2 CHO ko cell line
CHO细胞无血清培养基技术手册
(1)无血清培养基，为一般意义上无血清培养基，用各类可替代血清功能的生物材料配制细胞培养基，如牛血清白蛋白(BSA)、转铁蛋白、胰岛素等生物大分子物质，以及从血清中提取的去除蛋白质的混合脂类以及水解蛋白等。其特点是培养基中的蛋白含量较高,添加物质的化学成分不明确，其中含有大量的动物来源蛋白。
5 CHO细胞无血清无动物组分培养基（SAF-CHO-G-004）
其实个性化培养基并不新鲜，在国外生物制药企业普遍采用个性化培养基，世界著名的生物制药公司（如Amgen、Genetech）往往和培养基企业合作，通过细胞培养基的客户定制方式，研制最适合自身细胞特点的个性化细胞培养基应用于生产实践，用于提高细胞产率和生产效率。另有数据显示，国际上的细胞培养基生产企业，个性化、定制培养基占其培养基业务的90%以上，目录培养基不足10%。
ATCC提供的CHO/dhfr-细胞照片
2 CHO细胞表达系统的优势与特点
由于哺乳动物细胞结构、功能和基因表达调控的复杂性，外源基因在哺乳动物细胞中的表达与在原核生物中的表达存在较大差异，因而外源基因在哺乳动物细胞中高效表达所需的元件也不同于在原核生物细胞中的表达所需的元件。外源基因在哺乳动物细胞中的表达包括基因的转录、mRNA翻译及翻译后蛋白质的加工等过程，如蛋白质的糖基化、磷酸化、寡聚体的形成以及蛋白质分子内或分子间二硫键的形成等比较复杂，要表达具有生物学功能的蛋白质如膜蛋白、抗体和具有特异性催化功能的酶需要在哺乳动物细胞中进行。
其它
无水葡萄糖、HEPES、丙酮酸钠、亚油酸、硫辛酸、胰岛素、亚硒酸钠、植物蛋白等
2008-Cytoprotective effect of fucoxanthin isolated from brown=FX=细胞保护
ORIGINAL PAPERCytoprotective effect of fucoxanthin isolated from brown algae Sargassum siliquastrum against H 2O 2-induced cell damageSoo-Jin Heo ÆSeok-Chun Ko ÆSung-Myung Kang ÆHahk-Soo Kang ÆJong-Pyung Kim ÆSoo-Hyun Kim ÆKi-Wan Lee ÆMan-Gi Cho ÆYou-Jin JeonReceived:26March 2008/Revised:9June 2008/Accepted:23June 2008/Published online:22July 2008ÓSpringer-Verlag 2008Abstract In this study,the cytoprotective effect of fuco-xanthin,which was isolated from Sargassum siliquastrum ,against oxidative stress induced DNA damage was investi-gated.Fucoxanthin,a kind of carotenoid,was pretreated to the medium and the protective effect was evaluated via 20,70-dichlorodihydrofluorescein diacetate,3-(4,5-dimeth-ylthiazol-2-yl)2,5-diphenyltetrazolium bromide,and comet assays.Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H 2O 2),but the production was significantly reduced by the treatment with fucoxanthin.The fucoxanthin strongly enhanced cell via-bility against H 2O 2induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner.Furthermore,a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye.These results clearlyindicate that fucoxanthin isolated from S.siliquastrum possesses prominent antioxidant activity against H 2O 2-mediated cell damage and which might be a potential ther-apeutic agent for treating or preventing several diseases implicated with oxidative stress.Keywords Fucoxanthin ÁSargassum siliquastrum ÁCell damage ÁOxidative stress ÁReactive oxygen speciesIntroductionReactive oxygen species (ROS),which include free radi-cals such as superoxide anion radical (O 2-),hydroxyl radicals (ÁOH)and non free-radical species such as H 2O 2and singlet oxygen (1O 2),formed during normal metabolic processes,which can easily initiate the peroxidation of membrane lipids,leading to the accumulation of lipid peroxides.Free radical scavengers and antioxidants can reduce lipid peroxidation and the generation of ROS.The importance of antioxidants in human health has become increasingly clear due to spectacular advances in under-standing the mechanisms of their reaction with oxidants.Furthermore,interest in employing antioxidants from nat-ural sources to increase the shelf-life of foods is considerably enhanced by consumer preference for natural ingredients and concerns about the toxic effects of syn-thetic antioxidants [1–3].Algae,as photosynthetic organisms,are exposed to a combination of light and high oxygen concentrations what induces the formation of free radicals and other oxidative reagents.The absence of structural damage in the algae leads to consider that these organisms are able to generate the necessary compounds to protect themselves against oxidation [4].In this respect,algae can be considered as anS.-J.Heo ÁS.-C.Ko ÁS.-M.Kang ÁK.-W.Lee ÁY.-J.Jeon Faculty of Applied Marine Science,Cheju National University,Jeju 690-756,South Koreae-mail:skysjheaven@H.-S.Kang ÁJ.-P.KimKRIBB,52Eoeun-dong,Yuseong,Daejeon 305-806,South KoreaS.-H.KimDepartment of Food Bioengineering,Cheju National University,Jeju 690-756,South KoreaM.-G.ChoFood and Biotechnology,Dongseo University,Pusan 617-716,South KoreaY.-J.Jeon (&)Marine and Environmental Research Institute,Cheju National University,Jeju 695-814,South Korea e-mail:youjinj@cheju.ac.krEur Food Res Technol (2008)228:145–151DOI 10.1007/s00217-008-0918-7important source of antioxidant compounds that could be suitable for protecting our bodies against the ROS formed e.g.by our metabolism or induced by external factors(as pollution,stress,UV radiation,etc.).Fucoxanthin is found in edible brown algae and,along with b-carotene,is one of the most abundant carotenoids found in nature[5].There have recently been several reports that fucoxanthin showed biological activities such as radical scavenging,antiproliferative,antiobesity,anti-tumor,and antiangiogenic activities[6–10].Nonetheless, the protective effects of cell damages related to antioxidant activities of fucoxanthin have not yet been reported. Accordingly,the present study isolated the marine natural carotenoid fucoxanthin from Sargassum siliquastrum and evaluated its cytoprotective effects against H2O2-induced cell damage.Materials and methodsMaterialsThe marine alga S.siliquastrum was collected along the coast of Jeju Island,Korea,between October2005and March2006.The samples were washed three times with tap water to remove the salt,epiphytes,and sand attached to the surface,then carefully rinsed with fresh water,and maintained in a medical refrigerator at-20°C.Therefore, the frozen samples were lyophilized and homogenized with a grinder prior to extraction.General experimental proceduresOptical rotations were measured on a JASCO P-1020 polarimeter.The UV and FT-IR spectra were recorded on a Pharmacia Biotech Ultrospec3000UV/Visible spec-trometer and a SHIMAZU8400s FT-IR spectrometer, respectively.NMR spectra were recorded on a Varian INOVA400MHz NMR spectrometer.CD3OD was used as a solvent for the NMR experiments,and the solvent signals were used as an internal reference.ESI and HREI mass spectra acquired using a Finnigan Navigator30086and JMS-700MSTATION high resolution mass spectrometer system,respectively.The HPLC was carried out on a Waters HPLC system equipped with a Waters996photo-diode array detector and Millenium32software using C18 column(J’sphere ODS-H80,150mm920mm,4l m, YMC Co.).Extraction and isolationThe powdered S.siliquastrum(400g)was extracted with 80%aqueous MeOH,and was evaporated under vacuo.Then,the MeOH extract was partitioned with CHCl3.The chloroform extract(9g)was fractionated by silica column chromatography using stepwise elution with CHCl3–MeOH mixture(100:1?1:1)to afford separated active fractions.A combined active fraction was further subjected to a Sephadex LH-20column saturated with100%MeOH, and thenfinally purified by reversed-phase HPLC to give fucoxanthin(74.9mg).1H and13C NMR data were assigned in Table1and the structure was illustrated in Fig.1.Cell cultureCells of a monkey kidneyfibroblast line(Vero)were maintained at37°C in an incubator with humidified atmosphere of5%CO2.Cells were cultured in Dulbecco’s modified Eagle’s medium containing10%heat-inactivated fetal calf serum,streptomycin(100l g/mL),and penicillin (100U/mL).Intracellular ROS measurementFor detection of intracellular ROS,Vero cells were seeded in96-well plates at a concentration of19105cells/mL. After16h,the cells were treated with various concentra-tions of fucoxanthin,and incubated at37°C under a humidified atmosphere.After30min,H2O2was added at a concentration of1mM,and then cells were incubated for additional30min at37°C.Finally,20,70-dichlorodihy-drofluorescein diacetate(DCFH-DA;5l g/mL)was introduced to the