4 ℃ of refrigerators, placed 12～24 hours, promptly can be applicable to DNA and transform.

(8) get the fresh competent cell 100 μ l of intestinal bacteria DH-5 α in 1.5ml Eppendorf pipe, add 50～100ng plasmid pMsiI1-GFP DNA, rotate gently, in ice bath, placed 30 minutes with the mixed content thing.

(9) 42 ℃ of heat-shockeds 120 seconds are not shaken the Eppendorf pipe.

(10) add LB liquid nutrient medium 900 μ l, 37 ℃ are incubated 60 minutes.

(11) the LB solid medium is drawn bacterium: in the aseptic technique platform, with transfering loop 100 μ l conversion fluids are drawn bacterium and contain on the LB solid medium of kantlex (50 μ g/ml) 37 ℃ of constant temperature inversions, extremely single bacterium colony appearance (about 14-16 hour) in 1.5%;

(12) in the aseptic technique platform, single bacterium colony of growing on the picking LB solid medium is inoculated in 3.0mlLB (kantlex that the contains 50 μ g/ml) liquid nutrient medium, and 37 ℃, the 250rpm shaking culture is spent the night, and stops during for 0.4-0.5 cultivating to OD 600nm.

(13) in the aseptic technique platform, getting 100ml LB substratum (that penicillin of card that contains 50 μ g/ml) moves in the aseptic Erlenmeyer flask of 500ml, in the LB substratum, add 0.2ml bacterium liquid (1% inoculum size) again, 37 ℃, the 250rpm shaking culture is spent the night, and stops to cultivate (about 12-16 hour) during for 0.4-0.5 to OD 600nm;

(14) shake after bacterium finishes, the aseptic centrifuge tube of 50ml is put in the gradation of 100ml LB substratum, it is centrifugal to add room temperature 10000rpm, and 5min abandons supernatant, and centrifuge tube is inverted, and liquid is flow to end as far as possible.

4, the extraction of pMsiI1-GFP recombinant plasmid:

Step is summarized as follows:

(1) column equilibration step: (adsorption column is put into the 50ml collection tube) adds the balance liquid BL of 2.5ml in adsorption column CP5, and 10, centrifugal 2 minutes of 000rpm outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector;

(2) the bacterium liquid of getting the 100ml incubated overnight adds centrifuge tube, room temperature 10, and 000rpm collected bacterium in centrifugal 3 minutes, absorbed supernatant as far as possible;

(3) absorb supernatant as far as possible, all draw, please inhale the water droplet that goes on bottle wall with clean thieving paper for guaranteeing supernatant liquor;

(4) in the centrifuge tube that leaves bacterial sediment, add 7ml solution P1, use pipettor or the vortex vibrator bacterial cell precipitation that thoroughly suspends;

(5) add 7ml solution P2 in centrifuge tube, leniently spin upside down 6-8 time immediately, room temperature was placed 5 minutes;

(6) add 7ml solution P4 in centrifuge tube, leniently spin upside down 6-8 time immediately, fully mixing white flocks can occur at this moment.Room temperature was placed about 10 minutes then.10, the centrifugal 5-10 of 000rpm minute, solution is all poured among the strainer CS, slowly push away handle and filter, filtrate collection is in the pipe of clean 50ml;

(7) Virahol of 0.3 times of filtrate volume of adding in filtrate is transferred to behind the mixing that turns upside down (adsorption column is put into the 50ml collection tube) among the adsorption column CP5;

(8) room temperature 10, and centrifugal 2 minutes of 000rpm outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector;

(9) in adsorption column, add 10ml rinsing liquid PW, 10, centrifugal 2 minutes of 000rpm discards the waste liquid in the collection tube, adsorption column is relay reclaim in the collector;

(10) repetitive operation step 9.

(11) in adsorption column, add the 3ml dehydrated alcohol, room temperature 10, centrifugal 2 minutes of 000rpm outwells waste liquid;

(12) adsorption column CP5 is relay in the recovery collector, CP5 uncaps with adsorption column, places room temperature to place 5 minutes, and 10, centrifugal 5 minutes of 000rpm;

(13) adsorption column CP5 is placed a clean 50ml collection tube, to the unsettled dropping in the middle part of adsorption film 1.5ml elution buffer TB, room temperature was placed 5 minutes, and room temperature 10 then, centrifugal 2 minutes of 000rpm.Elutriant in the 50ml centrifuge tube is all moved into a clean 1.5ml centrifuge tube ,-20 ℃ of preservations.

5, the preliminary evaluation of the detection of plasmid concentration and plasmid DNA:

By following test:

(1) gets the plasmid order-checking;

(2) get plasmid and on spectrophotometer, survey OD 260nm and OD 280nm;

(3) 1% agarose gel electrophoresis.

Identify the concentration of plasmid, molecular weight size and purity.

