CN100480382C - Real time measurement of cellular responses - Google Patents

Abstract

Living cells can be stably modified to emit different colors from the cytoplasm and nucleus, thus permitting analysis of the status of said cells and the effect of agents on said cells either by visual or instrumentally-aided observation. These observations may be made, if desired, in real time. In addition, rates of proliferation and drug sensitivities can be determined in vitro in real time by the use of cells modified to express a single fluorescent protein and observing fluorescence intensity as a function of time.

Description

Real time measurement of cellular responses

Related application

The application is according to the right of priority of 35U.S.C. § 119 (e) claim U.S. series application 60/427,604 (being filed on November 18th, 2002) and 60/404,005 (being filed on August 16th, 2002), and it is incorporated herein by reference in full.

Technical field

The present invention relates to Real Time Observation cell proliferation (proliferation), drug susceptibility, cell cycle position and the reaction of other cell in vitro.The present invention relates to fluorescin serve as a mark thing direct viewing cell, particularly viable cell.The state of cell in the cell cycle can be by mark karyon and endochylema are monitored respectively.

Background technology

Green fluorescent protein (GFP) from Victoria's multitube jellyfish (Aequorea Victoria) is found in 1962.After cloning the GFP gene in 1992, the structure of GFP, mechanism and application development get very fast.After finding the heterogenous expression of GFP gene other organism, GFP becomes one of most widely used reporter gene.In 1999, the fluorescin of another family comprised that disk body worm (Discosoma) Red (DsRed) is cloned out from coral, was sea anemone (Anemonia) asRed in 2000 afterwards, was that HcRed is cloned out in calendar year 2001.These proteic biological actions expand to from the isolating new albumen of coral from the pure signal function as GFP the light of photosynthetic symbiosis animal are protected.For two kinds in these coloured aqueous soluble proteins, GFP and DsRed, its molecular weight are 25-27kD, and x ray structure analysis has shown homologous beta sheet structure.The common trait of these proteic primary structures is that 66 and 67 amino acid tyrosine and the glycine that have occupied the GFP position are guarded, and has participated in chromophoric formation in the autocatalytic modification after translation.

These albumen (wild-type and mutant) can be used as many colors reporter molecule.The spectral range of their fluorescence is about 180nm, the 640nm from spectrographic " blueness " peak 460nm to red color area.GFP is cytobiology (Gerdes, H.-H., and Kaether, C, FEBS Letters (1996) 389:44-47), immunology (Kawakami, N waits Immunology Lett (1999) 70:165-171) and transmissible disease research, for example host on the animal pattern-pathogenic agent repercussion study (Zhao, M waits Proc.Natl.Acad.Sci. (2001) 98:9814-9818) in one of the genetic marker of widespread use.GFP has been used as tumor growth, shift, and vasculogenesis (Yang, M wait Proc.Natl.Acad.Sci. (2002) 99:3824-3829; Yang, M waits Proc.Natl.Acad.Sci.USA (2000) 97:1206-1211; And Yang, M waits Proc.Natl.Acad.Sci.USA (2001) 98:2616-2621), genetic expression (Yang, M wait Proc.Natl.Acad.Sci.USA (2000) 97:12278-12282), and the whole body radiography (whole-body imaging) of infectation of bacteria (Zhao, M wait the same).

In all these were used, emission was red for minimum background emission and body scattering in, and analyzed particularly important for FRET (FRET (fluorescence resonance energy transfer)).The high extinction coefficient of fluorescin, quantum yield and free state be for they purposes as reporter molecule, comprises in the body using, and is important parameters very.Compare with the GFP that very little Dimerized trend is only arranged, relevant albumen has tangible trend to form oligomer, for example at the observed tetramer of DsRed, or even higher aggregate.Optical extinction coefficient also is relative low with quantum yield for red protein and for monomer DsRed newly developed.

Human histone H2B gene with the gene fusion of the GFP of coding Victoria multitube jellyfish, and transfection is produced stable clone to people's cell, this clone constitutive character expression H2B-GFP.Described H2B-GFP fusion rotein is mixed nucleosome and do not influence the cell cycle progress.H2B-GFP can make karyon with high-resolution imaging, and it comprises the chromatin of mitotic karyomit(e) and interval, and the latter has shown the multiple chromatin enrichment stage (Kanda, T wait Curr.Biol. (1998) 8:377-385) of viable cell.

It is disclosed in the article of being quoted this and is incorporated herein by reference.

These protein have been used for monitoring metastases, and monitor expression as reporter gene.See for example Kanda, T waits Curr.Biol. (1998) 8:377-385; Yang, M waits Clin.Exp.Metastasis (1999) 17:417-422; Yang, M waits Proc.Natl.Acad.Sci.USA (2001) 98:2616-2621; And Yang, M waits Proc.Natl.Acad.Sci.USA (2002) 99:3824-3829.Yet, known to the applicant, these protein are not used to monitor cell proliferation (cellularproliferation), cell cycle state so far, or external cell drug susceptibility, and the cell of double-tagging did not use in external or body yet up to now.

The technology of most of external monitoring propagation all causes the death of cell.For example, can pass through enzyme,, discharge, count with alpha counter then from plastics as trypsinase attached to the frosting proliferating cells.Normally used dyestuff such as tetrazolium dyestuff in addition, its is by the electron reduction from mitochondria enzyme activity, and the viability of pair cell is unfavorable.In addition, the lacZ gene may be introduced into described cell and be used as marker, but in order to observe activity, must the described cell of dyeing.

