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Establishment Of Stable EGFP –Expressing Osteosarcoma Cell Subseries With Different Metastatic Potential

 Zeng Heng * ,Chen Anming,Yang  Caihong

*Department of Orthopaedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030,China

Address for Correspondence:  

Zeng Heng M.D.
Orthopaedic surgeon, Tongji Hospital , Tongji Medical College ,
Huazhong University of Science and Technology, Wuhan 430030, China
Phone:   00862783663532
Mobile : 008613995655679


Objective:  To establish stable enhanced green fluorescent protein (EGFP)-expressing human osteosarcoma cell sublines with different metastatic potential and  investigate their biological characteristics. 
Method:  The pEGFP vector, which contains an enhanced GFP gene, was transfected into human osteosarcoma MG63 cell line .Then obtain two cell sublines of clone MG63-M6 and MG63-M8 with different metastatic potential . Per cell lines proliferation invitrosoft agar formationgrowth  curvenude mice tumor formation test aggregatedly analyze its biological behaviour.
Result:   M6 and M8 two lines both kept anthro-chromatoid karyotypethe tumor  formationed presents human osteogenic sarcoma epithelium organizational shape, among which the population doubling time of M6 is 38.4hsoft agar formation rate is 18.7%M8 population doubling time is 23.0hsoft agar formation rate is 29.3%. Hypodermically innoculate M6 and M8 on the back of nude micedetect  the  tumor formation time of M8 is shortcell proliferation is fastbut both don’t develop transfection in 4w. 
Conclusion:  Osteosarcoma cell subseries have different metastatic characteristicsthe integtation and expression of GFP have not posed any marked influence on the growth state of  MG63 cellit can be conducted and acted as the report gene to further comprehend the variability analyzation of osteosarcoma cell metastasis.

J.Orthopaedics 2008;5(1)e6

Osteosarcoma;  Neoplasm metastasis;  Green fluorescent protein


Green fluorescent protein (GFP) gene is a kind of newstyle report gene [1].The influence of human osteosarcoma MG63 cell which uses GFP “mark” on the bionomics of  MG63 cell and differentiation invitro,law of development is not reported yet domestically,so we progress comparative study on the bionomics of the two cell lines through the construction of vecter which contains enhanced green fluorescent protein (EGFP) gene to transfect MG63,together obtain two strains of MG63 monoclone sublines  which possess different metastasis potentiality

Material and Methods :

1 Material and instrument  Human osteosarcoma: MG63 cell line (No.ATCCCRL-1427) bought from Cell Conservation Center of Wuhan Universityenhancement type GFP eukaryotic expression plasmid pEGFP-N1 is favorably given by Doc. Rao Yaojian of our room. RPMI -1640fetal calf serumOPTI-MEM serum-free mediumliposome transfection kitLipofectamin2000),HEPES bought from Gibco Ltd. agar powder powderedSigma subpackage),pancreatintrypan blueDMSOG418PI bought from Sigma Ltd.CO2 gas incubator (USA)inverted microscope (OlympusJapan)EL 800 symase immunodetection instrument (Bio-Tek Ltd.USA)PACE5000 electrophoretic apparatusBeckManUSA. Nude mice BALB/c nu/nubought from Laboratory Animal Center o f Tongji Medical College46w’s ageweight 18~ 22g SPF conditional  breeding.

The extraction and identification of the plasmidthe preparationpurification of Bacillus coli competent cells and the plasmid identification are progressed according to routine methodthe plasmid extraction and purification is progressed according to the instruction of the kit.

