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cell culture, transfection and selection of targeted clones\nMouse R1 ES cells [49] were maintained on gelatin-coated plates with feeder layers of mouse embryonic fibroblasts at 37°C under 5% CO2 in ES cell medium (DMEM [high glucose] supplemented with 15% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 μg/ml amphotericin, and 1,000 U/ml of ESGro [Chemicon]). Fetal bovine serum from HyClone Laboratories Inc (Logan, Utah) was tested for its ability to support ES cell growth. Gelatin was from Sigma-Aldrich Canada (Oakville, Ontario). Unless otherwise specified, all other tissue culture reagents were from Gibco-BRL.\nThe targeting construct was linearized by NotI digestion and electroporated into R1 ES cells. Cells were plated without feeder layers on gelatin-coated plates and transformed clones were selected in the presence of 200 μg/ml G418 (Gibco- BRL) and 2 μM ganciclovir (Syntex, Inc.) for eight days. Drug-resistant clones were picked, expanded on gelatin-coated plates, and genotyped by Southern blotting and PCR analysis (see below)."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T17393","span":{"begin":155,"end":164},"obj":"UBERON:0000922"},{"id":"T17392","span":{"begin":149,"end":154},"obj":"NCBITaxon:10088"},{"id":"T17391","span":{"begin":105,"end":112},"obj":"CHEBI:5291"},{"id":"T17390","span":{"begin":72,"end":80},"obj":"CL:0002322"},{"id":"T17389","span":{"begin":17,"end":29},"obj":"GO:0009294"},{"id":"T17388","span":{"begin":0,"end":7},"obj":"CL:0002322"},{"id":"T17417","span":{"begin":1083,"end":1090},"obj":"CHEBI:5291"},{"id":"T17416","span":{"begin":1036,"end":1040},"obj":"CHEBI:23888"},{"id":"T17415","span":{"begin":993,"end":1004},"obj":"CHEBI:465284"},{"id":"T17414","span":{"begin":966,"end":970},"obj":"CHEBI:42768"},{"id":"T17413","span":{"begin":904,"end":915},"obj":"GO:0009294"},{"id":"T17412","span":{"begin":878,"end":885},"obj":"CHEBI:5291"},{"id":"T17411","span":{"begin":825,"end":833},"obj":"CL:0002322"},{"id":"T17410","span":{"begin":711,"end":719},"obj":"CHEBI:33893"},{"id":"T17409","span":{"begin":696,"end":702},"obj":"UBERON:0000479"},{"id":"T17408","span":{"begin":599,"end":606},"obj":"CHEBI:5291"},{"id":"T17407","span":{"begin":583,"end":590},"obj":"CL:0002322"},{"id":"T17406","span":{"begin":495,"end":500},"obj":"UBERON:0001977"},{"id":"T17405","span":{"begin":488,"end":494},"obj":"NCBITaxon:27592"},{"id":"T17404","span":{"begin":431,"end":443},"obj":"CHEBI:2682"},{"id":"T17403","span":{"begin":408,"end":420},"obj":"CHEBI:17076"},{"id":"T17402","span":{"begin":386,"end":396},"obj":"CHEBI:17334"},{"id":"T17401","span":{"begin":358,"end":375},"obj":"CHEBI:41218"},{"id":"T17400","span":{"begin":334,"end":349},"obj":"CHEBI:50144"},{"id":"T17399","span":{"begin":272,"end":277},"obj":"UBERON:0001977"},{"id":"T17398","span":{"begin":265,"end":271},"obj":"NCBITaxon:27592"},{"id":"T17397","span":{"begin":228,"end":235},"obj":"CHEBI:17234"},{"id":"T17396","span":{"begin":201,"end":208},"obj":"CL:0002322"},{"id":"T17395","span":{"begin":194,"end":197},"obj":"CHEBI:16526"},{"id":"T17394","span":{"begin":165,"end":176},"obj":"CL:0000057"}],"text":"ES cell culture, transfection and selection of targeted clones\nMouse R1 ES cells [49] were maintained on gelatin-coated plates with feeder layers of mouse embryonic fibroblasts at 37°C under 5% CO2 in ES cell medium (DMEM [high glucose] supplemented with 15% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 μg/ml amphotericin, and 1,000 U/ml of ESGro [Chemicon]). Fetal bovine serum from HyClone Laboratories Inc (Logan, Utah) was tested for its ability to support ES cell growth. Gelatin was from Sigma-Aldrich Canada (Oakville, Ontario). Unless otherwise specified, all other tissue culture reagents were from Gibco-BRL.\nThe targeting construct was linearized by NotI digestion and electroporated into R1 ES cells. Cells were plated without feeder layers on gelatin-coated plates and transformed clones were selected in the presence of 200 μg/ml G418 (Gibco- BRL) and 2 μM ganciclovir (Syntex, Inc.) for eight days. Drug-resistant clones were picked, expanded on gelatin-coated plates, and genotyped by Southern blotting and PCR analysis (see below)."}

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