cells,and20,70-dichlorofluorescein(DCF)fluorescence was detected at an excitation wavelength of 485nm and an emission wavelength of535nm,using a Perkin-Elmer LS-5B spectrofluorometer.The percentage of intracellular ROS scavenging activity was calculated in accordance with the following equation:Intracellular ROS scavenging activity%ðÞ¼1ÀC1=C0ðÞ½ Â100where C1is thefluorescence intensity of cells treated with H2O2and fucoxanthin,and C0is thefluorescence intensity of cells treated with H2O2and distilled water instead of fucoxanthin.Assessment of cell viabilityCell viability was then estimated via an3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide(MTT) assay,which is a test of metabolic competence predicated upon the assessment of mitochondrial performance.It is colorimetric assay,which is dependent on the conversion of yellow tetrazolium bromide to its purple formazan derivative by mitochondrial succinate dehydrogenase inviable cells[11].The cells were seeded in96-well plate at a concentration of19105cells/mL.After16h,the cells were treated with fucoxanthin at difference concentrations. Then,10l L of H2O2(1mM)was added to the cell culture medium,and incubated for24h at37°C.MTT stock solution(50l L;2mg/mL)was then applied to the wells, to a total reaction volume of200l L.After4h of incu-bation,the plates were centrifuged for5min at8009g,and the supernatants were aspirated.The formazan crystals in each well were dissolved in150l L of dimethylsulfoxide (DMSO),and the absorbance was measured via ELISA at a wavelength of540nm.Relative cell viability was evalu-ated in accordance with the quantity of MTT converted to the insoluble formazan salt.The optical density of the formazan generated in the control cells was considered to represent100%viability.The data are expressed as mean percentages of the viable cells versus the respective control.Determination of DNA damage by comet assayComet assay was performed to determine the oxidative DNA damage[12].The cell suspension was mixed with 75l L of0.5%low melting agarose(LMA),and added to the slides precoated with1.0%normal melting agarose. After solidification of the agarose,slides were covered with another75l L of0.5%LMA and then immersed in lysis solution(2.5M NaCl,100mM EDTA,10mM Tris,andTable1NMR spectroscopic data for fucoxanthin in CDCl3Position13C1H(mult.J=Hz)Position13C1H(mult.J=Hz) 135.81035.2247.1 1.36(1H,dd,J=8.7,14.2)1.49(1H,dd,J=14.2)2045.4 1.41(1H,dd,J=10.4,14.9)2.00(1H,dd,J=2.9,14.9)364.3 3.80(1H,m)3068.0 5.37(1H,tt,J=8.8,12.0)441.6 1.77(1H,dd,J=8.7,14.2)2.29(1H,dd,J=2.9,17.8)4045.2 1.53(1H,dd,J=10.4,14.9)2.29(1H,dd,J=2.9,17.8)566.25072.7667.160117.5740.8 2.59,3.64(2H,d,J=20.4)70202.48170.580103.4 6.04(1H,s)9134.590132.510139.17.14(1H,d,J=12.8)100128.5 6.12(1H,d,J=11.6) 11123.4 6.58(1H,m)110125.7 6.71(1H,t,J=12.0) 12145.0 6.66(1H,t,J=12.8)120137.1 6.34(1H,d,J=11.6) 13135.4130138.114136.6 6.40(1H,d,J=11.6)140132.2 6.26(1H,d,J=11.6) 15129.4 6.67(1H,m)150132.5 6.71(1H,t,J=12.0,14.2) 1625.0 1.37(3H,s)16029.2 1.37(3H,s)1728.10.95(3H,s)17032.1 1.06(3H,s)1821.2 1.34(3H,s)18031.3 1.34(3H,s)1911.8 1.80(3H,s)19014.0 1.80(3H,s)2012.8 1.98(3H,s)20012.9 1.98(3H,s)30OAc,CH321.4 2.03(3H,s)30OAc,C=O197.9Data were obtained using400MHz for1H and100MHz for13C1%sodium lauryl sarcosine;1%Triton X-100and10% DMSO)for1h at4°C.The slides were next placed into an electrophoresis tank containing300mM NaOH and 10mM Na2EDTA(pH13.0)for40min for DNA unwinding.For electrophoresis of the DNA,an electric current of25V/300mA was applied for20min at4°C. The slides were washed three times with a neutralizing buffer(0.4M Tris,pH7.5)for5min at4°C,and then treated with ethanol for another5min before staining with 50l L of ethidium bromide(20l g/mL).Measurements were made by image analysis(Kinetic Imaging,Komet5.0, UK)andfluorescence microscope(LEICA DMLB,Ger-many),determining the percentage offluorescence in the tail(tail intensity;50cells from each of two replicate slides).Nuclear staining with Hoechst33342The nuclear morphology of the cells was evaluated using the cell-permeable DNA dye,Hoechst33342.Cells with homogeneously stained nuclei were considered viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis[13,14].The cells were placed in24-well plates at a concentration of 19105cells/mL.Sixteen hours after plating,the cells were treated with various concentration of fucoxanthin,and further incubated for1h prior to expose to H2O2(1mM). After24h,1.5l L of Hoechst33342(stock10mg/mL),a DNA-specificfluorescent dye,were added to each well, followed by10min of incubation at37°C.The stained cells were then observed under afluorescence microscope equipped with a CoolSNAP-Pro color digital camera,in order to examine the degree of nuclear condensation.Statistical analysisThe data are expressed as the mean±standard error.A statistical comparison was performed via the SPSS package for Windows(Version10).p-Values of\0.05were con-sidered to be significant.Results and discussionCells are protected from ROS-induced damage by a variety of endogenous ROS-scavenging enzymes,chemical com-pounds,and natural products.Recently,interest has been increased in the therapeutic potential of natural products to be used as natural antioxidants in the reduction of such freeradical-induced tissue injuries,thereby suggesting that many natural products may prove to possess therapeuti-cally useful antioxidant compounds [15–17].H 2O 2has been extensively used as an inducer of oxi-dative stress in in vitro models.It readily crosses the cellular membranes giving rise to the highly reactive hydroxyl radical,which has the ability to react with mac-romolecules,including DNA,proteins,and lipids,and ultimately damage to whole cell [18,19].Several studies have shown that oxidative stress is a major cause of cellular injuries in a variety of human diseases including cancer,neurodegenerative,and cardiovascular disorders [20–22].Recently,many researchers have made considerable efforts to search natural antioxidants.Many studies have revealed that seaweeds have potential to be used as a candidate for natural antioxidant.Potent antioxidative compounds have already been isolated from seaweeds and identified as phylopheophytin in Eisenia bicyclis (arame),fucoxanthine in Hijikia fusiformis (hijiki),phlorotannin in Ecklonia stolonifera ,bromophenols in Polysiphonia urceolata ,and sulfated polysaccharides [6,23–27].Among these antiox-idants,fucoxanthine,a kind of carotenoid,showed radical scavenging activity.In this study,we investigated the antioxidant effects of natural carotenoid fucoxanthin,after the administration of H 2O 2in cell lines.20,70-Dichlorodihydrofluorescein diacetate was used as a probe for ROS measurement.DCFH-DA crosses cell membranes and is hydrolyzed enzymatically by intracel-lular esterase to nonfluorescent DCFH.In the presence of ROS,DCFH is oxidized to highly fluorescent DCF.It is well known that H 2O 2is the principal ROS responsible for the oxidation of DCFH to DCF [28].In the presentstudy,Fig.5Photomicrographs of DNA damage and migration observed under fucoxanthin isolated from S.siliquastrum where the tail moments were decreased.a control,b cells treated with 50l M H 2O 2,c cells treated with 5l M compound +50l M H 2O 2,d cells treated with 50l M compound +50l M H 2O 2,e cells treated with 200l M compound +50l M H 2O 2we investigated scavenging activity of fucoxanthin on intracellular ROS and the results are illustrated in Fig.2.Fluorescence intensity of control cells was recorded as 3398while H 2O 2treated