Therefore, the traditional method of monitoring propagation comprises destroys Normocellular metabolism, even causes necrocytosis.

The invention provides chance and come Real Time Observation propagation and other cytoactive in external or the body.These are observed also can expand in different steps and utilize the variation of karyon/endochylema ratio to come the observation of cell cycle.

Known to the applicant, be used to the mark karyon although be fused to the H2B gene of GFP, the protein of two kinds of different colours also of no use comes the karyon of difference mark viable cell and the proposal of endochylema.The invention provides the possibility of observing this viable cell.

Summary of the invention

The present invention relates to observation of cell propagation in the external and body and to the method for the susceptibility of its multiple compound.In addition, the present invention is directed to double labeling cells, wherein a kind of color is used to the mark karyon and distinct colors is used to the mark endochylema.

Therefore, one aspect of the present invention relates to the method for in-vitro measurements cell proliferation, and this method comprises with time being the step that function is observed the fluorescence intensity variation of the cells in culture of using fluorescent protein labeling.

The present invention relates to the method for external monitoring cell to the reaction of compound such as medicine on the other hand, it is by being function with time, when existing and lacking as test compound, measure the cell culture emitted fluorescence, wherein said cells in culture is modified express fluorescent protein.

The present invention also relates to the method for mark viable cell on the other hand, this method has the expression system that is retained in intracytoplasmic first kind of fluorescin with first kind of color by offering cell, with the expression system that is coupled to the proteic second kind of fluorescin of target karyon, wherein when described first kind of albumen and described second kind of expressing fusion protein, the karyon of described cell by a kind of color mark and endochylema by the distinct colors mark.

The present invention relates to the cell of preparation like this on the other hand, it at endochylema with first kind of fluorescin stable labelling and in karyon, be different from second kind of fluorescin stable labelling of first kind of fluorescin with color.

The present invention relates to the method for the viable cell that uses double-tagging on the other hand, and this cell obtains at culture or in live plant or animal with the inventive method.Also available these cells of the effect of multiple material cell cycle are determined.

The accompanying drawing summary

The human fibrosarcoma cell of outer high expression level GFP stably of Fig. 1 display body and RFP (HT-1080-two-1).In retroviral vector, use RFP and neomycin resistance gene, histone H2B-GFP and hygromycin gene transfection human fibrosarcoma cell (HT-1080).Select two transformant and set up stable clone with G418 and Totomycin.Bar mark (bar) is 50 μ m.

Fig. 2 be parental generation and express fluorescent protein the clone cell proliferation rate relatively.Count one week of cell count of three culture dish of each clone (parental generation HT-1080, HT-1080-RFP, HT-1080-two-1 and HT-1080-two-6) at each time point.The trypsinized cell with trypan blue dyeing, and is counted on hematimeter.Filled circles is represented every group average cell number.

Fig. 3 A-3B shows the process with fluorescently-labeled cells in vitro time-histories observation of cell propagation.In the PRMI 1640 that has replenished 10% FBS, cultivate two-1 cells of HT-1080-, and under fluorescent microscope, catch the time-histories image of identical viable cell at a plurality of time points.Fig. 3 A was with five minutes interval shootings.A:0 minute.B:5 minute.C:10 minute.D:15 minute.E:20 minute.F:25 minute.G:30 minute.Bar mark: 20 μ m.Fig. 3 B was with 30 minutes interval shootings.A:0 minute.B:30 minute.C:60 minute.D:90 minute.E:120 minute.Bar mark: 75 μ m.The representative area in proliferating cells is just observed in Lv Quan and white circle expression.Even mitotic cell has changed their relative position, also can easily observe them.

Fig. 4 A-4B has shown by the image of two-6 apoptosis of Staurosporine (staurosporine) inductive HT-1080-.Come two-6 cells of HT-1080-and 2 μ M Staurosporine incubations apoptosis-induced.Under blue light, observe described cell, and catch image with CCD photographic camera and fluorescent microscope.Fig. 4 A has shown 12 hours the image in 2 μ M Staurosporines processing back.The two cells of HT-1080-have been induced apoptosis in high proportion.Bar mark, 50 μ m.Fig. 4 B has shown the real-time high-amplification-factor image of two-6 apoptosis processes of following usefulness 2 μ M Staurosporine inductive HT-1080-: a: be untreated.B: Staurosporine was handled back 2 hours.C:4 hour.D:6 hour.E:8 hour.F:10 hour.G:12 hour.Can finely observe concentrating and nuclear fragment of endochylema and karyon.Bar mark, 10 μ m.

Fig. 5 A-5B shows is that the brain experiment that two-1 injection cells of HT-1080-occur behind the arteria carotis communis is immediately shifted.Skull by having skin lobe window is observed double labeling cells embolus (emboli of dual-coded cells) in the arteriole with the individual cells level.Observe described cellular form and each nuclear form.Fig. 5 A is the view of low magnification.Bar mark, 400 μ M, the wherein zone of the view that the magnification of white square presentation graphs 5B demonstration is high.Fig. 5 B is the high view of magnification.Bar mark, 100 μ M.

Fig. 6 A-6B has shown the realtime graphic of tumor cell proliferation in the mouse alive.Catch the realtime graphic of mitotic tumour cell in the intravital mouse after 12 hours at the injection cell.Fig. 6 A has shown the image that magnification is high.Bar mark, 50 μ M.Fig. 6 B is the sketch map of Fig. 6 A.

