transgenic mice bearing an o - glcnacase heterozygous allele were developed from oga floxed mice cross breeding with cre mice as described in . the resultant oga heterozygous mice were cross bred to derive mouse embryonic fibroblast ( mef ) cells for this study . mef cells were collected at embryonic day 12.5 following the protocol from the fred hutchinson cancer center ( www.fhcrc.org/science/labs/fero/protocols/mefs.html ) . cells were cultured in dmem media added with 10% fbs and 100  u / ml penicillin and 100  g / ml streptomycin . gene and protein expression patterns of wild type , heterozygous and ko mefs were evaluated using pcr and western blotting . genotypes were confirmed by pcr and increased o - glcnac modification in proteins observed in oga ko and heterozygous cell lines compared to wild type reflecting oga deletion ( fig . female mef cells from wild type ( wt ) , oga heterozygous ( het ) and oga knock out ( ko ) genotypes , collected from a single litter were used for the microarray study . total rna digested with rnase free dnase i , followed by column purification ( qiagen inc . , ca ) . rna quantified by nanodrop and quality assessed by bioanalyzer trace ( agilent technologies , ca ) and samples with rna integrity number ( rin # ) above 7.9 the were selected for the assay . the total rna from each sample ( 5  g ) was used for cdna synthesis and biotin labeling , in three technical replicates for each group . samples were processed using affymetrix kits according to the manufacturer 's instructions at the niddk genomic core facility . samples were hybridized to affymetrix 430.2 arrays for 16  h at 45  c rotating at 60  rpm . array chips were washed , and stained using fs450 fluidic stations ( protocol fs450 - 0004 ) using reagents from hws kits ( affymetrix inc . ) . the arrays were scanned using a 3000 g affymetrix scanner operated by genechip operating software ( gcos ) , which generated 9 data files . image files ( .cel ) were further analyzed by partek software ( partek inc . , st . quality control features indicated all arrays were good and signal intensity box plots are shown in fig . 2a . principle component analysis ( pca ) shows 66% of data could be described by genotype differences and well separated into 3 groups ( fig . the volcano plot from ko against wt comparison shows a large number of genes significantly affected even at 2 fold level ( fig . data statistically analyzed using partek anova and oga het and ko groups were compared with wt and also with each other , and considered significant at p  <  0.05 level . gene lists were generated for 1.5 fold or 2 fold differentially expressed transcripts with multiple test correction for false discovery ratio ( fdr ) set at 0.05 level . five transcripts interrogated oga ( mgea5 ) gene were significantly reduced in ko group compared to wt and intermediate levels were seen in het group . a total of 2534 transcripts from the ko group showed 1.5 fold change compared to wt with 1404 reduced transcripts and 1130 increased . 3a shows the number of transcripts passed at 1.5 fold or ( 2 fold ) level and unique or unknown transcripts for each comparison . the distribution of 1.5 fold significant transcripts in each comparison category is shown in a venn diagram ( fig . these results indicate that deletion of both oga alleles changed the gene expression significantly while partial deletion ( het ) had limited effects on gene expression . a heat map from hierarchical clustering of differentially expressed genes in ko and wt is shown in fig . 4 ( y axis sample type , x axis genes , blue reduced , and red increased gene expression ) . the cluster map shows single row for each sample and 3 replicates from each genotypes were very similar and tightly clustered ( raw colors ) . columns of gene clusters in the heterozygous group ( middle ) have shown higher similarity with wt while few gene clusters are similar to the ko group . gene enrichment analysis was performed using a gene ontology ( go ) database ( mm 2012 - 11 - 19 release ) to understand the biological effects . the group of 2534 transcripts ( ko vs wt ) showed the highest enrichment in genes associated with metabolic , and proliferation processes ( fig . het group compared to wt ( 959 ) shows enrichment in proliferation and growth associated genes ( fig . the same data set is analyzed with genomatix ( germany ) software using gene ranker analysis also showing similar categories while highest probability in enrichment is associated with innate immunity genes ( data not shown ) . pathway analysis conducted using 839 ( 2 fold change ) transcripts from ko versus wt comparison using geps ( genomatix ) and the highest 10 pathways listed are shown in table 1 . the highest number of genes are associated with the integrin pathway and shown in fig . transgenic mice bearing an o - glcnacase heterozygous allele were developed from oga floxed mice cross breeding with cre mice as described in . the resultant oga heterozygous mice were cross bred to derive mouse embryonic fibroblast ( mef ) cells for this study . mef cells were collected at embryonic day 12.5 following the protocol from the fred hutchinson cancer center ( www.fhcrc.org/science/labs/fero/protocols/mefs.html ) . cells were cultured in dmem media added with 10% fbs and 100  u / ml penicillin and 100  g / ml streptomycin . gene and protein expression patterns of wild type , heterozygous and ko mefs were evaluated using pcr and western blotting . genotypes were confirmed by pcr and increased o - glcnac modification in proteins observed in oga ko and heterozygous cell lines compared to wild type reflecting oga deletion ( fig . female mef cells from wild type ( wt ) , oga heterozygous ( het ) and oga knock out ( ko ) genotypes , collected from a single litter were used for the microarray study . total rna digested with rnase free dnase i , followed by column purification ( qiagen inc . , ca ) . rna quantified by nanodrop and quality assessed by bioanalyzer trace ( agilent technologies , ca ) and samples with rna integrity number ( rin # ) above 7.9 the were selected for the assay . the total rna from each sample ( 5  g ) was used for cdna synthesis and biotin labeling , in three technical replicates for each group . samples were processed using affymetrix kits according to the manufacturer 's instructions at the niddk genomic core facility . samples were hybridized to affymetrix 430.2 arrays for 16  h at 45  c rotating at 60  rpm . array chips were washed , and stained using fs450 fluidic stations ( protocol fs450 - 0004 ) using reagents from hws kits ( affymetrix inc . ) . the arrays were scanned using a 3000 g affymetrix scanner operated by genechip operating software ( gcos ) , which generated 9 data files . image files ( .cel ) were further analyzed by partek software ( partek inc . , st . quality control features indicated all arrays were good and signal intensity box plots are shown in fig . 