G418 Protocol and Selection Guide
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1 G418 Protocol and Selection Guide Background G418 is routinely used in gene selections to screen for resistant mammalian cells which express the neo gene. The neo gene encodes amino-glycoside 3 -phosphotransferase; an enzyme which renders the cell resistant to G418. For gene selection applications, a kill curve can be made to determine the minimum effective G418 concentration to kill non-resistant cells. Preparation and storage Generally it is easiest to dissolve the entire container of G418 at once (especially if it is a small 1g container). Calculate the amount of PBS to add to the vial to get a 200mg/ml 'active' concentration. o For example: 710μg/mg means that 710μg out of 1000μg are active, or that the Geneticin is 71% active. Therefore, to make a stock solution of 200mg/ml active Geneticin you need to add 1000/710*200= mg/ml of Geneticin. The vial contains 1g of Geneticin, so you need to dissolve the contents of the vial in 1000mg/ mg=3.55ml total volume. Sterile filter the drug using a syringe and a 0.45μm filter. Store stock at -80 C and working stock solution at 4 C Mammalian Cell Culture Selection procedure for mammalian cells G418 sulfate is normally used at a concentration of 400 μg/ml. After transfection with a plasmid containing the neo gene, cells are incubated in their regular growth containing G418 to select for stable transfectants hours post-transfection, pass cells (direct or diluted) in fresh containing G418 at the appropriate concentration. a) Note: Antibiotics work best when cells are actively dividing. If the cells become too dense, the antibiotic efficiency will decrease. It is best to split cells such that they are not more than 25% confluent. 2. Remove and replace antibiotic containing every 3-4 days 3. Evaluate cells for the formation of foci after 7 days of selection. Foci may require an additional week or more to develop depending on the host cell line and transfection/selection efficiency.
2 4. Transfer and pool 5-10 resistant clones to a 35mm cell culture plate and maintain on selection for an additional 7 days. This pooled culture will be expanded for subsequent cytotoxicity assays. General Selection Table Cell line Species Tissue Culture G Kidney; fetal 600 (μg/ml) 293 Kidney; fetal 143B bone; osteosarcoma 293T Kidney (embryonic) 32D Clone 3 bone marrow 600 (μg/ml) 3T3 embryonic fibroblast 3T3-L1 embryo 1 70Z/3 pre-b lymphoblast; methylnitrosourea-induced lymphoma A20 B lymphocyte; reticulum cell sarcoma A20 B lymphocyte; reticulum cell sarcoma A-204 muscle; rhabdomyosarcoma A253 epidermis; epidermoid carcinoma A-431 epidermis; epidermoid carcinoma essential A431NS epidermis; epidermoid carcinoma A-498 Kidney; carcinoma A549 Lungcarcinoma 700 (μg/ml) A9 / hybridoma AGS stomach; gastric adenocarcinoma AR42J Rat exocrine pancreas; tumor AtT-20 pituitary tumor 600 (μg/ml) BALB/3T3 clone A31 embryo BeWo placenta; choriocarcinoma BHK-21 (C- Syrian 13) golden Kidney Bing Kidney; amphotropic retroviral BJ skin (foreskin) BRL 3A Rat liver 200 (μg/ml) BT bovine turbinate BT-20 mammary gland; carcinoma essential 250 (μg/ml) BT-474 mammary gland; ductal carcinoma 200 (μg/ml) BT-549 mammary gland; ductal carcinoma 750 (μg/ml) C166 (transgenic) yolk sac; endothelial cell differentiation model; stem cell feeder layer
3 C2C12 muscle 4000 (μg/ml) C2C12 muscle C3H10T1/2 embryonic mesenchymal cell line C6 Rat brain; glioma Caco-2 colon; colorectal adenocarcinoma Caco-2 colon; colorectal adenocarcinoma 1 (mg/ml) Chang Liver HeLa contaminant 350 (μg/ml) CHO 600 (μg/ml) / F (μg/ml) + F12 essential essential Clone 9 Rat liver / F (μg/ml) COLO 320DM colon; colorectal adenocarcinoma 300 (μg/ml) COLO 320HSR colon; colorectal adenocarcinoma COS-1 monkey, African green Kidney COS-7 monkey, African green Kidney CRFK cat Kidney (cortex) 2000 (μg/ml) CRFK cat Kidney (cortex) CSM (μg/ml) CV-1 monkey, African green Kidney D17 dog bone; osteosarcoma 600 (μg/ml) DLD-1 colon; colorectal adenocarcinoma 600 (μg/ml) DLD-1 colon; colorectal adenocarcinoma DLD-1 colon; colorectal adenocarcinoma DO11.10 DT40 chicken bursa; lymphoma DT40 chicken bursa; lymphoma 350 (μg/ml) DT40 chicken bursa; lymphoma DU 145 brain (metastasis); carcinoma (prostate 200 (μg/ml) E11 (HCT-8 variant) colon