Fig. 7 A-7D has shown the cell cycle image of the PC-3 Human Prostate Cancer Cells of double-tagging, and it comprises G ₁Phase, S phase, G ₂Phase and M phase.Fig. 7 E has shown just in the PC-3 of apoptosis double labeling cells.

Fig. 8 has shown Taxol ^TMEffect time-histories to the PC-3 double labeling cells.

Fig. 9 has shown Taxol ^TMOther image of effect, it comprises the gel electrophoresis result displayed.

Figure 10 has shown the result who handles double-tagging PC-3 cell with vincaleucoblastine (vinblastin).

Figure 11 has shown the in-vivo image of the PC-3 double labeling cells of apoptosis.

Summary of the invention

Can be by mark karyon and endochylema strengthen the information that obtains from cell marking respectively. This double labelling side Method is not only monitored the event in cell proliferation but also the monitoring cells survival. Mark of the present invention especially easily Note, wherein karyon is marked as a kind of color of fluorescent emission, and endochylema is marked as another kind of color.

A feature of the present invention is simple by monitoring fluorescence, and the Real-Time Monitoring cell increases in external or body The method of growing. Although the cell of described double labelling can be used on this method, strictly speaking, double label method also Optional. Can use a mark single fluorescence labeling, its intensity can survey but cell is still survived Cell. Also can be in this way be used for monitoring to many by what determine medicine or material on cell proliferation Plant the reaction of processing or medicine.

Be applicable to that cell of the present invention is any Eukaryotic cell, described eucaryote such as yeast, true Bacterium, plant, vertebrate and invertebrate, it comprises people's cell. Be the observation unicellular organism, Such as yeast and for example, mould or fungi, preferred directly observation culture. For more high plant and Zooblast, also observable cell culture. Suitable animal comprises, be generally animal used as test such as rat, Mouse and other rodent, domestic animal (domestic animal) is such as livestock, fish and people's cell.

For the inventive method that does not need double labelling endochylema and karyon, except eucaryon discussed above thin Born of the same parents can use prokaryotic.

According to the inventive method, with the described cell of fluorescent protein labeling. As above used " fluorescin " refers to answer The suitable radiative albumen of stimulation meeting. Usually, when shining with excitation wavelength, fluorescin is transmitted in Visible range is namely at the light of the scope of about 400nm-800nm. When using instrument, available wider model Enclose. Also can use other to lure fluorescigenic mechanism; Example is the example of luciferase easily, wherein makes With metabolizable energy affect signal and produce. Therefore, " fluorescin " refers to can send out when appropriate energy is provided Penetrate the albumen of " as seen " scope light, this energy can be by exciting irradiation (excitation radiation), chemistry Luminous, or other provides necessary energy to affect photoemissive mechanism to provide.

Used fluorescin in a lot of embodiment of the present invention, such as those of above-mentioned background parts. Aobvious So, these fluorescins can produce multiple color; In some symbols (notation), described color is wrapped Drawing together in the abbreviation of protein, is that green fluorescent protein and RFP are red fluorescent protein such as GFP. So And, because the fluorescin of original separation is green, sometimes be generally called these fluorescence with abbreviation GFP The wavelength that albumen and not considering is launched. Like this, on the one hand, GFP for example can launch yellow, red or The light of blue wavelength region. By context can know know whether used GFP general implication also The light of emission in the green wavelength that refers to. For example, in the embodiment of giving an example, use red fluorescent protein The mark endochylema is also used the Green Fluorescent Protein karyon. During these cells of symbolically, GFP is actual to be referred to The emission of green wavelength and RFP refer to the emission of red wavelength.

The invention provides external or the interior observation of cell propagation of body, and the method for definite cell proliferation rate, this method is by with time being function observation emitted fluorescence intensity.Also can determine the heterogeneity of cloning with this technology.

The method of the transformant of acquisition generation fluorescin is known in the art.Can use the fluorescence of a variety of colors and produced, for example, United States Patent (USP) 6,232,523 described stable clones, the document is incorporated herein by reference.In embodiment of the present invention, do not observe these cells in vivo, but these cells are applied in the external real-time detection.

In the embodiment, with stable expression of fluorescent protein, as the stable culture dish that is inoculated into the 96-hole of the cancerous cell line of GFP.At the periodicity time point,, measure the fluorescence in each hole in the plate as Molecular DevicesGemini at the fluorescence plate reading machine.In each specific hole, when described cell proliferation, intensity of fluorescence strengthens.Therefore, by drawing or handling fluorescence intensity in addition and can directly read proliferation rate to the data of time.

For detecting the effect of various material on cell proliferation, the hole in the marking plate, so that one group of (set) hole is in contrast, and the hole of other group and purpose medicine incubation.In the suitable time period, usually every day or per two days, read the fluorescence intensity in every hole on the plate and obtain to contrast growth curve with the hole of drug treating.Culture does not need to handle or additive when being used for these mensuration.Because emitting fluorescence not after GFP is hydrolyzed in apoptosis of checkmating or non-viable non-apoptotic cell is so described GFP is the flash labelling thing of cell survival.Total fluorescence in every hole is relevant with viable count that can quantitative existence.Therefore, determine that easily test compound suppresses or improve the ability of propagation.

In front in method application, minimum cell derived from tumour or even individual cells are provided for each hole.By this way, the application of the invention method can be determined the tumour heterogeneity.Can be observed heterogeneity by the difference that obtains between the hole, each expression derives from one or the propagation of small amounts of cells of described tumour.