2 The cell culture and the plasmid transfection:   MG63 cell is cultured by using RPMI1640 medium equally assisted with 10% fetal calf serumPGstreptomycin 100ug/ml eachin the temp. of 37℃ 5%CO2 . Undertake G418 minimal lethal concentration test 2w’s before transfection. Crop the MG63 cells which are in the good condition of log phasetransfer to 12-well  board according to the density of 1×105cells/boreput in 37 5%CO2  incubator cultured for 24h. Throw away cell culture liquid as soon as cell adherence grows to 80% confluencecleaning cell for one time by serum-free medium, use the extractioned  purified pEGFP-N1 plasmid to transfect MG63 cell according to the instruction of liposome  transfection kitchange culture fluid 4h’s after transfection use G418 culture fluid which contains 600ug/ml to cultureafter 48h observe the condition of transient expressionchange to G418 which contains 400ug/ml to maintain screening when most part of  the control group’s cells are diedchange liquid to passage every 2~3d,resistance clone is initially formed 2 w’s laterobserve its intensity of green fluorescence under Fluophot. When cell conjugation reaches to 80%move the cells to 6-well board to cultureuntil passaging amplification revealed in culture flasktogether collect cells to do every item of experiments on the exponential phase of cell.

3 Monoplast clone separation:  Use limiting dilution assaytake 1×105/ml MG63 cell through trypsinization ft cell suspensionthe density is 10pieces /ml25pieces/ml innoculation 96- bore culture board add 100ul cell suspension in each boreaverage 0.5~2.0 pieces of cells /boretogether 5 culture boards add 100ul culture fluid in every bore. 4~5d after cloning choose the bore which only contains single cell growthadd mark  on the bore which is confirmed of containing single cellobserve monoplast colony formation in the mark bore. Change culture fluid every 4~5d. Remove cells to 24-well board to amplication culture as cell proliferation area reaches to 1/3 of the wellsuntil culture passage in the culture flask . Obtain 12 strains monoclone stocks which are denominated M1~M12.

4 The determination of cell electrophoresis module:   Take M1~M12 quaque monocell clone strain to ft cell suspension,density adjusted to 1.0×108/L( use trypan blue rejection test to demonstrate ultimately the suspension contains above 95% of living cells),admove into thawing fusing type quartacapillary electrophoresis instrument,in 15kV voltage, 10s pressure introduction,detect the speed of cell electrophoresis in the temp. of 37℃ ,therefrom obtain the two strains which has the highest electophoresis speed and the lowest electrophoresis speed as well as maternal plant to progress the following experiments .

5 Chromosome analysis:  Take 3 strains of above cells which have passaging grown for 48hadd Colchicine to the final concentration of 0.05ug/ml on experimental dayharvest after 6h’s effectper hypotensionfixationdropping sheetmake Giemsa and G strap coloration under the immersion objective each count 100 cells’ chromosome numbermoreover choose 20 pieces of scattered metaphase image to do chromosome karyotype analysis calculate mode numerusanalyse karyotype after G banding dyeing.

6  Cell morphological check: Dropwise three strains of cells onto glass slice culture for 48h in the plate which contains culture fluid HE dyeing light microscope observation after kryo-acetone fixation; obtain another  two strains of cells centrifugalized at the speed of 2000r/min,10min latercell mass fixed with 4.5% GA to make transmission electron microscope (TEM ) collection.

7 The observation of growth curve (MTT chromatometry) Inoculate three  strains of cells by the quantity of 1×103/bore into 96-well board 24 bore per strainculture in incubatoradd MTT(5mg/ml) dye 20ul every 24h in the temp. of 37 continue incubating 4h and then terminate culturesuction the supernate of every holeadd 150ul DMSOswing for 10minthen determine OD value of 490nm divisioncum time(day) to be  abscissa axisphoto-absorbtion valueAas YAS to draw growth curve.

8 The determination of clone formation:  Use soft agarose cloning techniquetake three  different density strains of cells which are incubated in the temp. of  37 ft suspension, take  9.4ml shift in tiny beaker, add 50 5% agar 0.6mlquickly misce beneimmediately spray into the two 24-bore culture boards which are paved by  bottom-layer agar (density 0.3%)the cells of every strain add into three bores each refer to the cell number of 100 and 50 add 0.8ml in each boreput in room temperature until agar coagulation culture in incubation box for 14d in the temp. of  37℃ 5CO2observe colony formation state under micro as well as counting the clone numberthe cell number of each colony50 is one clone.