cells showed 7452.However,the addition of fucoxanthin to the cells mixed with H 2O 2reduced intracellular ROS accumulation to 6079,4867,and 3149at 5,50,and 100l M,respectively.Moreover,the scavenging activity of fucoxanthin on intracellular ROS was dose-dependently increased as 18.4,34.7,and 57.7%at 5,50,and 100l M,respectively.Hence these results indicate that fucoxanthin has efficient antioxidant proper-ties which can be developed into a potential bio-molecular candidate to inhibit ROS formation of cellular.To evaluate whether fucoxanthin protect from cellular damage induced by H 2O 2,cells were pretreated with fuco-xanthin for 24h in the absence or presence of oxidative stress.After then,cell viability was measured via MTT assay.As shown in Fig.3,H 2O 2treatment without fuco-xanthin decreased cell viability to 43.7%,while fucoxanthin prevented cells from H 2O 2-induced damage,restoring cell survival to 63.6,69.4,78.5,and 89.2%at the concentrations of 5,50,100,and 200l M,respectively.Active mitochon-dria of living cells can cleave MTT to produce formazan,the amount of which is directly correlated to the living cell number [29].The results of MTT assay showed that for-mazan content was reduced due to H 2O 2treatment,however,significantly increased with addition of fucoxan-thin.These results suggest that fucoxanthin has ability to protect vero cells from oxidative stress-related cellular injuries.The protective effect of fucoxanthin on DNA damages was also confirmed by comet assay and was presented as tail DNA percent.This assay can reflect different types of DNA damage,such as DNA single strand breakage,or incomplete DNA repairing and shows high sensitivity in detecting oxidative stress [30].As shown in Fig.4,H 2O 2treatment increased the tail moments in the cells versus the control cells.However,the increased tail moments caused by H 2O 2induced oxidative DNA damage were decreased in the cells treated with fucoxanthin.The inhibitioneffectFig.6Protective effect of fucoxanthin isolated from S.siliquastrum against H 2O 2-induced apoptosis in vero cells.Cellular morphological changes were observed under afluorescence microscope after Hoechst 33342staining.a untreated,b 500l M H 2O 2,c 5l M compound +500l M H 2O 2,d 50l Mcompound +500l M H 2O 2,e 200l Mcompound +500l M H 2O 2.Apoptotic bodies are indicated by arrowsof fucoxanthin on DNA damage was38.8,52.5,and63.4% at the concentrations of5,50,and200l M,respectively. The DNA migration profiles of tested cells when treated with different concentrations of fucoxanthin are presented in Fig.5.The DNA was completely damaged and the amount of tail DNA was significantly increased in the group treated with only H2O2(Fig.5b),compared to the control(Fig.5a).However,the amount of tail DNA was increasingly decreased with increased concentrations of fucoxanthin(Fig.5c–e).In a previous study,Kang et al.[23]reported that some natural compounds from brown seaweeds have an ability to increase catalase located at peroxisome in cell that converts H2O2into molecular oxygen and water.In this study,we showed that fuco-xanthin has prominent effect on the H2O2-induced DNA damage.The inhibition activities on the DNA damage and the decrease of the amounts of tail DNA might be related to the ability of fucoxanthin to increase catalase.The protective effect of fucoxanthin on apoptosis induced by H2O2was investigated by nuclear staining with Hoechst33342.Hoechst33342dye specifically stains DNA,and is widely used to detect the nuclear shrinkage such as chromatin condensation,nuclear fragmentation, and the appearance of apoptotic bodies,which are all indications of apoptosis[31].The microscopic photograph in Fig.6,the negative control,which contained no com-pound or H2O2,possessed intact nuclei(Fig.6a),and the positive control(H2O2treated cells)exhibited significant nuclear fragmentation,indicating apoptosis(Fig.6b). However,the addition of fucoxanthin with H2O2reduced the apoptotic bodies(Fig.6c–e),which suggested the ability of fucoxanthin to protect against nuclear fragmen-tation amidst oxidative stress.In conclusion,the results obtained in the present study show that fucoxanthin isolated from S.siliquastrum can effectively inhibit intracellular ROS formation,DNA damage,and apoptosis induced by H2O2.Moreover, fucoxanthin showed evidence in cell survival rate.These results revealed that fucoxanthin can be used not only as the easily accessible source of natural antioxidants but also as an ingredient of the functional food and cosmetic agents related to the prevention and control oxidative stress. Acknowledgements This research was supported by a grant(M-2007-03)from the Marine Bioprocess Research Center of the Marine Bio21Center,funded by Ministry of Marine Affairs and Fisheries, Republic of Korea.References1.Schwarz K,Bertelsen G,Nissen LR,Gardner PT,Heinonen MI,Hopia A,Huynh-Ba T,Lambelet P,McPhail D,Skibsted LH, Tijburg L(2001)Eur Food Res Technol212:319–3282.Farag RS,El-Baroty GS,Basuny AM(2003)Int J Food SciTechnol38:81–873.Heo SJ,Park EJ,Lee KW,Jeon YJ(2005)Bioresour Technol96:1613–16234.Jimenez-Escrig A,Jimenez-Jimenez I,Pulido R,Saura-Calixto F(2001)J Sci Food Agric81:530–5345.Berhard K,Moss GP,Toth GY,Weedon BLC(1976)Tetrahe-dron Lett17:115–1186.Yan X,Chuda Y,Suzuki M,Nagata T(1999)Biosci BiotechnolBiochem63:605–6077.Hosokawa M,Kudo M,Maeda H,Kohno H,Tanaka T,MiyashitaK(2004)Biochim Biophys Acta1675:113–1198.Maeda H,Hosokawa M,Sashima T,Funayama K,Miyashita K(2005)Biochem Biophys Res Commun332:392–3979.Kotake-Nara E,Asai A,Nagao A(2005)Cancer Lett220:75–8410.Sugawara T,Matsubara K,Akagi R,Mori M,Hirata T(2006)JAgric Food Chem54:9805–981011.Mosmann T(1983)J Immunol Methods65:55–6312.Singh NP(2000)Mutat Res455:111–12713.Gschwind M,Huber G(1995)J Neurochem65:292–30014.Lizard G,Fournel S,Genestier L,Dhedin N,Chaput C,FlacherM,Mutin M,Panaye G,Revillard JP(1995)Cytometry21:275–28315.Lee SE,Ju EM,Kim JH(2002)Exp Mol Med34:100–10616.Jung WK,Rajapakse N,Kim SK(2005)Eur Food Res Technol220:535–53917.Ahn GN,Kim KN,Cha SH,Song CB,Lee J,Heo MS,Yeo IK,Lee NH,Jee YH,Kim JS,Heo MS,Jeon YJ(2007)Eur Food Res Technol226:71–7918.Satoh T,Sakai N,Enokido Y,Uchiyama Y,Hatanaka H(1996)JBiochem120:540–54619.Halliwell B,Clement MV,Long LH(2000)FEBS Lett486:10–1320.Al-Enezi KS,Alkhalaf M,Benov LT(2006)Free Radic Biol Med40:1144–115121.Zhang L,Yu H,Sun Y,Lin X,Chen B,Tan C,Cao G,Wang Z(2007)Eur J Pharmacol564:18–2522.Strazzullo P,Puig JG(2007)Nutr Metab Cardiovasc Dis17:409–41423.Kang HS,Chung HY,Jung HA,Son BW,Choi JS(2003)ChemPharm Bull51:1012–101424.Cahyana AH,Shuto Y,Kinoshita Y(1992)Biosci BiotechnolBiochem56:1533–153525.Li K,Li XM,Ji NY,Wang BG(2007)Bioorg Med Chem15:6627–663126.Zhang Q,Li N,Liu W,Zhao Z,Li Z,Xu Z(2004)Carbohydr Res339:105–11127.Kim SH,Choi DS,Athucorala Y,Jeon YJ,Senevirathne M,RhaCK(2007)Int J Food Sci Nutr12:65–7328.LeBel CP,Ischiopoulos H,Bondy SC(1992)Chem Res Toxicol5:227–23129.Hu Z,Guan W,Wang W,Huang L,Xing H,Zhu Z(2007)CellBiol Int31:798–80430.Senthilmohan ST,Zhang J,Stanley RA(2003)Nutr Res23:1199–121031.Kerr JF,Gobe GC,Winterford CM,Harmon BV(1995)MethodsCell Biol46:1–27。
中英文造纸专业词汇
Gu3ha ng2dia n4do ng4ji1带有)鼓行电动机
Troubleshoot ing
Gu4zha ng4pai2chu2故障排除
Channel doctor
Gua1dao1cao2刮刀槽
Doctor blade
Gua1dao1pia n4刮刀片
Doctor
Gua1dao1刮刀
Bia n4j in g4变径
Shifting fork
Bia n4su4cha1变速叉
Surface
Biao3mia n4表面
Peel off (sollevare, lift)
Bo1li2 (vetroe bo1li)
Lift-off hose
Bo1li2rua n3gua n3剥离软管
Glass fiber
Dao3gu n3导辊
Lead-i n roll
Dao3ru4gu n3导入辊
Collapse
Dao3ta1倒塌