The present invention also provides the cell of stable conversion, and it expresses the marker of endochylema and karyon.The expression vector of the second kind of fluorescin that the expression system of the fluorescin by no karyon target signal is provided to viable cell is different with comprising encoded colors and the nucleotide sequence of karyon target sequence link coupled fusion rotein is examined under a microscope and can be seen karyon and endochylema respectively.Therefore by the described cell of double-tagging, can conveniently monitor the incident in the cell cycle, and also can assess the effect of multiple material cell cycle.Can be at the described cell in the microscopically direct viewing culture, or can be complete animal, or even observe described cell in the living animal.

" color " of light of emission refers to the wavelength at the light place of launching.Karyon and endochylema must emission wavelength the different and light that can be measured respectively, so these light are called as " different colours ".The difference of color is not to differentiate by naked eyes; Although preferably there is the wavelength difference of this level, the wave filter (filter) and/or the wave-detector (detector) of different wave length susceptibility also can be arranged by use, auxiliary with suitable software, observe or even small wavelength difference.Therefore, for example, by using wide visual field microscope (widefield microscope), as United States Patent (USP) 6,444,992 described those (they are incorporated herein by reference) also can be used next-door neighbour's different wave length.

Yet, preferably make to observe and oversimplify by the fluorescin that uses color distortion with the naked eye to differentiate.If enough differentiate wavelength difference machine operation is oversimplified by vision.

Because being stabilized to be converted, described cell produces two kinds of fluorescins, so can live and observe described albumen during through a plurality of stage of cell cycle at cell.Can observe described cell in culture or that be present in live organism.For example, United States Patent (USP) 6,251,384,6,235,968 and 6,235,967 have described use is come living animal is made the whole body Real Time Observation by the cell of green fluorescent protein stable conversion, and it is incorporated herein by reference.For use the albumen of preferred high fluorescence whole organism; When therefore carrying out the whole body observation, only be that faint albumen of fluorescence such as luciferase are unpractical.

Certainly, if foreign cell is implanted laboratory animal, these animals fully non-responsiveness (sufficiently immunocompromised) thus can not repel described cell.Make the technology of multiple animal non-responsiveness known in this field; The experimenter easily of non-responsiveness comprises nude mice or SCID mouse, and other rodent such as the rat of similar modification.

Described fluorescin can be the fluorescin that any those this areas can get usually, the fluorescin above-mentioned as background parts.The multiple modification that original isolating A.victoria green fluorescent protein is done has made albumen have multiple color, and albumen is easily expressed in the cell category of wide range.The selection of emission multi-wavelength's fluorescin is a lot of and be known.

The cell that any modification is studied all is suitable with the method that comprises expression system.Method for transformation depends on cellularity and includes but not limited to, for example, and lipofection, electroporation, and virus infection.For vegetable cell, also can use agrobacterium-mediated conversion, and the modification of protoplastis.The selection of control sequence of expression system that comprises the nucleotide sequence of code for said proteins also can change to some extent, and selects suitable contrast and carrier also to depend on the character of cell and the mode of modifying cell.

Can use any suitable karyon target signal; It is following that give an example is histone H2B; Yet, known other the target karyon sequence and can replace with it.

Therefore, the cell that modify is by the suitable carriers transfection, and this carrier comprises the expression system that is used for each described fluorescin, and an expression system and a unique fluorescin are coupled on the other aminoacid sequence that makes that targeting proteins karyon.The carrier that is used to transform can be other carrier of branch for the fluorescin of the fluorescin of the final arrival endochylema final arrival karyon different with color, and perhaps two cover expression systems can be contained in the identical expression vector.Can use described karyon target sequence earlier, make described cell comprise the expression system of the fluorescin of wanting the mark endochylema by transfection then, thereby the stability of preferably guaranteeing described clone between the transfection incident guarantees stability.Also can put upside down the order of transfection by using the protein expression system of endochylema earlier.Perhaps, two expression vectors are exposing cell simultaneously, preferably uses different selected marker things to guarantee cotransfection.Also can use the bicistronic mRNA expression system to two kinds of protein.

For guaranteeing stable the modification, comprising relevant expression system is incorporated into genomic example, makes described cell through selective pressure.The character of suitable selected marker thing meeting dependent cells; G418 or hygromycin resistance are markers easily to a lot of cells; Other optional system of selection comprises for the use based on the toxin of the system of DHFR, as methotrexate.Those persons skilled in the art will appreciate that the selection kind that will use.

Therefore, on the one hand, infect suitable cell with retroviral vector, this carrier comprises expression system, and wherein the nucleotide sequence of the fluorescin of coding emission blue light merges with the aminoacid sequence of coding karyon target signal.Described virus vector also comprises the hygromycin resistance as the selected marker thing.Make handled cell when Totomycin exists, accept selective pressure then, select the back to obtain stable transformant several the wheel.Handle the cell of described stable conversion with electroporation with DNA then, wherein said carrier comprises the protein with DHFR link coupled transmitting green fluorescence.Make described cell accept several the wheel with Totomycin and methotrexate then and select to obtain clone, wherein its karyon dyed for blueness and endochylema for green.

The coloured differently that obtains with the inventive method is an available, because the various piece of cell cycle makes karyon different with the radiation profile and/or the intensity of endochylema emission.Therefore, for example, the ratio of intensity can be determined the position of cell cycle.In addition, the form of karyon is changed when apoptosis occurring, and this is detected easily.Can detect the effect of multiple material by the effect of observing these material cell cycle or form, comprise multiple small-molecule drug, protein, antisense (antisense) or form nucleic acid or the inhibition RNA of triploid (triplex).Multiple material is to the also observation of available the inventive method of different targets of endochylema or karyon.Described cell can evaluated character comprise dormancy, apoptosis, cell cycle phase, multiple other character that the position of material target and technician are familiar with.Ideal words, the material self that is used to handle cell can be labeled.