The observation of cell after the transfection of embodiment 2 recombinant plasmids and the transfection

1, plamid vector transfection:

In inoculating cell to 6 well culture plate, 2x105 cell inoculated in every hole when inducing the 3rd day.Cell attachment after 24 hours carries out the vector plasmid transfection under liposome-mediated, uses LipofectamineTM2000 reagent to carry out according to the following steps:

(1) discards the old liquid for the treatment of transfectional cell, wash one time, discard PBS with 0.1M PBS;

(2) in the cell of 6 orifice plates, add 0.5ml Opti-MEM I, simultaneously with aseptic 1.5ml centrifuge tube preparation:

The A agent: pMSI1-GFP plasmid vector DNA 2 μ g, being supplemented to volume with Opti-MEM I is 250 μ l;

B agent: LipofectamineTM2000 transfection reagent 8 μ l ten Opti-MEM I 242 μ l;

A agent and B agent are mixed, after 20 minutes, add together in the 6 orifice plate cells, form the rotaring redyeing system of 1ml with existing 0.5ml Opti-MEM I.

(3), hatch 6h in the cell culture incubator of 5%CO2 at 37 ℃;

(4) remove transfection reagent, 0.1M PBS washes 1 time, adds the 2ml substratum and continues to cultivate.

2, the observation of cellular form after the transfection:

(1) expression of green fluorescence in the usefulness fluorescence inverted microscope observation of cell;

Noble cells with pMsi1-GFP and empty plasmid pAcGFP1-1 transfection mESCs different times.According to the Musashi1 mRNA expression rule of mESCs noble cells, select mESCs, the 7th day differentiation cell, the 10th day differentiation cell to carry out transfection.For the better green fluorescence of observing, after 24 hours,, under fluorescent microscope, observe transfectional cell then with Hochest staining cell nuclear at transfectional cell.Shown in Fig. 2,3, transfection can see a spot of green fluorescence expression in the differentiation cell at the 8th day, and the 11st day differentiation cell was seen more green fluorescences after 24 hours.And in the empty plasmid transfection group, 3 kinds of cells there is no luciferase expression.Cell after each group transfection is carried out flow cytometer detect the GFP positive cell rate, the 11st day differentiation cell GFP positive cell rate of visible transfection pMsi1-GFP is the highest.

(2) adopt selected by flow cytometry apoptosis GFP positive component;

PMsi1-GFP is transfected into mESCs differentiation in the 10th day cell, carries out sorting with flow cytometer after 24 hours.GFP positive cell after the sorting is still expressed green fluorescence.As shown in Figure 4, behind the selected by flow cytometry apoptosis 1 hour, cell was not adherent as yet, and cell is small circular, and all green fluorescence appears in cells.

(3) use real-time quantitative PCR and western blots measure the expression of Musashi1 in the GFP positive cell, and compare with the GFP negative cells.

MESCs differentiation in the 10th day cell transfecting pMsi1-GFP is after 24 hours, the Musashi1 mRNA relative expression quantity of (being called for short not sorting cells) in mESCs after the GFP positive cell that selected by flow cytometry apoptosis goes out, GFP negative cells and unsorted transfection differentiation in the 11st day cell, the Musashi1 mRNA relative expression quantity in the sorting cells does not compare, and the Musashi1 mRNA relative expression quantity of GFP positive cell and GFP negative cells is respectively 15.9 ± 3.40 and 0.8 ± 0.20.Musashi1 mRNA relative expression quantity in the GFP positive cell that sub-elects is apparently higher than not sorting cells group and GFP negative cells group.

The GFP positive that extraction sub-elects and the total protein of GFP negative cells, the differential expression in 2 groups of cells that after BCA is quantitative, sub-electing with western blot method detection Musashi1 albumen.The result shows, can detect the Musashi1 protein expression in the GFP positive cell, and do not see Musashi1 protein expression (as shown in Figure 6) in the GFP negative cells.Verified that further constructed pMsi1-GFP expression can reflect the proteic expression of the endogenous Musashi1 of cell synchronously.Can think that the GFP positive cell that sub-elects is the Musashi1 positive cell.

Embodiment 3, propagation, the research of differentiation capability with the isolated Musashi1 positive cell of pMsi1-GFP recombinant plasmid:

(1) describing of cell growth curve:

Under the culture condition of EB substratum, every hole inoculation 1 * 10 in 12 orifice plates ⁴Cell, count every group 1 hole attached cell every day, and method is: at first remove old nutrient solution with suction pipe, wash 1 time with 0.1M PBS, every hole adds 0.25% trypsinase/0.02%EDTA Digestive system 0.5ml, digestion 1min, the trypsinase neutralizer of adding equivalent, piping and druming orifice plate bottom is taken off wall to all cells becomes cell suspension, move to the 1.5ml centrifuge tube, carry out conventional cell counting,, describe cell growth curve according to the count results of respectively organizing cell every day.As shown in Figure 7, most cells can be adherent, and can continue propagation, do not see that cell contamination occurs.Sorting cells, Musashi1 positive cell and negative cells multiplication capacity are not seen notable difference.Initial cell is 1x104, and cell proliferation in preceding 3 days is slower, after this obviously accelerates, and being cultured to differentiation beginning in the 16th day cell proliferation rate has downtrending.