Plasma device
Den g3li2zi qielgeliil等离子切割机
Baseme nt (floor)
dilcha n3低产
Zone
Di4dai4地带
An chor bolt
Scraper
Gua1shui3ba n3刮水板
Pipe
Gua n3dao4管道
Pipi ng
gua n3lu4管路
Grout ing
gua n4jia ng1灌浆
Tank
Guan4zi
Finish (fin itura)
Gua ng1jie2du4光洁度
Fiber optics
Gua ng1xia n1光纤
(完整版)基于CHO细胞的单抗生产2
细胞库的相关法规要求
细胞库的检定
细胞库检定-CHO细胞
检测项目
细胞鉴别 细菌、真菌检查
MCP
+ +
WCP
+ +
分歧杆菌检查
+
+
支原体检查
(+)
(+)
细胞形态观察及血吸附试验
+
+
内 体外不同细胞接种培养法
+
+
、 动物和鸡胚体内接种法
+
-
源 病
逆转录病毒检查
+
-
毒 种属特异性病毒检查（鼠源）
(+)
荧光强度值能反映细胞
目标的相对表达水平
筛选方法
➢ ClonePix
Semi solid media
筛选方法
➢ Limiting dilution
有限稀释法(limiting dilution cloning, LDC)是一种通过梯度稀释以获得单 克隆的方法。 它的应用范围广, 对于大多数细胞类型, 如杂交瘤细胞、 CHO细胞及干细胞等都有较好的克隆分离效果。 LDC的操作过程大致 分为接种(铺板)、 筛选和扩增。 接种的细胞液经过逐级稀释后加到96 孔板中, 每个孔所含的细胞个数理论上不大于1
载体的一般性构成
1、IRES：即内部核糖体进入位点，是一段核酸序列，它的存在能够使蛋白质翻
重组人促红素β注射液(CHO细胞)
重组人促红素β注射液(CHO细胞)【药品名称】通用名称：重组人促红素β注射液(CHO细胞)英文名称：Recombinant Human Erythropoietin β Injection (CHO Cell)【成份】主要组成成份:重组人红细胞生成素-β辅料包括：尿素，氯化钠，聚山梨醇酯-20，二水合磷酸二氢钠，十二水合磷酸氢二钠，二水合氯化钙，甘氨酸，左旋亮氨酸，左旋异亮氨酸，左旋苏氨酸，左旋谷氨酸，左旋苯丙氨酸，注射用水。
【适应症】本品适用于因慢性肾衰竭引致贫血，包括行血液透析、腹膜透析和非透析治疗者。
治疗接受化疗的非髓性恶性肿瘤成人患者的症状性贫血。
本品用于治疗贫血时，仅在出现贫血症状时方可使用。
【用法用量】应由在上述适应症领域中有丰富经验的医生指导下进行罗可曼治疗。
由于在个别的病例中观察到过敏样反应，建议在医学监护下进行首次给药。
应注意本品只有在溶液澄清或呈轻微乳状，无色且几乎无可见颗粒时方可用于注射。
欲重拾注射器中的药品是无菌的，但未作防腐处理。
在任何情况下，每支注射器都不可以多次注射。
治疗成人和儿童慢性肾功能衰竭患者的症状性贫血。
患者贫血的症状和后遗症随着年龄，性别和疾病总负荷的不同而有所不同；医生有必要对个体患者的临床病程和状态进行评价。
可以通过皮下或静脉注射罗可曼来升高血红蛋白的水平，使其不超过12g/dl(7.5mmol/l)。
对于未进行血液透析的患者，首选皮下注射给药以避免刺穿外周静脉。
如果静脉注射给药，应在约2分钟内完成，例如，对于血液透析患者在透析结束时经动静脉瘘管注入。
因为病人的个体差异，临床上可以观察到个别患者的血红蛋白值偶尔超过或低于预期的血红蛋白水平。
考虑到血红蛋白靶浓度在10g/dl （6.2mmol/l）到12g/dl（7.5mmol/l）之间，可以通过剂量管理达到不同的血红蛋白水平。
应该避免出现持续的血红蛋白水平超过12g/dl(7.5mmol/l)；当血红蛋白值超过12g/dl(7.5mmol/l)时，可根据以下指导原则调整剂量：应当避免出现用药4周后血红蛋白水平升高幅度超过2g/dl(1.25mmol/l)。
无血清培养CHO细胞的研究进展
Science &Technology Vision 科技视界随着生物技术的飞速发展,单克隆抗体、细胞因子、疫苗和各种生物活性蛋白等生物制品在诊断和治疗中应用日益广泛,其需求量大大增加,因此,通过体外大规模培养动物细胞生产生物制品技术的研究越来越受到青睐。
CHO 细胞是生产蛋白类生物制品最重要的表达系统,采用无血清培养CHO 细胞相比含血清培养,能避免对实验动物的伤害、潜在的污染源影响、不同批次血清间的生物活性和因子的不一致及价格高等诸多弊端,因而受到生物制药领域的广泛关注。
1CHO 细胞CHO 细胞即中国仓鼠卵巢细胞(Chinese Hamster Ovary Cell),是最具代表性的动物细胞表达系统,被广泛应用于生物制药领域。
它建株于1957年,由美国科罗拉多大学Theodore T.Puck 从一成年雌性中国仓鼠卵巢中分离得到,是一种连续细胞系,能传百代以上,具有永生性。
后来陆续又有很多科学家用药物加压、极端条件筛选和诱导突变等方法筛选得到了一系列不同表型的CHO 细胞株[1],如DUKX-B11、DG-44和CHO-K1等。
CHO 细胞能用于表达各种复杂的重组蛋白。
据统计,70%以上的动物细胞表达药物产品都是以CHO 细胞为表达宿主[2]。
2011年6月批准的27种单克隆抗体中,其中13种就是通过CHO 细胞表达的,占了约50%的比例。
CHO 细胞之所以成为最受欢迎的宿主细胞,主要在于其易于培养以及基因操作,而且相比其他表达系统,它还具有许多优点[3-4]:①具有准确的转录后修饰功能,表达的糖基化药物蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分子;②具有产物胞外分泌功能,便于下游产物分离纯化;③具有外源重组基因的高效扩增和表达能力;④既可贴壁生长也可以进行悬浮培养,且有较高的耐受剪切力和渗透压能力,外源蛋白表达水平较高;⑤CHO 细胞属于成纤维细胞,很少分泌自身的内源蛋白,利于外源蛋白的后分离。
康天晟合生物技术有限公司QuaCell
Version 3.0Revision date: Aug.09,2019产品说明书简介QuaCell ®CHO CD02培养基可为中国仓鼠卵巢细胞(CHO 细胞)的高密度增殖提供丰富的营养成分。
此产品是一款无血清、无动物成分、化学限定培养基，适用于悬浮培养CHO 细胞以表达抗体及蛋白产物。
只需简单的驯化甚至不用驯化，就可从含血清培养基或其他无血清培养基中，直接传代接种至QuaCell ® CHO CD02培养基中。
此培养基配方中不含次黄嘌呤、胸苷及L-谷氨酰胺，适合二氢叶酸还原酶、谷氨酰胺合成酶（GS）筛选系统。
组分L-谷氨酰胺 不含，用前按需添加 葡萄糖 6.0 g/L 次黄嘌呤&胸苷 不含 酚红 不含 碳酸氢钠 1.90g/L 水解产物不含产品用途在处理或补充培养基时使用无菌技术。
本产品用于研究或进一步制造使用。
警告：不用于人类或动物治疗用途。
超出规定范围的使用可能会触犯当地法律。
安全信息阅读物料安全数据表（MSDS）并依据相关的安全操作规范，佩戴适当的护目镜，洁净服，口罩和手套等。
用前准备• QuaCell ®CHO CD02培养基的使用需要无菌• 产品不含L-谷氨酰胺；用于正常表达GS 基因的宿主细胞的增殖培养时，建议在使用前添加4mM L-谷氨酰胺；根据细胞株GS 基因编辑情况，可按需调节L-谷氨酰胺浓度。
• 不推荐使用抗生素。
• 开封后未用完的培养基应进行分装，使用封口膜封口，在2～8℃避光保存。
产品外包装副标提供一组印有产品信息的不干胶便签，可撕下使用，便于分装标记及实验记录。
培养条件培养基：完全的QuaCell ®CHO CD02 细胞系：CHO cells 培养类型：悬浮培养容器：摇瓶/TPP/反应器 温度范围：37℃±0.5培养箱气体要求：5～8% CO 2的加湿培养注意：确保适当的气体交换和最小化的曝光培养。
细胞复苏1. 在37℃水中快速解冻（<2分钟）冻存管中的细胞液；2. 将细胞液转移至15 ml 离心管中，加入10ml 预热的QuaCell ®CHO CD02培养基，1000rpm 离心3分钟，丢弃上清液，使用5 ml QuaCell ®CHO CD02培养基重悬，计数；3. 将冷冻管的全部细胞液转移到装有15 ml 预热的完全QuaCell ®CHO CD02培养基的125 ml 摇瓶中，稀释至所需细胞密度；4. 在含有5～8% CO 2，37℃，加湿的培养箱或摇床进行培养，培养时拧松瓶盖或使用通气盖以进行气体交换； 5. 细胞复苏后培养2～5天处于对数生长中期时传代。
基于CHO细胞的单抗生产PPT
单克隆拍照系统
FDA目前对于单克隆证明方法意见
克隆性
FISH不但可用于已知基因或序列的染色体定位，而且也可用于未克隆基因或遗传 标记及染色体畸变的研究。在基因定性、定量、整合、表达等方面的研究中颇 具优势。
筛选检定
细胞代次的规定
细胞库的相关法规要求
细胞库的相关法规要求
➢ ClonePix /Cell Xpress TM 克隆筛选及细胞表达系统
➢ Limiting dilution 有限稀释法
筛选方法
➢ Fluorescence-Activated Cell Sorting (FACS)-Based Screening
The cold capture method used in FACS where fluorescently labeled antibodies bind to secreted
载体的一般性构成
1、IRES：即内部核糖体进入位点，是一段核酸序列，它的存在能够使蛋白质翻
译起始不依赖于5‘帽结构，从而使直接从信使RNA（mRNA）中间起始翻译成 为可能。通常来讲，真核生物翻译只能从mRNA的5‘端开始，因为翻译起始必 须依赖于5’端的帽子结构。
2、Promoters：即启动子，控制基因表达（转录）的起始时间和表达的程度，启 动子本身并不控制基因活动，而是通过与称为转录因子（transcription factor）的这种蛋白质（proteins）结合而控制基因活动的 抗体序列需使用强启动子如SV40启动子等 而抗性基因需要使用削弱的启动子
基于CHO细胞的单抗生产（第一版）
汪洋 2018/02
目录
• CHO细胞的构建 • CHO细胞的表达 • CHO细胞表达后的产物纯化
CHO-K1 (仓鼠卵巢细胞亚株) 说明书
碧云天生物技术/Beyotime Biotechnology订货热线：400-168-3301或800-8283301订货e-mail：******************技术咨询：*****************网址：碧云天网站微信公众号CHO-K1 (仓鼠卵巢细胞亚株)产品编号产品名称包装C7750 CHO-K1 (仓鼠卵巢细胞亚株) 1支/瓶产品简介：Organism Tissue Morphology Culture Properties Cricetulus griseus(Hamster, Chinese)Ovary Epithelial Adherent本细胞株详细信息如下:General InformationCell Line Name CHO-K1 (Hamster Ovary Cell Substrain)Synonyms CHO K1; CHOK1; CHO cell clone K1; GM15452Organism Cricetulus griseus (Hamster, Chinese)Tissue OvaryCell Type －Morphology EpithelialDisease －Strain －Biosafety Level* 1Age at Sampling AdultGender FemaleGenetics －Ethnicity －Applications This cell line is suitable as a transfection hostCategory Spontaneously immortalized cell line* Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.CharacteristicsKaryotype Chromosome Frequency Distribution 50 Cells: 2n = 22. Stemline number is hypodiploid.Virus Susceptibility Vesicular stomatitis, Orsay (Indiana) Vesicular stomatitis, Glasgow (Indiana) Getah virusDerivation The CHO-K1 cell line was derived as a subclone from the parental CHO cell line initiated from a biopsy of an ovary of an adult Chinese hamster by T. T. Puck in 1957.Clinical Data Female Antigen Expression －Receptor Expression －Oncogene －Genes Expressed －Gene expression databases GEO: GSM1968555; GSM1968563; GSM1968571; GSM1968572; GSM1968569; GSM1968570; GSM1968594; GSM1968596; GSM1968598; GSM1968589; GSM1968600; GSM1968599; GSM1968591; GSM1968593; GSM1968595; GSM1968597; GSM1968565; GSM1968566; GSM1968560; GSM1968559; GSM1968558Metastasis －Tumorigenic －Effects －Comments The cells require proline in the medium for growth.2 / 5 C7750 CHO-K1 (仓鼠卵巢细胞亚株)400-1683301/800-8283301 碧云天/BeyotimeCulture Method Doubling Time~24 hrs Methods for Passages Wash by PBS once then 0.05% trypsin-EDTA solution and incubate at room temperature (or at 37ºC), observe cells under an inverted microscope until cell layer is dispersed (usually within 1 to 5 minutes) Medium RPMI-1640+10% FBS Special Remarks － Medium Renewal Once or twice between subculture Subcultivation Ratio 1:4 to 1:8 Growth Condition 95% air+ 5% CO 2, 37ºC Freeze medium DMEM (high glucose)+20% FBS+10% DMSO ，也可以订购碧云天的细胞冻存液(C0210)。
GS系统简介
一、GS系统的原理 二、GS系统的优势 三、GS系统的应用 四、GS研究评估协议
GS系统是什么
• GS系统是瑞士的LONZA公司开发出来的一种细胞基因表 达系统。 LONZA（龙沙） 瑞士的制药化工巨头，龙沙集团是 一家以生命科学为主导，在生物化学、精细化工、功 能化学等行业均处于领先地位的全球性跨国公司，具 有一百多年历史，总部位于瑞士巴塞尔。
GS系统的原理
•即： 用含单抗蛋白等的目的基因和GS基因标记的
表达载体，转染宿主细胞（如CHO），再通过 MSX等化合物选择加压，促使GS基因和目的蛋白 基因扩增，筛选获得重组细胞株。可在不含有Lglu的培养基中生长。
GS系统的优势
•产量高 •速度快 •稳定性好 •可比性高
产量高
•蛋白质产品的制造成本高,特别是对于治疗性蛋白 质,意味着如果生产流程已经建立起来的话，体积 最大化时的生产率至关重要。使用GS系统™可靠 结果的高产细胞系细胞早期建设项目。最大表达 水平的实现取决于产品，但产量超过5 g / L的重组 抗体的细胞系已经创建,而某些特定的蛋白，其生 产率在15 - 65 pg /细胞/天。
GS系统的原理
在不含有谷氨酰胺的培养基中，谷氨酰胺酶在哺 乳动物细胞的生长中扮演着非常重要的角色： • 一些哺乳动物细胞系,如小鼠骨髓瘤细胞, 在 没有添加谷氨酰胺的培养基中，无法表达足够的 谷氨酰胺来维持生存。在这些细胞中,转染GS基因 可以在无谷氨酰胺培养基中作为选择标记来获得 增殖。 • 其他细胞系,如中国仓鼠卵巢(CHO)细胞,表达 足够的GS离不开外生谷氨酰胺。在这些情况下,GS 抑制剂,L-氨基亚砜蛋氨酸（Methionine Sulphoximine MSX）,可用于抑制内源性GS活性,只 有转染了额外GS基因可以生存。
第二章细胞生物学研究方法
第二章细胞生物学研究方法第四节细胞培养与细胞工程一、细胞培养高等生物是由多细胞构成的整体，在整体条件下要研究单个细胞或某一群细胞在体内（in vivo）的功能活动是十分困难的。
但是如果把活细胞拿到体外（in vitro）培养进行观察和研究，则要方便得多。