Therefore, if, described material directly can be added in the culture by the described cell in the microscopic examination culture.If in the animal or plant of living, observe described cell, directly give this animal or plant with described medicine usually.

In the embodiment, with the histone H2B-GFP and the DsRed-2 double-tagging cancer cells of only expressing at karyon, wherein DsRed-2 is introduced by the retroviral vector of only expressing at endochylema.Like this, can determine the ratio of karyon-endochylema by the ratio of green fluorescence and red fluorescence.This measurement can determine to be in the relative populations of cell of the vegetative state of cell cycle.Determine S and G by higher karyon/endochylema ratio or higher green and the ratio of red fluorescence ₂Phase is because the DNA in cell multiplicative process center is synthetic than non-proliferative cell height.Described double-colored cell inoculation in the culture dish of 96-hole, and is determined the intensity and the ratio of red and green fluorescence at different time.By this ratio, can determine the state in the cell cycle.

With regard to propagation was observed self, described mensuration can be fit to detect the result with multiple these cells of compound treatment.This is seeded in control cells in one group of hole in using, and detection material is seeded in the hole of other group.Afterwards plate is inserted in the fluorescence plate reading machine of energy measurement dual wavelength, measure relative growth and ratio thereof green and red fluorescence.This can determine relative proliferation rate and in position and the effect of medicine in these processes of cell cycle.

Present method also can by the cell that uses every Kongzui small number determine described cell from the heterogeneity of any tissue.

Following embodiment to be in order setting forth but not to be in order to limit the present invention.Image under all situations is all used Hamamatsu C5810 3CCD camera, and (Hamamatsu Photonics, Bridgewater NJ) directly take.Be macroscopical imaging (macro-imaging), used fluorescence lamp box (fluorescence light box) (Lightools Research, Encinitas, CA).Be micro-imaging (micro-imaging), used Leica fluorescent and stereo microscope (fluorescence stereo microscope) (model LZ12) with the CCD camera.This microscope is equipped with the mercury lamp of GFP bank of filters (set) and power 50W.Also analyze with contrast and brightness processed image with software I mage ProPlus 3.1.At IBM PC 40) on directly capture the image of 1024 * 724 pixel high-resolutions.

Preparation scheme one preparation RFP retrovirus

For producing RFP retrovirus 22), HindIII/NotI fragment (CLONTECH Laboratories with pDsRed2, Inc., Palo Alto, CA), it contains the red fluorescent protein cDNA of total length, inserts the HindIII/NotI site (Clontech) of the pLNCX2 that has neomycin resistance gene and sets up the pLNCX2-DsRed2 plasmid.Be derived from packing (packaging) the clone PT67 of the expression 10A1 peplos of NIH3T3, available from CLONTECH Laboratories, Inc.Replenishing 10% heat-inactivated fetal bovine serum (FBS) (Gemini Bio-products, Calabasas, DME substratum CA) (Irvine Scientific, Santa Ana, CA) the middle PT67 cell of cultivating.For producing carrier, make the PT67 cell and the LipofectAMINE of 70% degree of converging (confluence) ^TM(Life Technologies, Grand Island is NY) with the precipitation mixture incubation of the pLNCX2-DsRed2 plasmid of saturation capacity 18 hours for reagent.Replenish fresh substratum in this time.After transfection 48 hours with the described cell of fluorescence microscopy.For selecting to produce the clone of a large amount of RFP retroviral vectors (PT67-DsRed2), there is 200-1, the G418 of 000 μ g/ml cultivated described cell 7 days when (Life Technologies).Sometimes, medicament selection is checked the clone of survival and is isolated GFP or RFP male colony under fluorescent microscope after 15 days.Select several clones, and amplification is a clone after making viral titration experiments with the 3T3 cell.

Preparation scheme two is produced histone H2B-GFP carrier

For producing histone H2B-GFP retrovirus, Geoff Wahl professor (Salk Institute) friendship provides histone H2B gene.This gene does not have the termination codon, thereby makes described H2B gene be connected to the 5 ' coding region (Clontech) 24 of Victoria's multitube jellyfish EGFP gene).Then this histone H2B-GFP fusion gene is inserted into the HindIII/Cal I site of the pLHCX (Clontech) of hygromycin gene.For setting up the clone who produces a large amount of histone H2B-GFP retroviral vectors, use the method identical, with described pLHCX histone H2B-GFP plasmid transfection PT67 cell to PT67-DsRed2.When the Totomycin that has 200-400 μ g/ml (Life Technologies), cultivate described cells transfected 15 days, finally set up PT67-histone H2B-GFP cell.Sometimes, medicament selection was checked the colony of survival after 15 days under fluorescent microscope, and selected GFP or RFP male colony.Select several clones, and amplification is a clone after making viral titration experiments with the 3T3 cell.

Embodiment 1 expresses histone H2B-GFP and expresses the people of pLNC DsRed at endochylema at karyon Prostate cancer cell

Separate double-colored PC-3 cell, this cell is expressed GFP and is only expressed RFP at endochylema at karyon because GFP and histone H2B (21) only merge.These cells show and make the double-colored imaging of prostate cancer cell alive easily.

Step 1: the cell of DsRed is expressed in preparation

Use the pLNC DsRed-2 that produces by the PT67 packing cell to transform the PC-3 cell.The DsRed-s of monitoring in the PC-3 cell expresses under fluorescent microscope.Measure and select by increasing G418.