9  The experiment of nude mice tumor formation  in vivo:   Use  RPMI1640 which contains no serum to prepare the cell suspension with the density of 1×107pieces/ml  after the digestion and ablution of the two lines and stock plantinnoculate neoplastic cell on the back of nude mice subcutaneously 0.3ml each 6 mice in eachcontinually observe for 6 w from the injection dayrecord the formation of neoplastic cell and the general state.6 w later unifily executethe tumor tissue is established light microscope blade as a routineobserve under micro.

Results :

1.  The survey of cell transfection result     48h after cell transfection,use Fluophot micro to observe the transient expression of green flourescence fusion protein in cellsas well as calculate the transfection efficacy which is 18%,see Fig 1.

Fig. 1  Expression of Green fluorescent protein in MG63 Cells by transfection after 48h

2. The separation of monoplast clone   Obtain 12 lines of MG63 cell subseries which are numbered M1-M12 respectively through limiting dilution assay.

3. The velocity of cell electrophoresis   Process cell electrophoresis censorship on each cell line of M1-M12 divided into  highly metastasis M8 and lowly metastasis M6the result see Fig 2the electrophorogram of M6 and M8 see Fig 3



























Fig 2    The electrophoresis velocity of MG63 cell substrain


                                 M6                                                        M8

Fig 2   The cell electrophoregram of M6 and M8

4. Chromosome analysis   Chromosome modal number disposition and karyotype analysis,the modal number of M8 and M6 is centered between 61~65  separately which are both hypotriploid ,the appearance are still anthropo-chromatoid,the three kinds of cells chromosome have plesiomorphism.

5. Cell morphological check    Under contrast phase microscope,cultured cell appearance is aligned epidermically invitro,cell monolayer-poietic has the competence of overlapping growth. Under light microscope the cells of each group have plesiomorphism,kytoplasm is coorespondencely less,cell heteromorphism is significant,it is thus clear minority tumor giant cell,can be seen caryomitosis figure accidently,cells are assumed suffusion distribution. Under TEM the cells of every group are plesiomorphism,there is major RI in periplast,density is high,coloring is deep,can be seen comparatively large mitochondria,turgescent poly-lysosome and poly-mitochondria,the quantity of endocytoplasmic reticulum is little,some distension,some short and with Ves shape.Compared with M6,M8 endoplast is larger and irregular,there is  evident Ves shape protrution on the surface of M8 cell,microvilli is long and much,there is no marked morphological difference between M6,M8 and M.

 6.  Growth curve observation(MTT chromatometry)   The characteristics of cell proliferation    3~6d after cell substrain postinoculation it is exponential growing,after 6d cell growth decreased,living cells decreased by the 8th day.The morphous of growth curve is basi-uniformal with parental generational cells,M6 and M8 cell population doubling time is 38.4h and 23.0h respectively in logathmic growth phase.The double time of maternal plant cell is M8 32.65h. It hints M8 growing  active, M6 growing comparetively slow.










Fig. 3  Growth curve of MG63 Cells by MTT method

7. Cloning efficiency M8 plastidogenetic clone is more M6 plastidogenetic clone is lesspresented spindle shape just like maternal plant.

The cloniescloning efficiency % of M6M8 and the number of maternal plant

cell subline

Number0f colonies

number of cells

100     50


 cloning efficiency/%


18      10



29      15



23      11


* P<0.05 chi square test Marked difference in M8 vs M6

Table 1: the Potential of Forming Colonies of MG63 Cell Lines in Soft Agar

8. The growth state of bearing cancer in nude mice in vivo  M6M8 and maternal plant M are subsutaneously inoculated on the back of nude mice 3 mice have tumor formation after 5d M8 inoculationon 15dthe average diameter of tumor is 0.69cm × 0.55cm 2 mice have tumor formation after 8d M6 innoculation on 15d the average diameter of tumor is 0.29cm × 0.77cm 2 mice  have tumor formation after 7d M innoculationon 15d the average diameter of tumor is 0.32cm × 0.61cm execute 6 w after operationthe three lines have no pulmonary metastasis in anatomy. The organization of tumor-forming is accredited caryomegaly, thick dye,multiple mucleoli, a lot of karyokinesis phase, unclean circumsciption between cells by HE pathological section.