活细胞离体后要在一定的生理条件下才能存活和进行生理活动，特别是高等动植物细胞要求的生存条件极其严格，稍有不适就要死亡。
所以细胞培养技术（cell culture）就是选用最佳生存条件对活细胞进行培养和研究的技术。
动物细胞的生存环境与植物细胞差别很大，因而二者的培养方法很不相同。
（一）动物细胞培养细胞培养方式大致可分为两种（图2-25）：一种是群体培养（mass culture），将含有一定数量细胞的悬液置于培养瓶中，让细胞贴壁生长，汇合（confluence）后形成均匀的单细胞层；另一种是克隆培养（clonal culture），将高度稀释的游离细胞悬液加入培养瓶中，各个细胞贴壁后，彼此距离较远，经过生长增殖每一个细胞形成一个细胞集落，称为克隆（clone）。
一个细胞克隆中的所有细胞均来源于同一个祖先细胞。
此外，为了制取细胞产品而设计了转鼓培养法，使用大容量的圆培养瓶，在培养过程中不断地转动，使培养的细胞始终处于悬浮状态之中而不贴壁。
图2-25 群体培养（左）和克隆培养（右）表2-3 目前实验室中常用的几种细胞系正常细胞培养的世代数有限，只有癌细胞和发生转化的细胞才能无限生长下去。
所谓转化即是指正常细胞在某种因子的作用下发生突变而具有癌性的细胞。
目前世界上许多实验室所广泛传用的HeLa细胞系就是1951年从一位名叫Henrietta Lacks的妇女身上取下的宫颈癌细胞培养而成。
此细胞系一直延用至今。
1. 原代培养（primary culture）：从动物机体取出的进行培养的细胞群。
原代培养的细胞生长比较缓慢，而且繁殖一定的代数后（一般10代以内）停止生长，需要从更换培养基。
cho细胞培养基础知识
cho细胞培养基础知识CHO细胞（Chinese Hamster Ovary cells）是一种在生物医药领域中广泛应用的哺乳动物细胞系。
CHO细胞最早于1957年从中国仓鼠卵巢中分离出来，并在实验室中久经培养和改良。
CHO细胞系具有许多优点，如较高的细胞生长速度、易于培养和维持、对病毒感染的抵抗性等。
因此，CHO细胞系被广泛用于生物医药领域中的蛋白质表达、病毒疫苗生产和药物筛选等方面。
CHO细胞培养基是用于细胞培养的基本培养液。
它提供了细胞所需的营养物质和生长因子，同时维持细胞在体外的自由生长状态。
CHO细胞培养基的主要组成部分包括：基础培养液、补充物和 pH 调节剂。
基础培养液是细胞生长和繁殖所需的基本营养物质的来源。
CHO细胞需要一定的糖类、氨基酸、无机盐和维生素等营养物质来满足其生长和代谢的需求。
常用的CHO细胞培养基基础液包括 Dulbecco's Modified Eagle Medium（DMEM）和Iscove's Modified Dulbecco's Medium（IMDM）等。
CHO细胞培养基的补充物主要包括血清和其他添加剂。
血清是CHO细胞培养中最常用的补充物之一，它提供了细胞生长所需的营养物质和生长因子。
然而，由于血清存在着传染病的风险、批次之间的变异性较大等问题，越来越多的研究者开始使用无血清培养基来代替传统的血清培养基。
无血清培养基通常采用人血浆蛋白替代血清，或添加其他生长因子、激素和细胞因子等来维持细胞生长。
除了血清外，CHO细胞培养基中还可以加入一些其他的添加剂来提高细胞的生长和产物表达。
例如，普通培养基PI值较低，而加入polyethyleneimine（PEI）或lipofectamine（LF）等转染试剂能够提高细胞的转染效率。
此外，亲和调节因子、抗氧化剂和基因选择素等也是常用的培养基添加剂。
pH 调节剂是控制CHO细胞培养基 pH 值的重要组成部分。
重组人促红素注射液(CHO细胞)
重组人促红素注射液（CHO细胞）Chongzu Ren Cuhongsu Zhusheye (CHO Xibao)Recombinant Human Erythropoietin Injection (CHO Cell)本品系由高效表达人红细胞生成素（简称人促红素）基因的中国仓鼠卵巢(CHO)细胞，经细胞培育、分离和高度纯化后制成。
含适宜稳固剂，不含防腐剂和抗生素。
1大体要求生产和检定用设施、原料及辅料、水、器具、动物等应符合“凡例”的有关要求。
2制造工程细胞2.1.1名称及来源重组人促红素工程细胞系由带有人促红素基因的重组质粒转染的CHO-dhfr-(二氢叶酸还原酶基因缺点型细胞)细胞系。
2.1.2细胞库成立、传代及保留由原始细胞库的细胞传代，扩增后冻存于液氮中，作为主细胞库；从主细胞库的细胞传代，扩增后冻存于液氮中，作为工作细胞库。
每次传代不超过经批准的代次。
细胞系冻存于液氮中，检定合格后方可用于生产。
2.1.3主细胞库及工作细胞库细胞的检定应符合“生物制品生产用动物细胞基质制备及检定规程”规定。
2.1.3.1外源因子检查细菌和真菌、支原体、病毒检查均应为阴性。
2.1.3.2细胞辨别实验应用同工酶分析、生物化学、免疫学、细胞学和遗传标记物等任一方式进行辨别，应为典型CHO细胞。
2.1.3.3人促红素表达量应不低于原始细胞库细胞表达量。
2.1.3.4目的基因核苷酸序列检查（工作种子批可免做）目的基因核苷酸序列应与批准序列相符。
原液2.2.1细胞的苏醒与扩增从工作细胞库来源的细胞苏醒后，于含灭能小牛血清培育液中进行传代、扩增，供转瓶或细胞培育罐接种用。
小牛血清的质量应符合规定（附录ⅩⅢ D）。
2.2.2细胞培育液生产用细胞培育液应不含牛血清和任何抗生素。
2.2.3细胞培育细胞培育全进程应严格依照无菌操作。
细胞培育时刻可依照细胞生长情形而定。
2.2.4分离纯化搜集的培育液按经批准的纯化工艺进行，采纳经批准的超滤法或其他适宜方式进行浓缩，多步色谱纯化后制得高纯度的重组人促红素，即为人促红素原液，除菌过滤后保留于适宜温度，并规定其有效期。
Cayman Chemical 产品说明书
Caution: For Laboratory Use. A research reagent for research purposes onlyhuman Dopamine D2L Receptor Aequorin Cell LineProduct No.: ES-171-ALot No.: 1839203Material ProvidedCells: 2 x 1 mL frozen aliquots (ES-171-AV)Format:~2.5 x 106 cells / mL in freezing mediumProduct InformationCellular Background: CHO-K1Cell Line Development:Our proprietary bicistronic expression plasmid containing thesequence coding for the human Dopamine D2L receptor wastransfected in CHO-K1 cells stably expressing the mitochondriallytargeted Aequorin and Gαq/i(5). Geneticin-resistant clones wereobtained by limit dilution and compared for their response to areference agonist using the AequoScreen® assay.DNA Sequence: Identical to coding sequence of GenBank NM_000795.3.Corresponding Protein Sequence: Identical to GenBank NP_000786.1.Receptor expression level (B MAX): N/D.K D for the above radioligand: N/DShipping Conditions: Shipped on dry ice. Please ensure dry ice is still present in thepackage upon receipt or contact Customer Support.Storage Conditions: Store in liquid nitrogen (vapor phase)immediately upon receipt.Quality ControlThe EC50for a reference agonist was determined in an AequoScreen®assay performed on the MicroBETA®JET (PerkinElmer) instrument. A mycoplasma test was performed using MycoAlert®Mycoplasma (Lonza) detection kit. We certify that these results meet our quality release criteria.Bromocriptine (EC50): 28.24 nMStability: Cells were kept in continuous culture for at least 60 days and showed nodecrease in functional response (EC50, E max).Mycoplasma:This cell line tested negative for mycoplasma.Assay ProceduresWe have shown for many of our GPCR cell lines that freshly thawed cells respond with the same pharmacology as cultured cells. All of our products validated in this way are available as frozen ready-to-use cells in our catalogue. PerkinElmer also offers a custom service for the preparation of large quantities of frozen cryopreserved cells either from a catalogue cell line or a customer’s own cell line. This demonstrates that cells can be prepared and frozen in advance of a screening campaign simplifying assay logistics.Recommended Cell Culture Conditions (CHO-K1)∙The recommended media catalogue number and supplier reference information are listed in this Product Technical Data Sheet (last page). Media composition is specifically defined for each cell type and receptor expression selection. The use of incorrect media or component substitutions can lead to reduced cell viability, growth issues and/or altered receptor expression.∙Cells undergo major stress upon thawing, and need to adapt to their new environment which may initially affect cell adherence and growth rates. The initial recovery of the cells, and initial doubling time, will vary from laboratory to laboratory, reflecting differences in the origin of culture media and serum, and differences in methodology used within each laboratory.∙For the initial period of cell growth (i.e. until cells have reached Log-phase, typically 4-10 days), we strongly recommend removal of the antibiotics (G418, Zeocin™, Puromycin, Blasticidin, Hygromycin, Penicillin and Streptomycin) from the culture media. Immediately after thawing, cells may be more permeable to antibiotics, and a higher intracellular antibiotic concentration may result as a consequence. Antibiotics should be re-introduced when cells have recovered from the thawing stress.Growth Medium: Ham's F-12, 10% FBS, 0.4 mg/mL Geneticin (receptor expressionselection), 0.25 mg/mL Zeocin (Aequorin expression selection), 5 µg/mlPuromycin (Gαq/i(5)expression selection).Freezing Medium:Ham's F-12, 10% FBS with 10% DMSO, without selection agents. Thawing Cells:Using appropriate personal protective equipment, rapidly place the frozen aliquot in a 37°C water bath (do not submerge) and agitate until its content is thawed completely. Immediately remove from water bath, spray aliquot with 70% ethanol and wipe excess. Under aseptic conditions using a sterile pipette, transfer content to a sterile centrifuge tube containing 10 mL growth medium without antibiotics, pre-warmed at 37°C, and centrifuge (150 x g, 5 min). Discard supernatant using a sterile pipette. Resuspend cell pellet in 10 mL of pre-warmed growth medium without antibiotics by pipetting up and down to break up any clumps, and transfer to an appropriate culture flask (e.g. T-25, T-75 or T-175, see recommended seeding density below). Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2.Recommended Seeding Density: Thawing: 15 000 – 33 000 cells/cm2Log-phase: 11 000 – 15 000 cells/cm2Troubleshooting: Initial doubling time can vary between 18 and 96 hours (Average = 25 hours). If cells are still not adhering after 48 hours or grow very slowly, we recommend maintaining the cells in culture and not replacing the media before 5-6 days (cells secrete factors that can help with adherence and growth). If confluence is still <50% after 5-6 days, it is recommended that you replace the media with fresh media (without antibiotics). Do not passage the cells until they reach 80-90% confluence (Log-phase). If cells have not recovered after 10-12 days, please contact our Technical Support.Culture Protocol: Under aseptic conditions, cells are grown to 80% confluence (Log-phase) and trypsinized (0.05% trypsin / 0.5 mM EDTA in calcium and magnesium-free PBS). See recommended seeding density for Log-phase above.Banking Protocol: Cells are grown to 70-80% confluence (Log-phase). Under aseptic conditions, remove medium and rinse the flask with an appropriate volume of calcium and magnesium-free PBS (example 10 mL for T-175). Trypsinize (0.05% trypsin / 0.5 mM EDTA in calcium and magnesium-free PBS) to detach cells (example 5 mL for T-175), let stand 5-10 min at 37°C. Add fresh, room temperature growth medium (without antibiotics) to stop trypsinization and dilute EDTA (example 10 mL for T-175). Transfer cells to a sterile centrifuge tube and centrifuge (150 x g, 5 min). Discard supernatant using a sterile pipette. Resuspend cell pellet in ice-cold freezing medium by pipetting up and down to break up any clumps. Count cells and rapidly aliquot at the selected cell density (e.g. 2.5 x 106 cells/mL) in sterile polypropylene cryovials. Use appropriate material to ensure slow cooling (about -1°C/min) until -70°C. Transfer vials into a liquid nitrogen tank (vapour phase) for storage.Typical Product Data – AequoScreen® AssayFigure 1: Agonist Response in AequoScreen® assayAn agonist dose-response experiment was performed in 96-well format using 5 000 cells/well. Luminescence was measured on a MicroBeta Jet (PerkinElmer). Data from a representative experiment are shown.Figure 2: Antagonist Response in AequoScreen® assayAn antagonist dose-response experiment was performed in 96-well format using 5 000 cells/well with the reference agonist (Bromocriptine) injected at a final concentration equivalent to the EC80 (265 nM). Luminescence was measured on a MicroBeta Jet (PerkinElmer). Data from a representative experiment areshown.AequoScreen® Assay Procedure (MicroBeta® JET)Assay Buffer:DMEM / HAM’s F12 with HEPES, without phenol red (Invitrogen # 11039-021) +0.1 % protease-free BSA (from 10% solution sterilized by filtration at 0.22 µm).Store at 4°C.Coelenterazine h:To prepare a 500 µM Coelenterazine h stock solution, solubilize 250 µg ofCoelenterazine h (Promega # S2011 or Invitrogen # C6780) in 1227 µLmethanol. Store at –20°C in the dark.Digitonin: To prepare a 50 mM Digitonin stock solution, dissolve 1 g of Digitonin (Sigma #D5628) in 16.27 mL of DMSO. Aliquot and store at -20°C.Important Notes:∙Temperature should remain below 25°C during the coelenterazine loading of the cells, and until using the cells for the readings. Excessive heating by the cell stirrer for example will result in signal loss.∙Depending on (1) sensitivity of the reader used, (2) plate format used, and (3) assay characteristics wanted, it is possible to load cells at (a) different concentrations of cells and coelenterazine, (b) with different subsequent dilution factors, and (c) using different cell numbers per well. This is part of the validation work when importing an assay to a new reader.∙For tips and examples on running AequoScreen®assays on different readers, please refer to theAequoScreen® Starter Kit Manual available at /CellLines.References1. Dupriez VJ, Maes K, Le Poul E, Burgeon E, Detheux M. (2002) Aequorin-based functional assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors. Receptors Channels 8:319-302. Rizzuto R. Simpson AWM, Brini M, Pozzan T. (1992) Rapid changes of mitochondrial Ca2+ revealed byspecifically targeted recombinant aequorin. Nature 358:325-327.3. Stables J., Green A., Marshall F., Fraser N., Knight E., Sautern M., Milligan G., Lee M., Rees S. (1997) Abioluminescent assay for agonist activity at potentially any G-protein-coupled receptor. Anal. Biochem.252:115-126.4. Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR. (1993) Substitution of three amino acids switchesreceptor specificity of Gq alpha to that of Gi alpha. Nature 363:274-276.5. Gardner B, Strange PG. (1998) Agonist action at D2 (long) dopamine receptors: ligand binding andfunctional assays. Br. J. Pharmacol. 124: 978-984.6. Lin H, Saisch SG, Strange PG (2006) Assays for enhanced activity of low efficacy partial agonists at theD2 dopamine receptor. Br. J. Pharmacol. 9: 291-299.7. Martelle JL, Nader MA. (2008) review of the discovery, pharmacological characterization, and behavioraleffects of the dopamine D2-like receptor antagonist eticlopride. CNS Neurosci. Ther. 14: 248-262.8. Tadori Y, Miwa T, Tottori K, Burris KD, Stark A, Mori T, Kikuchi T. (2005) Aripiprazole's low intrinsicactivities at human dopamine D2L and D2S receptors render it a unique antipsychotic. Eur. J. Pharmacol.515: 10-19.PerkinElmer, Inc. 940 Winter StreetWaltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 Materials and InstrumentationThe following tables provide the references of compounds and reagents used for the characterization of the human Dopamine D 2L Receptor Aequorin cell line, as well as some advice on how to use these compounds:Table 1. References of compounds used for functional characterization assaysTable 2. References of cell culture media and additives.Note: The table below lists generic media and additives typically used for PerkinElmer cell lines. For product specific media and additives, please refer to the “Recommended Cell Culture Conditions ” section.Please visit our website: /CellLines for additional information on materials, microplates and instrumentation.This product is not for resale or distribution except by authorized distributors.。
吉满生物 H_ROR2 CHO-K1 Cell Line说明书
产品手册H_ROR2 CHO-K1 Cell LineH_ROR2 CHO-K1细胞系For research use only！本品仅供科研使用，严禁用于治疗！版本号：V2.8目录一、产品基本信息及组分 (3)二、包装、运输及储存 (3)三、材料准备 (3)1.细胞培养、冻存、复苏试剂准备 (3)2.试剂耗材准备 (3)四、细胞培养、复苏、冻存 (4)1.H_ROR2 CHO-K1 Cell Line细胞复苏 (4)2.H_ROR2 CHO-K1 Cell Line细胞传代 (4)3.H_ROR2 CHO-K1 Cell Line细胞冻存 (5)五、验证结果 (6)1.流式检测蛋白表达 (6)使用许可协议： (7)附录1 H_ROR2氨基酸序列 (8)一、产品基本信息及组分基本信息产品编号产品名称规格GM-C19164H_ROR2 CHO-K1 Cell Line5E6 Cells/mL 组成成分产品编号产品名称规格数量储存Fetal Bovine Serum500 mL Thermo/10099141F12K 500 mL BOSTER/PYG0036Human ROR2 Antibody / R&D SYSTEMS/MAB20641重要仪器细胞计数仪ThermoFisher Scientific/Countess 3流式细胞仪常州必达科生物科技有限公司/BeamCyte-1026四、细胞培养、复苏、冻存1.H_ROR2 CHO-K1 Cell Line细胞复苏a)细胞冻存密度为5 × 106 cells/mL，冻存管分装1 mL。
b)在37℃水浴锅预热培养基，加入预热完全培养基5 mL到15 mL离心管。
c)从液氮中取出冻存的细胞并迅速放入37℃恒温水浴锅，将细胞液面浸至水面以下不断摇动至融化。
d)用70%乙醇擦拭冻存管外部以降低污染的几率。
e)在生物安全柜或超净台中将冻存管中的细胞悬液转移到预先加有预热好的15 mL离心管中，轻轻混匀，1000 rpm，离心5 min使细胞沉淀，弃上清。
cho细胞培养工艺开发
cho细胞培养工艺开发English Answer:CHO cell culture process development is a complex and challenging process that requires careful planning and execution. The following are some key considerations for CHO cell culture process development:1. Cell line selection: The choice of cell line is critical to the success of a CHO cell culture process. The cell line should be stable, high-producing, and easy to culture.2. Media optimization: The media used to culture CHO cells must provide all of the nutrients that the cells need to grow and produce the desired product. The media should also be free of contaminants and toxins.3. Culture conditions: The culture conditions, such as temperature, pH, and dissolved oxygen, must be carefullycontrolled to ensure optimal cell growth and product production.4. Bioreactor selection: The type of bioreactor used to culture CHO cells will depend on the scale of the process and the desired product.5. Harvesting: The cells and product are harvested from the bioreactor using a variety of methods, such as centrifugation, filtration, and chromatography.CHO cell culture process development is a complex and challenging process, but it is essential for the production of biologics. By following the steps outlined above, you can increase the chances of success for your CHO cell culture process.中文回答：CHO细胞培养工艺开发是一个复杂而富有挑战性的过程，需要精心的计划和执行。