Step 2: the H2B GFP and the DsRed-2 cell of preparation double-tagging

Use LipofectAMINE Plus ^TMWith pLHC H2B-GFP DNA transfection DsRed-2PC-3 cell.Behind the incubation 24 hours, select to express the cell of H2B GFP and DsRed-2 by the amount that increases Totomycin and G418.

Analyze the cell cycle position of viable cell than the area ratio of red endochylema with green karyon.Determine apoptosis with the nuclear morphology in the viable cell.

The RFP of embodiment 2 fibrosarcoma cells and histone H2B-GFP gene transfer

For RFP and histone H2B-GFP gene transfer, (Rockville ML) buys the HT-1080 cell that is derived from the human fibrosarcoma, and its degree of converging is 70% from American type culture collection (American Type Culture Collection).For setting up double-colored cell, at first be based upon the HT-1080 clone who expresses RFP (HT-1080-RFP) in the endochylema.In brief, with the HT-1080 cell with go up cleer and peaceful RPMI1640 (Mediatech, Inc., Herndon, mixture incubation VA) 72 hours that comprises 10% foetal calf serum with the sedimentary PT67-RFP cell reversal of 1:1 record virus.Replenish fresh culture this moment.Transduce back 72 hours with trypsinase/EDTA harvested cell, and with the ratio subculture of 1:15 in the selective medium of the G418 that comprises 200 μ g/ml.The level of G418 is increased to 800 μ g/ml gradually.Use conventional cultural method, (Bel-Art Products, Pequannock NJ) separate HT-1080-RFP and amplification by trypsinase/EDTA with clone's cylinder (cloning cylinder).

Two clones' subbreed (HT-1080-two-1 and HT-1080-two-6) is expressed RFP at karyon stably express GFP and at endochylema.Obtain the other clone who only comprises RFP or GFP in contrast (HT-1080-RFP, HT-1080-GFP).

The two clones' of embodiment 3 parental generation HT-1080-RFP and HT-1080-cell proliferation rate

Each fluorescently-labeled HT-1080 clone (HT-1080-RFP, HT-1080-two-1 or HT-1080-two-6) and parental generation are cloned (HT-1080) with density 1 * 10 ³Cell/culture dish is seeded in the 100mm culture dish, and the RPMI (first day) that contains the 10%FBS substratum is wherein arranged.Described culture dish is remained on 37 ℃ and 5%CO ₂Incubator.(2-7 days) do the cell counting with three culture dish of each clone every other day afterwards.In brief, collected re-suspended cell dyes with trypan blue (Sigma) after the trypsinized.(Reichert Scientific Instr μ Ments, Buffalo NY) only count the cell of living to use hematimeter afterwards.

Fig. 1 shows that the double-colored cell of selected HT-1080-has bright external GFP and RFP fluorescence.Green fluorescence is sitting in the karyon.All cells shows described genetically modified stability in the colony of expression GFP and RFP.Fig. 2 is presented at the parental generation HT-1080 that the monolayer culture thing is determined, HT-1080-RFP, HT-1080-two-1 or two-6 clones' of HT-1080-proliferation rate indistinction.

In the 150mm culture dish, use two-1 cells of the described HT-1080-of RPMI 1640 culture medium culturing that contains 10%FBS.Every 5-30 minute, described culture dish is placed the fluorescent and stereo microscopically, and catch the time-histories image from identical viable cell.

Fig. 3 A shows HT-1080 two-1 time-histories image series with 5 minutes intervals when mitotic division.Fig. 3 B shows that with 30 minutes mitotic cells at interval, even the position of wherein described mitotic cell in a big cluster changes, they were also tracked easily.

Embodiment 4 external Real Time Observation apoptotic processes

For catching the realtime graphic of apoptotic process, use two-6 clones of HT-1080-.With the Staurosporine that is dissolved in being stored among the DMSO-80 ℃ (Alexis, San Diego, CA) come apoptosis-induced.With 3 * 10 ⁵Cell inoculation to 25cm ²Culturing bottle, the RPMI 1640 that contains 10%FBS is arranged in the culturing bottle.Added Staurosporine at second day with concentration 2 μ M.Per 2 hours, described culturing bottle is placed the fluorescent and stereo microscopically and catches realtime graphic at each time point.

Induce the apoptosis of two-6 cells of HT-1080-with the Staurosporine of 2 μ M.(Fig. 4 A and 4B).Can observe gradual nuclear fragment in per two hours.

In embodiment 5 bodies Real Time Observation two-cell of mark

Take care of principle and the method listed according to the U.S.'s state-run commune hospital laboratory animal and carry out all zooscopies (social security number or taxid A3873-1) with using rules (National Institutes ofHealth Guide for the Care and Use of Laboratory Animals).Animal is remained in the filterable barrier equipment of HEPA (barrier facility).(Orange CA) feeds mouse for Tecldand LM-485, WesternResearch Products with autoclaved rodent diet.