2a . principle component analysis ( pca ) shows 66% of data could be described by genotype differences and well separated into 3 groups ( fig . the volcano plot from ko against wt comparison shows a large number of genes significantly affected even at 2 fold level ( fig . data statistically analyzed using partek anova and oga het and ko groups were compared with wt and also with each other , and considered significant at p  <  0.05 level . gene lists were generated for 1.5 fold or 2 fold differentially expressed transcripts with multiple test correction for false discovery ratio ( fdr ) set at 0.05 level . five transcripts interrogated oga ( mgea5 ) gene were significantly reduced in ko group compared to wt and intermediate levels were seen in het group . a total of 2534 transcripts from the ko group showed 1.5 fold change compared to wt with 1404 reduced transcripts and 1130 increased . 3a shows the number of transcripts passed at 1.5 fold or ( 2 fold ) level and unique or unknown transcripts for each comparison . the distribution of 1.5 fold significant transcripts in each comparison category is shown in a venn diagram ( fig . these results indicate that deletion of both oga alleles changed the gene expression significantly while partial deletion ( het ) had limited effects on gene expression . a heat map from hierarchical clustering of differentially expressed genes in ko and wt is shown in fig . 4 ( y axis sample type , x axis genes , blue reduced , and red increased gene expression ) . the cluster map shows single row for each sample and 3 replicates from each genotypes were very similar and tightly clustered ( raw colors ) . columns of gene clusters in the heterozygous group ( middle ) have shown higher similarity with wt while few gene clusters are similar to the ko group . gene enrichment analysis was performed using a gene ontology ( go ) database ( mm 2012 - 11 - 19 release ) to understand the biological effects . the group of 2534 transcripts ( ko vs wt ) showed the highest enrichment in genes associated with metabolic , and proliferation processes ( fig . het group compared to wt ( 959 ) shows enrichment in proliferation and growth associated genes ( fig . the same data set is analyzed with genomatix ( germany ) software using gene ranker analysis also showing similar categories while highest probability in enrichment is associated with innate immunity genes ( data not shown ) . pathway analysis conducted using 839 ( 2 fold change ) transcripts from ko versus wt comparison using geps ( genomatix ) and the highest 10 pathways listed are shown in table 1 . the highest number of genes are associated with the integrin pathway and shown in fig . the lack of a mammalian model system limits the understanding of o - glcnac signaling . deletion of the oga enzyme which removes o - glcnac modification has also shown about 89% neonatal lethality in mice , . mefs isolated from oga ko , het and wt embryos were used for the microarray to facilitate the understanding of global effects . oga deletion in mef cells is confirmed by gdna pcr and as a result increased o - glcnac modification were observed in het and ko cell types compared to wt . the present data shows o - glcnac modification affects not only cellular signaling but also gene expression . differentially expressed genes under oga deletion indicate the effects in multiple cellular functions such as metabolism , cell proliferation , and growth . perturbation of o - glcnac cycling affects metabolic pathways crucial for survival and corroborate with the observed neonatal lethal phenotype of oga ko pups . defects of cell proliferation and cytokinesis are also shown through other scientific evidence in oga disrupted cells . tyrosine kinase pathways are involved in downstream signaling associated with immunoreceptors in b cell receptors . many cytokine and immunity associated genes were largely down regulated in ko mefs compared to wt . integrin pathway associated with extracellular matrix signaling and are significantly affected by oga deletion . observed gene regulation in oga ko group could be explained by lack of oga activity directly , or due to increased o - glcnac modification of proteins . dysregulation of the o - glcnac cycling could also have effects on protein phosphorylation ( slowson and hart ) , ubiquitin pathway , transcription factor binding , or localization . the present microarray data provide insight into global effects on multiple cellular functions associated with disruption of o - glcnac modification .
adding a single o - glcnac moiety to a ser / thr molecule of a protein by o - glcnac transferase and transiently removing it by o - glcnacase is referred to as o - glcnac cycling ( or o - glcnacylation ) . 
 this o - glcnac modification is sensitive to nutrient availability and also shows cross talk with phosphorylation signaling , affecting downstream targets . 
 a mouse model system was developed and evaluated to show genome wide transcriptional changes associated with disruption of o - glcnac cycling . 
 mouse embryonic fibroblast cells derived from o - glcnacase ( oga ) knock out ( ko ) , heterozygous ( het ) and wild type ( wt ) embryos were used for an affymetrix based microarray . 
 results are deposited in geo dataset gse52721 . 
 data reveals that oga ko mefs had 2534 transcripts differentially expressed at 1.5 fold level while oga heterozygous mefs had 959 transcripts changed compared to wt mefs . 
 there were 1835 transcripts differentially expressed at 1.5 fold het versus wt comparison group . 
 gene ontology analysis indicated differentially expressed genes enriched in metabolic , growth , and cell proliferation categories .