Discussion :

The infestation of malignant tumor and the regulation mechanizm of metastatic molecule are the hot spot topics at present in the investigation of tumor. The formation of metastasis is not only the accomodation of tumor cells to certain organic environment but also include primary tumor has oncocyte subseries which possesses different metastatic potential [2]they present heterogeneous phenomenon on the biological characteristics. Heterogeneity of tumor is roughly displayed on the difference of immunogenicitygrowth kineticsmetabolic capabilitykaryotypemobilityinvasionmetastasiscell membrane receptorthe sensitivity on radiotherapy and chemotherapythe foundation of heterogeneity production is due to considerable transformed cellsit is independent of the original of PT monoclone and polyclone. Fidler and etc. refer the hypothesis of tumor cell heterogeneitythey consider the metastasis of tumor is not randomthere are only very few tumor cells in primary tumor which possess the metastatic potential can ultimately metastasize, 99% of circulation cancer cells are going to dieonly less than 1% of tumor cells have the possibility of survival and transfectionthis is maily decided by the existence of hyp-metastatic ability clone cell in tumor cell colonies [3]the heterogeinicity of mono-clone cell subseries extracted from heterogeinicity maternal cell colony can be reduced to mn and its expression of metastatic capability is stablethat is extremely make for comparative experiment.So it has significant pratical importance in the investigation of tumor metastasis mechanism, the instruction of clinical treatment by extraction of these cells to do the comparason of their biological characteristic difference .

Osteosarcoma is one of the most viscious tumor in bone tumorit’s difficult to cure radically and completely for its easy metastasis and local infiltrationrecently the study on the mechanizm of osteosarcoma metastasis is more [4,5]MG63 cell line is a human osteosarcoma cell line which possesses high metastatic potential [6,7]the experiment is through the method of cloning in vitro separating two lines of clone cells which have similar modal number with maternal line , still holding human chromatoid karyotype M8 compare with M6cell proliferation phase is shortercloning efficiency is higher in soft agarOn morphosisM8 cell volume is largerpheno-plasmosome is evident under electron microscopecell surface vec pustute is much and manifest.

GFP is a kind of photoprotein extracted from medusa which can erupt green light under the optical excitation of 450~490nm blue lightcan be sighted directly under Fluophot .Because of Green Fluorescent Protein is easily observed and detectedso it has already become an important indication molecule in the study of transgene. GFP conducts and actions as the tumor marker of gene expression stably existing in cellstracing the expression level of fusion partner molecule and fixing detecting environment changes or protein interactive marks. The consequence is real and reliable and followed with great interest by the people [1,8].This experiment is using the characteristics of GFP gene to finish the construction of green fluorescence protein gene expression bearerthe bearer can use GFP as report gene to observe the expression fixing  and time series changes of MG63 cells in animal invivo. We use EGFP as the marker gene to transfect human osteosarcoma MG63 cell48h transient transfection efficacy is 18%can stably expressed through G418 screening to form resistance clone.

Meanwhilce our comparason results display the integration and stable high performance of exogenous gene GFP in MG63 cells have no marked influence on the basic biological characteristics of MG63growth ratepoorly differentiated statekaryotype and the capability of multi-directional differentiation in vivoproviding A/W for effective utilizing GFP to progress tracing in MG63 cell in vivo.