CHO细胞表达体系特点及CHO细胞表达疫苗
CHO细胞表达体系特点及CHO细胞表达疫苗CHO细胞表达体系特点及CHO细胞表达疫苗来源：易生物实验浏览次数：533 网友评论0 条CHO细胞表达体系特点及CHO细胞表达疫苗关键词：细胞疫苗CHO细胞表达体系CHO细胞表达分子生物学、分子免疫学等学科的发展使基因工程疫苗具有越来越重要的地位。
在基因工程疫苗研究的动物细胞表达系统中，最具代表性的就是中国仓鼠卵巢细胞(Chinese Hamster Ovary，CHO)。
它是用来表达外源蛋白最多也最成功的一类细胞。
本文就CHO细胞表达系统在疫苗研制中的应用做一综述。
1、CHO细胞表达体系及其特点CHO细胞属于成纤维细胞，既可以贴壁生长。
也可以悬浮生长。
目前常用的CHO细胞包括原始CHO和二氢叶酸还原酶双倍体基因缺失型(DHFR-) 突变株CHO。
近年来，为降低生产成本和减少血制品带来的潜在危害性，动物细胞生产开始使用无血清培养基(SFM)，但SFM往往导致细胞活力差，贴壁性差，分泌外源蛋白的能力差等缺点。
另有研究者尝试将类胰岛素生长因子IGF基因和转铁蛋白基因转入CHO细胞获得能自身分泌必需蛋白的“超级CHO”，无需在培养基中转铁蛋白和胰岛素，细胞可在sFM 中生长良好。
与其他表达系统相比，CHO表达系统具有以下的优点：(1)具有准确的转录后修饰功能，表达的蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分子；(2)既可贴壁生长，又可以悬浮培养，且有较高的耐受剪切力和渗透压能力；(3)具有重组基因的高效扩增和表达能力，外源蛋白的整合稳定；(4)具有产物胞外分泌功能，并且很少分泌自身的内源蛋白，便于下游产物分离纯化；(5)能以悬浮培养方式或在无血清培养基中达到高密度培养。
且培养体积能达到1000L以上，可以大规模生产。
2、CHO细胞表达疫苗（1）乙肝疫苗CHO细胞表达疫苗的种类不多，多数处于研究阶段。
目前只有CHO表达乙肝疫苗已投入生产，这是除酵母表达乙肝疫苗以外，唯一已用于人类使用的基因工程亚单位疫苗。
第二章动物细胞培养的基本知识
培养细胞分化状态的变化
体内细胞--三方面的影响与调节: 体外环境的影响 体内神经—体液因素 的调节 细胞与局部微环境的相互作用
控制-- 维持着细胞的分化特征。
体外培养时， 失去了-分化特征逐渐减弱
去分化(脱分化): 各种分化细胞，逐渐失去各自的形态与 功能“个性”，表现出某种趋同性 如:培养的成纤维细胞 在适当刺激下， 可分化为 其它结缔组织细胞和肌组织细 胞，表现出类似未分化的间充质细胞的 特性，‘返祖’ 。
3、游走细胞型
常具有胞质突起（伪足），呈散在生长， 一般不连成片，呈活跃游走或变形运动， 方向不规则。此型细胞不稳定，有时难 以和其他细胞相区别。
单核巨噬细胞系统的细胞
4、多形细胞型
细胞一般分胞体和胞突两部分，胞体为 细长型，似丝状伪足。
有一些细胞，如神经细胞难以确定其规 律和稳定的形态，可统归于此类。
接触抑制和密度依赖性生长仰制
运动的接触抑制
密度依赖性生长仰制
增殖的密度抑制 实验 方 法 ： 3T3 细 胞 ， 接 种 105 于 6cm 培 养 皿 ，
5d细胞计数
于 加 入 10 ％ 、 20 ％ 、 30 ％ 牛 血 清 的 培养液中，结果：
结果：
①10％血清： 20hr后铺满 以后至5d 细胞数不再增加(细
胞 数约106／6cm皿)
30%血清： 以后经常换液至5d，细胞密度继续增加 (可达6xl06／6cm皿)
说明: 密度抑制---铺满后不再增殖(分裂停 止)血清可降低密度抑制
只加入于3T3停止生 长的培养液中 细胞不生长
○ 当再加入血清 细胞又生长
说明 血清可消除密度抑制
(2)转化细胞的密度抑制降低
措施（因素）
CellLineDevelopment：细胞系的发展
Cell Line DevelopmentProducing high-expressing,stable cell banks from DNASelection of initial stable producing poolsTransfected cells are grown in selective media to generate stable pools. For CHO-DG44 cells, the pools can also be subject to additional MTX selection pressure to increase expression levels. Once the pools are fully recovered, the productivity and quality of the pools will be evaluated to select the top three pools for cloning.Construction of high-expression vectorsWuXi AppTec o ers gene-speci c cloning into commercial vectors (e.g., Invitrogen), or client-speci ed vector systems. The integrity of the target gene cloned into the vector will be con rmed by sequencing prior to transfection into a host cell line.Construction of high-expression vectorsTransfectionSelection of initial stable producing poolsCloningFed-batch evaluation & top clones selectionLong-term productivity & genetic stability studyFinal reportsThe WuXi AppTec ProgramTransfectionTransfection into one of WuXi AppTec’s platform cell lines(e.g., CHO-DG44) or a client-speci ed host cell line is typicallyconducted via lipid-based transfection method. Other transfection methodologies are available upon request, such as electroporation, polyethylenimine (PEI) mediated transfection, etc. Paralleltransfections will be performed to increase the probability of success and to generate the necessary number of clones for screening.CloningThree pools are plated into semi-solid medium in 6-well plates for clone-picking. High-expressing clones can be captured andvisualized by a uorescently labeled antibody. Approximately 300 clones in 96-well plates are screened utilizing the ClonePix TM system. An HTRF assay is performed to evaluate the expression of these clones, and 20 to 30 clones from each pool are selected and expanded into shake asks.Fed-batch evaluation & top clones selectionThe top 20 to 30 clones from each pool are further evaluated based on growth rates. A few vials of a research cell bank (RCB) are made from these clones. The productivity of the selected clones isevaluated in fed-batch culture with a platform process. The top 4-6 clones are determined according to the titer and product quality data and are transferred to the cell culture process development group for analysis in bioreactors. The nal clone and the back-up clone are selected based on several rounds of bioreactor screening data. The primary cell bank (PCB) is made for the nal clone and the back-up clone. The master cell bank (MCB) and working cell bank (WCB) can be produced for the nal clone under GMP conditions.Long-term productivity & genetic stability studyPCB protein expression and product quality stability is evaluated for a minimum of 60 generations. (Duration can be extended uponrequest.) The genetic stability of the target gene(s) for PCB, MCB and WCB is performed by sequencing the full length cDNA. Additional genetic stability studies (e.g., copy number determination, detection of insertions/deletions) can also be conducted. The banks are subjected to adventitious agent detection (e.g., bacteria, fungi,mycoplasma, and viruses such as MVM). Full cell line characterizationcan be conducted on the MCB and WCB.Final reportsReports of cell line development and stability studies are provided to clients for review. After review and acceptance of the reports, cell banks and process will be transferred to clientsCELL LINE DEVELOPMENT TEAMWuXi AppTec’s cell line development team has over 30 years’ combined experience in developing high- yielding cell lines and comprises experts who are familiar with regulatory requirements of the US FDA, EMA, ICH, and SFDA. The team has developed numerous mammalian cell lines expressingmonoclonal antibodies (MAbs) and recombinant proteins yielding multiple grams of target protein per liter in standard fed-batch bioreactors as well as continuous perfusion bioreactors.The depth and breadth of experience includes: • Construction of high-expression vectors • High-throughput clone screening • High-throughput clone selection and evaluation • Cell banking• Cell line stability analysis• Cell culture process developmentEQUIPMENT / INSTRUMENTATIONOur well-equipped molecular biology and cell biology labs o er a full range of services required for successful cell line development processes. Instruments include: • Beckman Coulter Vi-CELLs: cell counting • INFORS HT incubators: shake flasks • Kuhner incubators: high-throughput cell culture in spin tubes and 24 well plates • Beckman Coulter Biomek® NX: automated high-throughput clone screening • Molecular Devices ClonePix TM : automated clone picking• SpectraMax M5e plate reader: high-throughput HTRF and for glucose/Lac assay•Guava: high-throughput cell countingCell Line Development(1)651-675-2000/888-794-0077•*******************• S S 9 1 4 -1 3 -0 4 . 1 1 . 1 3WuXi AppTec, a leading global contract services organization, provides services ofexceptional quality, leveraging more than 20 years of experience in the discovery,development, manufacturing and testing of mammalian-derived protein therapeutics.This single-source strategy can reduce the time-to-clinic and decrease signi cantly thecost of our customers’ drug development e orts.⑥Proven high-yielding cell line development platforms accepted by global regulatory agencies.⑥Quick turnaround time and competitive costs.⑥Well-equipped molecular biology and cell line development labs.