Project description:Splenic Transitional Type-1 B-cells from CBA wild-type mice, X-linked immunodeficiency mice and Bruton's tyrosine kinase knock-out mice. Two replicates where run on Affymetrix 420 2.0 arrays for CBA wild-type, Xid samples and the Btk KO samples. Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional type 2 (T2) cells are also located in distinct anatomic locations. Based on a limited number of markers it has previously been reported that the earliest phenotypic sign of Btk deficiency is manifested at the T2 stage in mice. Here, we report on distinct genome-wide transcriptomic signature differences found in T1 B-lymphocytes from Btk-defective compared to normal mice and demonstrate that Btk deficiency is visible already at this stage. 2 replicates of T1 B-cells from CBA (WT), Xid samples and the Btk KO samples
Project description:Splenic Transitional Type-1 B-cells from CBA wild-type mice, X-linked immunodeficiency mice and Bruton's tyrosine kinase knock-out mice. Two replicates where run on Affymetrix 420 2.0 arrays for CBA wild-type, Xid samples and the Btk KO samples. Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional type 2 (T2) cells are also located in distinct anatomic locations. Based on a limited number of markers it has previously been reported that the earliest phenotypic sign of Btk deficiency is manifested at the T2 stage in mice. Here, we report on distinct genome-wide transcriptomic signature differences found in T1 B-lymphocytes from Btk-defective compared to normal mice and demonstrate that Btk deficiency is visible already at this stage.
Project description:Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (KO) and immunocompetent CBA mice, were used in microarrays containing more than 12,000 genes and expressed sequence tags (ESTs). We found 4515 transcripts expressed in duplicate experiments in all three strains. Out of these, 38 were differentially expressed genes (21 up-regulated >2 fold and 17 down-regulated <-2 fold) between CBA and Btk defective mice. Ten out of these genes were selected and quantitative Real-Time PCR was conducted for validation and further investigation. Real-Time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes, whose expression differed (>2 fold) between the two strains. Moreover, when the 38 genes, which differed between immunocompetent CBA and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant negative form. Experiment Overall Design: 6 Affymetrix U74Av2 GeneChip arrays was run. Three initially with CBA, Xid and Btk KO mice and then additional three chips with new RNA preperations from new CBA, Xid and Btk KO mice.
Project description:Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (KO) and immunocompetent CBA mice, were used in microarrays containing more than 12,000 genes and expressed sequence tags (ESTs). We found 4515 transcripts expressed in duplicate experiments in all three strains. Out of these, 38 were differentially expressed genes (21 up-regulated >2 fold and 17 down-regulated <-2 fold) between CBA and Btk defective mice. Ten out of these genes were selected and quantitative Real-Time PCR was conducted for validation and further investigation. Real-Time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes, whose expression differed (>2 fold) between the two strains. Moreover, when the 38 genes, which differed between immunocompetent CBA and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant negative form. Keywords: Btk, DNA microarray, Xid, Btk KO, Real-Time PCR
Project description:Global gene expression profiling of the avian B-lymphoma DT40 cell line was used as a model to differentiate among Btk KO and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell line, subclones are selected, which have multiple changes in their gene expression pattern (p<0.01). Experiment Overall Design: DT40 cells along with the mutants were grown at various time points in different batches of fetal calf serum with or without any stimulation. All experiments were repeated ten times and then polled together as one sample. In total 28 samples were used RNA extraction and hybridization on Affymetrix microarrays.