For injection cell is arrived arteria carotis communis, by subcutaneous injection ketamine (ketamine) mixture (vetatar 10 μ l, xylazine (xylazine) 7.6 μ l, acetypromazin maleate (acepromazinemaleate) 2.4 μ l, water 10 μ l) the anesthesia nude mice.At first, on neck, make the skin longitudinal cut.Cut at the middle part after detecting glandula submandibularis, handle each limit then.Separate right sternohyoideus and right nutator and reticular tissue with blunt.After detecting right common carotid artery, gently this artery and on every side reticular tissue are separated.(Foster City is CA) to the slight tractive of the near-end of artery for Fine ScienceTools, Inc. with the hook of tack.200 μ l are altogether contained 2 * 10 ⁵The substratum of two-1 cells of individual HT-1080-is expelled in the artery with the pin (Fine Science Tools) of 33G.Pin injection site for some time with swab immediately after the injection and avoid the tumour cell seepage of bleeding or injecting.After confirming hemostasis, with the airtight skin of 6-0 suture.(Deerfield carries out under IL) all above-mentioned operating procedures for MZ6, Leica at 7 * dissecting microscope.

Behind the arteria carotis communis injection HT-1080-two-1, in the arteriole of brain, even can both observe embolus cell (Fig. 5 A and 5B) through skull by scalp lobe (scalpflap).The skull of described mouse is transparent relatively.Observing described tumour cell extends as white corpuscle and adapts to capillary blood vessel.In the tumour cell of seeing in blood vessel, the karyon of tumour cell can be stretched very longly.

Observe the karyon-endochylema kinetics of tumour cell in embodiment 6 bodies

For observing the karyon endochylema kinetics of tumour cell in the body, with ketamine mixture anesthetized mice.Under blue light, use fluorescent and stereo microscope direct observing ear surface.

For observing the karyon endochylema kinetics in lung shifts, with 1 * 10 ⁶Two-1 cells of HT-1080-be expelled to the tail veins of six nude mices with volume 300 μ l.Put to death mouse and take out lung at a plurality of time points.Directly under blue light, observe the colony that shifts in the lung.For making histologic analysis, the lung of excision is embedded in organizes in the freezing substratum (Triangle Biomedical Sciences, Durham NC) and be stored in-80 ℃.Prepare frozen section with freezing-microtome LEICA model C M-1850 with thickness 4 μ m.Having the prepared section of fluorescent and stereo microscopically direct viewing of GFP filter set.

The micrometastasis (micrometastases) of observation on the lung that cuts out finds that each karyon all abuts one another (data not shown).It seems it is that cell is the distortion shape so that karyon is in contact with one another.

It is interior and external in cell levels Real Time Observation cell karyon-endochylema kinetics that this has researched and developed body, comprises the possibility of apoptotic process.After the mouse ear injection of intravital mouse, observe mitotic cell easily.Behind tail vein injection, observe double-colored micrometastasis at the lung of excision.

Embodiment 7 whole body fluorescent optics imagings

The two back realtime graphics that captured at the mitotic cell of intravital mouse in 12 hours of-1 (Fig. 6 A and 6B) of HT-1080-can injected.As if shown cell exosmoses (extravasated) and is rounded, the positive splitted cell in the similar culture.Need not fixing or dyeing, can observe the situation and the described cell boundaries of described each karyon of cell at living animal.

The double-colored PC-3 of embodiment 8 preparations

Growth PC-3 cell in being supplemented with the RPMI1640 substratum of 10% FCS.There are 8 μ gml ^-1During Polybrene, with the cell (in the 10cm culture dish) of exponential growth and viral supernatant incubation from PT67/pLHCXH2B-EGFP.Spend the night behind the incubation, change substratum, the Totomycin that the cell that amplification is infected is used to do is progressively selected.After the medicament selection 15 days, under fluorescent microscope, observe the colony of survival and separate the positive colony of GFP.Select clone single, high level expression H2B-EGFP.

Afterwards, when having 8 μ g/ml Polybrene, with the cell of the expression H2B-EGFP of exponential growth and the supernatant incubation that comprises virus from PT67/pLNCX DsRed-2.Spend the night behind the incubation, change substratum, after the infection in 48 hours, the G418 that the cell that amplification is infected is done progressively selects.After the medicament selection 15 days, under fluorescent microscope, observe the colony of survival and separate the positive colony of RFP.Select single, high level expression H2B-EGFP and RFP (PC3-be'ss two) clone.

For determining growth rate, cultivate culture dish (Petri dish) at 60mm and cultivate 1 * 10 ⁵Individual cell, and count totally one week every day.The triplicate viable count of determining every hole of fixed time does not comprise the painted dead cell of trypan blue.

When no Totomycin B and G418 selection cultivation, stablize high expression level H2B-EGFP and endochylema RFP in this clone above three months.Described double-colored cells in vitro rate of propagation is identical with its parental cell PC-3.These results show that double-colored PC-3 can be in the body and the useful model of in vitro study.

Embodiment 9 observation of cell cycle and apoptosis

By can easily finding the position of described double-colored cell in the cell cycle at them at viable cell observed " karyon endochylema " ratio.For using these cells of fluorescence microscope, use the Leica fluorescent and stereo microscope (model LZ12) of the mercury lamp of having equipped power supply 50W.For observing GFP and RFP fluorescence simultaneously, by D425/60 bandpass filter (band pass filter), 470 DCXR dichroic mirrors (dichroicmirror) excite, and by long pass filter (long pass filter) GG475 (ChromaTechnology, Brattleboro VT) collects emitted fluorescence.

Fig. 7 A-7D represents the phase of cell cycle; Fig. 7 E represents apoptosis.

The relative endochylema of karyon can easily be identified G greatly ₂The phase cell, in contrast to this, G ₁The karyon of phase cell-endochylema ratio is little a lot.The karyon endochylema ratio of S phase cell compares G ₁Big but compare G ₂Little.Concentrate by karyomit(e) and to identify and to enter mitotic prophase of cell.Observe equatorial plate (metaphase plate) bar mark shape easily and arrange the cell of (line up), and can observe them and enter the anaphase.Comprise that by unusual karyon form fracture can identify apoptotic cell.