Cell electrophoresis  behavior is decided by cell surface structure and its functional  statusafter cell cancerationthe series of the changes of cell surface structure and its function necessanly can affect its electrophoresis behaviormeanwhile can change its invasion transfection capability, charge density on the cell surface increasedthe repelling force between both increased which maybe promote the abjunction of tumor cells from maternal nuclide. Because of different cell subseries has different electrophoresis behaviorthis method can be taken as the screening route of the tumor cells which possess different metastatic characteristicscan initially prefractionated on tumor cells. Generally believe the cell electrophoresis rate is increased on strong invasion and high metastatic cells. Take cell electrophoresis rate as the chief indicator for screening different metastatic capability tumor cell clone cell lines [9].A great deal of experiments have shown growth advantage possibly can amplicate cell subseries in vitro which have high degree of malignance or gradually develop into the major component of the whole tumor tissuesearching  the difference of growth metastasis advangage has pratical significance in the approach of tumor invasive metastasis molecular mechanizm and treatment [10,11]. This experiment is utilizing the correlation of cell electrophoresis velocity with tumor metastasis potential to preliminarily screen human osteosarcoma cell seriesobtain 2 monoclone cell subseries.Among which the electrophoresis velocity of M8 is 50% higher than M6this demonstrates the variability of tumor cell subseries is manifest.We observe from the experiment the growth doubling time of M8 subseries is markly faster than M6 subseriesthis demonstrates progressing variability analyzation by the utilyzation of electrophoresis velocity difference on the osteosarcoma cells which have good growthactive functional status is feasibethis has established a better foundament for the further investigation of osteosarcoma invasion metastasis mechanism.

Reference :

  1. Rosochacki SJ, Matejczyk M. Green Fluorescence Proteinasa molecular marker in microbiology. Acta Microbiol Pol, 2002,51(3):205~216.

  2. Gao Jin. The infestation and metastasis of cancerfundament and clinic [M].Beijin:Beijin Medical College, China Union Medical University ,Union Publishing House1996:2~3.

  3. Fidler IJ. Tumor heterogentity and the biology of cance rinvastion and metastasis. Cancer Res,1978,38:2651

  4. Jung SY, Kwak JO, Kim HW, et al. Calcium sensing receptor forms complex with and is up-regulated by caveolin -1 in cultured human osteosarcoma (Saos-2) cells. Exp Mol Med. 2005 ,37(2):91-100.

  5. Serra M, Reverter-Branchat G, Maurici D, et al. Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells. Ann Oncol. 2004 Jan;15(1):151-60.

  6. Rex C, Hay D, Lan Z, et al. Nuclear Receptor Agonists as potential differentiation therapy agents for human osterosarcoma. Clin Cancer Res 2002, 8:1288~1294.

  7. Masahiko K, Noriko S, Akihiro F, et al. Laminin r2-Chain Fragment in the Circulation: A Prognostic Indicator of Epithelial Tumor Invasion. Cancer Res, 2003,63:222~229.

  8. Liu MefangWang Enduo.Green Fluorescence Protein.  The development of biochemistry and biophysics200027(3):238243.

  9. Gao Jin, Liu Yaqin, Han Liqun and etc.. The separation and appreciation of different metastatic potentiality cancer cell subseries as well as the modeling of cell clone and the application of metastatic mechanizm investigation.China Tumor,1997,6(2),20~21

  10. Calvo A, Xiao N, Kang J, et al. Alterations in gene expression profiles during prostate cancer progression: functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors. Cancer Res. 2002 ,62(18):5325~5335.

  11. Fan DG, Fan QY, Zhang HZ,et al. Study on metastasis-associated gene in osteosarcoma by cDNA microarry. J Fourth Mil Med Univ, 2003,24(9):816~819.


This is a peer reviewed paper 

Please cite as : Zeng Heng : Establishment Of Stable EGFP –Expressing Osteosarcoma Cell Subseries With Different Metastatic Potential

J.Orthopaedics 2008;5(1)e6





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