(2) formation of NOD/SCID mouse subcutaneous transplanting thing experiment:

For research Musashi1 positive cell in the intravital differentiation situation of NOD/SCID mouse, cell that following three state is transplanted in this experiment is subcutaneous to the NOD/SCID mouse back: 1. unsorted transfection mESCs differentiation in the 11st day cell (not sorting cells) of pMsi1-GFP; 2. from transfection the GFP positive cell (Musashi1 positive cell) that sub-elects in mESCs the 11st day differentiation cell of pMSI1-GFP; 3. from transfection the GFP negative cells (Musashi1 negative cells) that sub-elects in mESCs the 11st day differentiation cell of pMsi1-GFP.

The Transplanted cells method is: with aseptic 0.1M PBS re-suspended cell, regulating cell density is 1 * 10 ⁷/ ml, after routine is wiped out NOD/SCID mouse back hair, sterilization, subcutaneous with the above-mentioned cell suspension of 1ml injector to inject 0.3ml to mouse, according to institute's transplanted cells type, with picric acid and liquor epinephrinae bitartratis ophthalmicus mark mouse, and the record transplant time.

Observe mouse graft growing state every day, when treating that graft grows to diameter 0.8-1.2cm size, live and kill mouse, complete separation back graft, after half-and-half cutting open, 2 3mm are got on every limit ³Size tissue (as shown in Figure 8) carries out total RNA and extracts and quantitative PCR detection, and all the other are put into 4% Paraformaldehyde 96 immediately and carry out histology and immunohistochemistry research.Detect the expression of respectively organizing neurocyte marker gene (Nestin, Tubulin β III), mesoderm marker gene (Pax6, PDGFR-α) and intestinal epithelial cells sign (Villin, FABP2, ChA, Lyz, TFF3) in the graft with fluorescent quantitative PCR technique; Expression with immunohistochemical methods technology for detection Nestin, Villin, FABP2.

Under the interior transplanting condition of body, Musashi1 positive cell group graft neurocyte marker gene (Nestin, Tubulin β III), intestinal epithelial cells marker gene (Villin, FABP2, Lyz, ChA) mRNA express and are higher than Musashi1 negative cells group graft, and mesoderm marker gene (Pax6, PDGFR-α) mRNA expression is lower than Musashi1 negative cells group graft.

With respect to Musashi1 positive cell group, sorting cells group and Musashi1 negative cells group graft do not contain more pseudocartilage tissue.Nestin, the Villin of sorting cells group and Musashi1 positive cell group graft and FABP2 positive cell are not expressed more than Musashi1 negative cells group graft.

Shown in Fig. 9-13, experimental result result shows: 1. constructed Musashi1 promotor report carrier pMsi1-GFP can follow the trail of Musashi1 expression of gene in the mESCs atomization, and can be used for sub-electing the Musashi1 positive cell from the mESCs noble cells.2. the Musashi1 positive cell ratio Musashi1 negative cells that sorts out in the mESCs noble cells forms more neural sample and enteric epithelium like cell, and forms cartilage like cell still less.

Should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.

Claims

1. expression vector, it comprises mouse Musashi1 gene promoter and by the reporter gene of this mouse Musashi1 gene promoter regulating and expressing, wherein, described mouse Musashi1 gene promoter is for comprising the dna sequence dna of mouse Musashi1 gene translation starting point (ATG).

2. expression vector according to claim 1, wherein, described mouse Musashi1 gene promoter is the 823bp sequence that comprises mouse Musashi1 gene translation starting point (ATG) upstream 5 ' non-translational region, and the 316bp gene fragment in translation starting point (ATG) downstream.

3. expression vector according to claim 1, wherein, described mouse Musashi1 gene promoter has the sequence of SEQ ID NO:1.

4. expression vector according to claim 1, wherein, described reporter gene is green fluorescence (GTP) reporter gene.

5. recombinant plasmid, it comprises as the described expression vector of one of claim 1～4.

6. recombinant plasmid according to claim 5, wherein, described recombinant plasmid also comprises polyclone restriction enzyme site and kan/neo resistant gene.

7. recombinant plasmid according to claim 6, wherein, the polyclone restriction enzyme site is arranged in described mouse Musashi1 gene promoter and the mouse Musashi1 gene promoter insertion zone at this recombinant plasmid at least.

8. recombinant plasmid according to claim 6, wherein, described polyclone restriction enzyme site is Ecl136II and BamHI double enzyme site.

9. host cell, it comprises one of described expression vector of one of claim 1～4 or claim 5～8 described recombinant plasmid.

10. the application of the described reorganization matter of one of the described expression vector of one of claim 1～4 or claim 5～8 in preparation Musashi1 positive cell and research Musashi1 positive cell biological function.