Therefore, successive cell cycle progress that can the real-time follow-up individual cells, and with frequent interval shooting Photomicrograph.

The apoptosis that embodiment 10 Real Time Observation are drug-induced

Use Taxol ^TM(0.8 μ g/ml); Thymus pyrimidine (2mM), and vincaleucoblastine (60nM) is handled the two cells of PC-3.0,12,24,36 and 48 hours real time imageries.Collecting cell extracts DNA and does agarose gel electrophoresis (1.8%) analysis.Paclitaxel (Taxol ^TM) (0.8 μ g/ml) have very special effect to karyon, makes their obviously form ring structure, because chromatin has been attached on the nuclear membrane.Fig. 8 shows that this structure is easy visible by the endochylema background of the karyon contrast RFP mark of GFP mark.Ring structure by the end of 24 hours described karyons is survived.By the end of 48 hours, the visible described apoptotic cells that enters had very unusual nuclear structure and fracture.

Fig. 9 is presented at and begins to handle back 24 hours observed Taxol ^TMThe inductive ring structure is before dna break occurs, and can observe by gel electrophoresis.By the end of 48 hours, when described cell enters apoptosis, can be observed dna break.

Other tubulin material, vincaleucoblastine (60nM) and Taxol ^TMIt is different to compare nuclear effect, and karyon becomes more concentrated and endochylema presents expansion as described in wherein showing as Figure 10.

Observe in embodiment 11 bodies

Take care of principle and the method listed according to the U.S.'s state-run commune hospital laboratory animal and carry out all zooscopies (social security number or taxid A3873-1) with using rules.Male nude mice (NCr-nu) between age in 5-6 week is remained in the shielding unit on the filterable feed shelf of HEPA (rack).With described double-colored PC-3 Human Prostate Cancer Cells (2 * 10 ⁶) be expelled to nude mice foot pad.After 20 days mouse is implemented euthanasia.Tumour and lymphoglandula and lung all pass through to handle and are used for fluorescence microscopy.

Figure 11 shows under the fluorescent microscope observed mitotic double-colored PC-3 cell in lymphoglandula.

Claims (18)

1. the method for preparing viable cell, wherein said cell comprise the second kind of fluorescin that is positioned at nuclear first kind of fluorescin and is positioned at endochylema,

Wherein said first kind and second kind of fluorescin are launched the light of different wave length, thereby this method comprises that modifying viable cell comprises it:

(a) first kind of expression system of the described first kind of fluorescin of expression, wherein said first kind of fluorescin merges with the aminoacid sequence that comprises karyon target signal, with second kind of expression system of expressing described second kind of fluorescin, it lacks karyon target signal; Or

(b) not only expressed described first kind of fluorescin but also expressed the expression system of described second kind of fluorescin, wherein said first kind of fluorescin merges with the aminoacid sequence that comprises karyon target signal, and described second kind of fluorescin lacks karyon target signal;

And from described adorned cell, select the cell that is stabilized modification.

2. the process of claim 1 wherein that in step (a) described cell is at first modified then with described first kind of expression system and modified with described second kind of expression system, otherwise or.

3. the process of claim 1 wherein that described karyon target signal is histone H2B.

4. the process of claim 1 wherein that described selection is by cultivating when microbiotic or toxin exist.

5. by each the adorned viable cell of method of claim 1-4.

6. determine the method for the cell cycle position of viable cell, this method comprises the ratio of the karyon area and the endochylema area of the cell of assessing claim 5.

7. the method for claim 6, wherein said assessment is that function carries out with time.

8. the method for claim 6, wherein said cell is observed in living animal.

9. determine the method for the effect of material pair cell, this method comprises

With first sample of the cell of described mass treatment claim 5, and observe described processing for from the distribution of described cell emitted fluorescence albumen ray and/or the effect of intensity.

10. the method for claim 9, it comprises that also observation never uses the distribution and/or the intensity of the cell institute emitted fluorescence albumen ray of described mass treatment, and

Relatively to first kind of sample and second kind of observation that sample is done.

11. the method for claim 9 or 10, wherein said distribution and/or intensity are the features of dormancy.

12. the method for claim 9 or 10, wherein said distribution and/or intensity are features of apoptosis.

13. the method for claim 9 or 10, wherein said distribution and/or intensity are the features in the stage of cell cycle.

14. determine the method for the target location of material, this method comprises the cell with described mass treatment claim 5, and observe distribution and/or intensity from described cell emitted fluorescence albumen ray, wherein said material is labeled self, and described method also comprises the position of the described mark of direct viewing.

15. determine the method for the proliferation rate of cell culture, this method comprises the viable cell of cultivating claim 5, and is the described cell emitted fluorescence of function measurement with time, thereby determines the proliferation rate of described cell.

16. the method for claim 15, wherein said culture is grown from individual cells.

17. determine the method for the effect of testing compound on cell proliferation, this method comprises

There is and lacks the viable cell of cultivation claim 5 when no at described compound;

Existing and lack when no with time at described compound is thereby that the definite described compound of function mensuration fluorescence intensity exists and lack the proliferation rate when no;

And there is and lacks the proliferation rate when no in more described compound;

Wherein said compound exists with the change that lacks no hourly growth rate identifies that described compound is the instrumentality of cell proliferation.

18. the method for claim 17, wherein said cultivation is from individual cells.

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