4 ES-E14TG2a embryonic stem cell; pluripotent; HGRPT deficient 200 (μg/ml) FRTL Rat thyroid 600 (μg/ml) GH3 Rat pituitary tumor 1 (μg/ml) GH4 Rat GLC-82 lung GP+E-86 GP+envAM- 12 H1299 lung HeLa cervix; adenocarcinoma HeLa cervix; adenocarcinoma + F (μg/ml) HeLa cervix; adenocarcinoma essential HeLa S3 cervix; adenocarcinoma 300 (μg/ml) HeLa S3 cervix; adenocarcinoma 700 (μg/ml) Hep 3B2.1-7 liver; hepatocellular carcinoma essential 2000 (μg/ml) Hep 3B2.1-7 liver; hepatocellular carcinoma 2 (mg/ml) Hep 3B2.1-7 liver; hepatocellular carcinoma Hep G2 liver; hepatocellular carcinoma 700 (μg/ml) HEp-2 HeLa contaminant Hs27 skin (foreskin) 850 (μg/ml) HT-1080 connective tissue; fibrosarcoma 250 (μg/ml) HuT 78 T lymphocyte; cutaneous; lymphoma JAR placenta; choriocarcinoma JB6 Cl 41-5a skin (epidermis); chemically transformed essential JEG-3 placenta; choriocarcinoma Jurkat, Clone E6-1 T lymphocyte; acute T cell leukemia 750 (μg/ml) K562 lymphoblastoid K-562 pleural effusion (metastatic); chronic myelo-genous leukemia (bone 850 (μg/ml) marrow KB HeLa contaminant LLC-PK1 pig Kidney Charcoal-stripped fetal bovine serum 1 LMH chicken liver; hepatocellular carcinoma / F 12 LNCaP prostatic adenocarcinoma 1 LS 174T colon; colorectal adenocarcinoma M1 myeloblast; myeloid leukemia 100 (μg/ml) M1 myeloblast; myeloid leukemia 600 (μg/ml) MC3T3-E1 bone (metastasis); osteosarcoma
5 Subclone 4 (femur MCF 10A mammary gland; fibrocystic disease MCF-12A mammary gland 600 (μg/ml) MCF7 pleural effusion (metastasis); adenocarcinoma (mammary gland 800 (mg/ml) pleural effusion (metastasis); MDA-MB- adenocarcinoma (mammary gland (μg/ml) MDA-MB- 468 pleural effusion (metastasis); adenocarcinoma (mammary gland 2 (μg/ml) MDCK (NBL- 2) dog Kidney ME-180 omentum (metastasis); epidermoid carcinoma (cervix MG-63 bone; osteosarcoma MV-4-11 peripheral blood; biphenotypic B myelomonocytic leukemia NCI-H1299 lymph node (metastasis); large cell neuroendocrine carcinoma (lung 700 (μg/ml) Neuro-2a brain; neuroblast; neuroblastoma NG /Rat brain; glial cell; neuron; glioblastoma; neuroblastoma (μg/ml) (hybrid) NIH/3T3 embryo NIH3T3 embryo 100 (μg/ml) NRK Rat Kidney 200 (μg/ml) NRK-52E Rat Kidney OK opossum Kidney (cortex); proximal tubule + F12 P19 embryo; teratocarcinoma; embryonic carcinoma PA317 PA (μg/ml) PA317 PANC-1 pancreas (duct); epithelioid carcinoma PANC-1 pancreas (duct); epithelioid carcinoma 550 (μg/ml) PC-12 Rat adrenal gland; pheochromocytoma PC-12 Rat adrenal gland; pheochromocytoma 700 (μg/ml) PC-3 bone (metastasis); adenocarcinoma (prostate PLC/PRF/5 liver; Alexander cells; hepatoma PT67 + F12
6 PT67 RD muscle; rhabdomyosarcoma 100 (μg/ml) Saos-2 bone; osteosarcoma 1 (mg/ml) Saos-2 bone; osteosarcoma 300 (μg/ml) Saos-2 bone; osteosarcoma SC peripheral blood; macrophage; monocyte SCC-25 tongue; squamous cell carcinoma + F12 Sf9 armyworm, fall SF900 II serum free 350 (μg/ml) SH-SY5Y bone marrow (metastasis); neuroblastoma (brain SiHa cervix; squamous cell carcinoma SIRC (Statens Seruminstitut Rabbit rabbit eye (cornea) 600 (μg/ml) Cornea) SK-HEP-1 ascites (metastasis); adenocarcinoma (liver SK-N-MC supraorbital area (metastasis); neuroepithelioma (brain SK-N-SH bone marrow (metastasis); neuroblastoma (brain STO embryo SW 1353 bone; chondrosarcoma essential SW480 colon; colorectal adenocarcinoma T2 (174 x CEM.T2) / lymphoblast cell hybrid 600 (μg/ml) T24 urinary bladder; transitional cell carcinoma 50 (μg/ml) T-47D pleural effusion (metastasis); ductal carcinoma (mammary gland T-47D pleural effusion (metastasis); ductal carcinoma (mammary gland U-2 OS bone; osteosarcoma 700 (μg/ml) U-2 OS bone; osteosarcoma 600 (μg/ml) U-937 macrophage; histiocytic lymphoma U-937 macrophage; histiocytic lymphoma essential WEHI-231 B lymphocyte; lymphoma 600 (μg/ml) References: Davies J & Jimenez A A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg 29(5 Suppl):
7 Bar-Nun S et al G-418, an elongation inhibitor of 80 S ribosomes. Biochim Biophys Acta. 741(1): Beck E et al Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19(3):
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