⑥Capability to develop MAb clones at 2-3 g/L prior to cell culture process optimization.⑥Robust cell lines with high productivity and exceptional product quality and stability.⑥LIMS system for tracking cell line history and seamless linkage between cell line development,bioprocess development and PAS groups.⑥Cell line characterization, including biosafety, genetic and expression stability studiesDiscovery ServicesWuXi AppTec o ers a full spectrum of discovery services for the generation of high potency novel monoclonal antibody therapeutics. Discovery technologies include hybridoma generation, phage display libraries, antibody humanization and a nity maturation. Through partnership with OMT, WuXi AppTec became the rst CRO inChina with access to transgenic animal models capable of generating fully human monoclonal antibodies.Cell Banking & Cell LIne CharacterizationIndependent cell banking, cell line biosafety testing and cell line characterization services are also o ered.Cell banks are manufactured to client speci cations, under exacting standards and QA oversight, in secure, monitored cell banking suites. Long-term storage is available at WuXi AppTec sites in highly controlled and regulated GMP facilities. Complete biosafety, identi cation and characterization assessment of cell banks andcells at the limit of in-vitro cell age is conducted under FDA and ICH guidelines in our in-house laboratories. Process DevelopmentTop selected clone(s) can be transferred to our cell culture process development team for further process development and optimization. WuXi AppTec’s upstream cell culture process technology platform provides for optimization of media, seeding densities, feeding and bioreactor process control strategies to maximize expression, enhance process reproducibility and facilitate scale-up.。
CMV启动子和RSVLTR启动子表达sIL_6R效率的比较
CMV 启动子和RSV LTR 启动子表达sIL -6R效率的比较于占洋 任蕴芳1 沈倍奋1(军事医学科学院野战输血研究所 北京 100850)摘要 利用分别含有CM V 启动子和RSV LT R 启动子的两种真核表达载体,在CHO-K 细胞中成功地表达了人可溶性白细胞介素-6受体(hsIL -6R ),比较了两种启动子在表达效率上的差异,结果表明,CM V 启动子的效率略高于RSV LTR 启动子。
关键词 hsIL-6R;CMV 启动子;RSV LT R;CHO-K 细胞Comparision of CMV promotor and RSV LTR in expressionof human soluble IL -6R geneYu Zhanyang ,Ren Yunfang ,Shen Beifen(I ns titute of T r ansf us ion M ed icine ,Beij ing 100850)Abstract U sing CM V prom otor and RSV LTR ,human so luble IL -6R gene w as ex pressed in CHO-K cell line.The efficiencies of the two promo to rs w ere compared.T he results sho wed that CMV pr omotor ′s efficiency is higher than that o f RSV LT R.Key words hsIL -6R ;CMV promo to r ;RSV LT R ;CHO -K cell line 1军事医学科学院基础医学研究所 白细胞介素-6(IL-6)是一种多功能细胞因子,参与免疫调控、造血、急性期反应等生物学过程,其表达失调与一些疾病密切相关[1]。
IL -6受体由A 、B 两条链组成,A 链为配基特异性受体(一般称为IL -6R ),它与IL -6以低亲和力结合,但不能传导信号。
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CHO-敲除细胞系简介：CHO细胞又称中国仓鼠卵巢细胞，是一种来源于中国仓鼠卵巢的上皮细胞系。
中国仓鼠卵巢（CHO）细胞以其独特的特性，已成为制备治疗性重组蛋白的主要工具。
这种细胞系通常用于生物学和医学研究。
同时，它也是最广泛使用的哺乳动物宿主在工业生产治疗蛋白方面。
应用程序：基因打靶法构建基因敲除CHO细胞系由于CHO细胞系是治疗性抗体产生的主要宿主，构建高效的CHO细胞系具有重要意义。
在构建CHO细胞系中，常用的转染方法有两种，一种是随机整合法，另一种是基因靶向法。
前者导致抗体产生率的变化，通常会影响转基因表达水平。
然而，在基因打靶方法中，外源基因被插入特定的染色体区域。
这种方法是基于利用针对宿主细胞特定基因组区域的序列进行同源重组。
研究人员利用CRISPR/Cas9系统作为基因靶向方法，通过引导RNA和Cas9诱导双链断裂，提高同源重组效率。
因此，CRISPR/Cas9载体系统可以有效地将外源基因插入CHO细胞系中。
利用谷氨酰胺合成酶基因敲除细胞提高CHO细胞系的产生效率尽管中国仓鼠卵巢（CHO）细胞以其独特的特性，已经成为制造治疗性重组蛋白的主要工具，但如何有效地鉴定这些稀有的CHO细胞系是CHO细胞系产生的主要挑战之一，在大量低产和低产无性系中的高产无性系。
目前，有两种主要的CHO表达系统被广泛应用，基于二氢叶酸还原酶（DHFR）的甲氨蝶呤（MTX）选择和基于谷氨酰胺合成酶（GS）的甲硫氨酸硫氧胺（MSX）选择。
为了研究内源性GS表达对选择效率的潜在影响，采用锌指核酸酶（ZFN）技术，以内源性CHO-GS基因为靶点，构建了GS基因敲除CHOK1SV细胞株。
双等位基因修饰CHO-GS基因的高效性（∼2%）支持了ZFN技术的独特优势，特别是在CHO 细胞中。
通过对所有GS基因敲除细胞系谷氨酰胺依赖性生长的观察，证实了GS 酶功能的破坏。
谷氨酰胺合成酶介导的CHO细胞单克隆抗体筛选利用GS基因敲除CHO细胞系12和启动子工程，即使在没有MSX1的情况下，基于GS的系统也能有效地产生细胞系。
利用两个不同的宿主细胞系（CHO-K1和GS-knockout-CHO，GS-KO），在不同MSX浓度下，通过单轮筛选产生mAb的rCHO细胞克隆。
GS基因敲除CHO细胞系具有改进的选择严格性。
使用GS基因敲除CHO宿主细胞系有助于在没有MSX的情况下快速产生高产克隆，降低乳酸和氨的产量。
mAb克隆产生和长期培养的生产稳定性试验流程示意图。
CRISPR/Cas9基因敲除HEK293细胞系的策略敲除HEK293细胞株的工作流程参考文献:Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC, Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.Aga, M., Yamano, N., Kumamoto, T. et al. Construction of a gene knockout CHO cell line using a simple gene targeting method. BMC Proc 9, P2 (2015).Noh, S.M., Shin, S. & Lee, G.M. Comprehensive characterization of glutaminesynthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8, 5361 (2018).CHO ko cell lineIntroduction:CHO cells are also called Chinese hamster ovary cells, which are an epithelial cell line that originated from the ovary of the Chinese hamster. Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins. This cell line is usually used in biological and medical research. Also, it is the most widely used mammalian host in terms of the industrial production of therapeutic proteins.Application:Construction of a gene knockout CHO cell line using gene targeting methodSince CHO cell line is a major host for therapeutic antibody production, constructing productive CHO cell lines is important. There are two major transfection methods that are commonly used in building characterized CHO cell lines, one is random integration and the other one is gene targeting. The former one, which causes variation in antibody productivity, would usually affect transgene expression levels. However, in gene-targeting methods, exogenous genes are inserted into a specific chromosomal region. This method is based on homologous recombination using sequences targeting a specific genomic region of the host cell. Researchers used CRISPR/Cas9 system as a gene-targeting method, which induces double-strand breaks(DSBs) via guide RNA and Cas9, which increases the efficiency of homologous recombination. Therefore, CRISPR/Cas9 vector system can efficiently insert exogenous genes into CHO cell lines.Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cellsAlthough Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. Currently, there are two main CHO expression systems that have been widely used, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection.To study endogenous GS expression's potential impact on selection efficiency,GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of the glutamine-dependent growth of all GS-knockout cell lines.Characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodiesWith a GS-knockout CHO cell line12 and promoter engineering, the GS-based system can be effective in cell line generation even in the absence of MSX1. Using two different host cell lines (CHO-K1 and GS-knockout CHO (GS KO), mAb producing rCHO cell clones were generated by a single round of selection at various MSX concentrations.GS-knockout CHO cell lines with an improved selection stringency. The use of the GS-knockout CHO host cell line facilitates the rapid generation of high producing clones with reduced production of lactate and ammonia in the absence of MSX.A schematic diagram of the process for mAb producing clone generation andlong-term culture for testing the production stability.A schematic diagram of the process for mAb producing clone generation and long-term culture for testing the production stability.CRISPR/Cas9 knockout HEK293cell line strategiesWork flow of knockout HEK293cell lineReference:Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC, Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.Aga, M., Yamano, N., Kumamoto, T. et al. Construction of a gene knockout CHO cell line using a simple gene targeting method. BMC Proc 9, P2 (2015).Noh, S.M., Shin, S. & Lee, G.M. Comprehensive characterization of glutaminesynthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8, 5361 (2018).。