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maintenance and differentiation of CbCln3 cerebellar neuronal precursor lines\nCln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T5658","span":{"begin":28,"end":43},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T5659","span":{"begin":47,"end":49},"obj":"UBERON:0002037"},{"id":"T5660","span":{"begin":49,"end":53},"obj":"PR_EXT:000005591"},{"id":"T5661","span":{"begin":54,"end":64},"obj":"UBERON:0002037"},{"id":"T5662","span":{"begin":54,"end":73},"obj":"CL:1001611"},{"id":"T5663","span":{"begin":90,"end":94},"obj":"PR_EXT:000005591"},{"id":"T5664","span":{"begin":110,"end":114},"obj":"NCBITaxon:10088"},{"id":"T5665","span":{"begin":163,"end":167},"obj":"NCBITaxon_UBERON_EXT:pup"},{"id":"T5666","span":{"begin":282,"end":292},"obj":"UBERON:0002037"},{"id":"T5667","span":{"begin":282,"end":307},"obj":"CL:0001031"},{"id":"T5668","span":{"begin":336,"end":341},"obj":"GO:0007567"},{"id":"T5669","span":{"begin":353,"end":362},"obj":"UBERON:0002037"},{"id":"T5670","span":{"begin":397,"end":410},"obj":"CHEBI_EXT:saline_solution"},{"id":"T5671","span":{"begin":450,"end":457},"obj":"CHEBI:17234"},{"id":"T5672","span":{"begin":459,"end":463},"obj":"UBERON:0002415"},{"id":"T5673","span":{"begin":497,"end":508},"obj":"SO_EXT:genomic_DNA"},{"id":"T5674","span":{"begin":505,"end":508},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T5675","span":{"begin":523,"end":532},"obj":"SO_EXT:genotype_or_entity_with_genotype"},{"id":"T5676","span":{"begin":543,"end":550},"obj":"PR_EXT:000027795"},{"id":"T5677","span":{"begin":551,"end":555},"obj":"CHEBI_EXT:EDTA"},{"id":"T5678","span":{"begin":578,"end":585},"obj":"GO_PR_EXT:deoxyribonuclease_I"},{"id":"T5679","span":{"begin":643,"end":652},"obj":"UBERON:0002037"},{"id":"T5680","span":{"begin":697,"end":706},"obj":"CHEBI_EXT:ornithine"},{"id":"T5681","span":{"begin":770,"end":779},"obj":"UBERON:0002037"},{"id":"T5682","span":{"begin":816,"end":819},"obj":"CHEBI:16526"},{"id":"T5683","span":{"begin":824,"end":838},"obj":"CL:0000120"},{"id":"T5684","span":{"begin":839,"end":845},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5685","span":{"begin":926,"end":932},"obj":"NCBITaxon:27592"},{"id":"T5686","span":{"begin":933,"end":938},"obj":"UBERON:0001977"},{"id":"T5687","span":{"begin":980,"end":983},"obj":"CHEBI:32588"},{"id":"T5688","span":{"begin":1043,"end":1053},"obj":"NCBITaxon:11632"},{"id":"T5689","span":{"begin":1054,"end":1065},"obj":"GO:0009293"},{"id":"T5690","span":{"begin":1110,"end":1117},"obj":"CHEBI_EXT:59132"},{"id":"T5691","span":{"begin":1134,"end":1142},"obj":"CHEBI:7507"},{"id":"T5692","span":{"begin":1154,"end":1162},"obj":"SO_EXT:0005853"},{"id":"T5693","span":{"begin":1270,"end":1276},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5694","span":{"begin":1327,"end":1333},"obj":"GO_EXT:biological_growth_entity_or_process"},{"id":"T5695","span":{"begin":1365,"end":1369},"obj":"CHEBI:42768"},{"id":"T5696","span":{"begin":1519,"end":1525},"obj":"SO_EXT:sequence_cloned_entity"},{"id":"T5697","span":{"begin":1555,"end":1563},"obj":"SO_EXT:genotype_or_entity_with_genotype"},{"id":"T5698","span":{"begin":1624,"end":1626},"obj":"UBERON:0002037"},{"id":"T5699","span":{"begin":1626,"end":1630},"obj":"PR_EXT:000005591"},{"id":"T5700","span":{"begin":1631,"end":1635},"obj":"CL_GO_EXT:cell"},{"id":"T5701","span":{"begin":1643,"end":1645},"obj":"UBERON:0002037"},{"id":"T5702","span":{"begin":1650,"end":1654},"obj":"CL_GO_EXT:cell"},{"id":"T5703","span":{"begin":1691,"end":1700},"obj":"CHEBI_EXT:ornithine"},{"id":"T5704","span":{"begin":1752,"end":1755},"obj":"CHEBI:16526"},{"id":"T5705","span":{"begin":1859,"end":1867},"obj":"CL:0000540"},{"id":"T5706","span":{"begin":1868,"end":1883},"obj":"GO_RO_EXT:developmental_differentiation_process"},{"id":"T5707","span":{"begin":1936,"end":1944},"obj":"CHEBI:60004"},{"id":"T5708","span":{"begin":1955,"end":1960},"obj":"PR_EXT:000007479"},{"id":"T5709","span":{"begin":1969,"end":1973},"obj":"CHEBI:48518"},{"id":"T5710","span":{"begin":1982,"end":1985},"obj":"CHEBI:37537"},{"id":"T5711","span":{"begin":1993,"end":2002},"obj":"CHEBI:42471"},{"id":"T5712","span":{"begin":2009,"end":2017},"obj":"CHEBI:18243"}],"text":"Generation, maintenance and differentiation of CbCln3 cerebellar neuronal precursor lines\nCln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}

{"project":"2_test","denotations":[{"id":"15588329-12374761-12950162","span":{"begin":147,"end":149},"obj":"12374761"},{"id":"15588329-8248201-12950163","span":{"begin":327,"end":329},"obj":"8248201"},{"id":"15588329-3023876-12950164","span":{"begin":1164,"end":1166},"obj":"3023876"},{"id":"15588329-9671979-12950165","span":{"begin":1194,"end":1196},"obj":"9671979"},{"id":"15588329-11092756-12950166","span":{"begin":1913,"end":1915},"obj":"11092756"}],"text":"Generation, maintenance and differentiation of CbCln3 cerebellar neuronal precursor lines\nCln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma)."}