tet2 mouse model  (New England Biolabs)

Journal: bioRxiv

Article Title: Epigenetic Regulation of Inflammation by Dopamine in Primary Human Macrophages

doi: 10.64898/2026.01.21.700899

Figure Lengend Snippet: Primary human monocyte-derived macrophages (hMDM) were treated with dopamine (10 -6 M) for 3 hours, and gene expression was assessed by qPCR. ( A ) Dopamine significantly increased TET2 mRNA expression (n=20 donors, *p<0.05). ( B ) DNMT1 mRNA expression was not significantly altered by dopamine treatment (n=21 donors). ( C ) DNMT3A mRNA expression was not significantly altered by dopamine treatment(n=18 donors). ( D ) DNMT3B mRNA expression was not significantly altered by dopamine treatment (n=17 donors).

Article Snippet: TaqMan Fast Universal Master Mix, and PCR assay probes for DRD1-5 (Hs00265245_s1, Hs00241436_m1, Hs00364455_m1, Hs00609526_m1, Hs00361234_s1), IL1B (Hs01555410_m1), DNMT1 (Hs00945875_m1), DNMT3A (Hs01027166_m1), DNMT3B (Hs00171876_m1), TET2 (Hs00325999_m1), HDAC2 (Hs00231032_m1), HDAC6 (Hs00997427_m1), KDM6B (Hs00996325_g1), KAT2A (Hs00904943_gH), and 18S (4319413E) genes were purchased from Applied Biosystems.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Epigenetic Regulation of Inflammation by Dopamine in Primary Human Macrophages

doi: 10.64898/2026.01.21.700899

Figure Lengend Snippet: Data from were stratified by donor sex and age to assess potential sources of inter-donor variability. ( A ) Dopamine significantly increased TET2 mRNA expression in hMDM from female donors but not male donors (n=10 per group, *p<0.05). ( B ) Dopamine did not significantly alter DNMT1 gene expression in either male (n=10) or female (n=11) hMDM. ( C ) Dopamine treatment showed a decreasing trend in DNMT3A expression in male hMDM (n=9; p=0.0712) and a non-significant increase in female hMDM (n=9). ( D ) Dopamine treatment showed a decreasing trend in DNMT3B expression in male hMDM (n=11; p=0.0823) and a non-significant increase in female hMDM (n=6). ( E ) Dopamine treatment showed an increasing trend in TET2 gene expression in donors under 40 years of age (n=10, p=0.0645), but not donors aged 40 years or older (n=10). ( F-H ) No age-associated differences were observed for dopamine-induced changes in (F) DNMT1, (G) DNMT3A, or (H) DNMT3B expression.

Article Snippet: TaqMan Fast Universal Master Mix, and PCR assay probes for DRD1-5 (Hs00265245_s1, Hs00241436_m1, Hs00364455_m1, Hs00609526_m1, Hs00361234_s1), IL1B (Hs01555410_m1), DNMT1 (Hs00945875_m1), DNMT3A (Hs01027166_m1), DNMT3B (Hs00171876_m1), TET2 (Hs00325999_m1), HDAC2 (Hs00231032_m1), HDAC6 (Hs00997427_m1), KDM6B (Hs00996325_g1), KAT2A (Hs00904943_gH), and 18S (4319413E) genes were purchased from Applied Biosystems.

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: Epigenetic Regulation of Inflammation by Dopamine in Primary Human Macrophages

doi: 10.64898/2026.01.21.700899

Figure Lengend Snippet: Donors from were combined with an independent archival cohort to assess correlations between dopamine receptor expression and epigenetic enzyme expression by qPCR. DRD1 expression was significantly correlated with ( A ) TET2 (n=32, *p<0.05), ( B ) DNMT1 (n=27, **p<0.01), and ( C ) DNMT3A (n=30, ***p<0.001), but not with ( D ) DNMT3B expression (n=27, p=0.0532). DRD5 expression was not correlated with ( E ) TET2 (n=32, p=0.0642) or ( H ) DNMT3B (n=27), but was significantly correlated with ( F ) DNMT1 (n=29, *p<0.05) and ( G ) DNMT3A (n=30, *p<0.05).

Article Snippet: TaqMan Fast Universal Master Mix, and PCR assay probes for DRD1-5 (Hs00265245_s1, Hs00241436_m1, Hs00364455_m1, Hs00609526_m1, Hs00361234_s1), IL1B (Hs01555410_m1), DNMT1 (Hs00945875_m1), DNMT3A (Hs01027166_m1), DNMT3B (Hs00171876_m1), TET2 (Hs00325999_m1), HDAC2 (Hs00231032_m1), HDAC6 (Hs00997427_m1), KDM6B (Hs00996325_g1), KAT2A (Hs00904943_gH), and 18S (4319413E) genes were purchased from Applied Biosystems.

Techniques: Expressing

Journal: bioRxiv

Article Title: Systematic functional dissection of germline noncoding risk variants impacting clonal hematopoiesis

doi: 10.64898/2026.01.21.700897

Figure Lengend Snippet: (A) Schematic of lentiviral MSI2 overexpression constructs and Western blot confirming MSI2 overexpression in K562 cells. (B) Setup of MSI2 overexpression experiments in murine Tet2 -/- hematopoietic stem and progenitor cells (HSPCs). (C) Colony replating assays of sorted GFP + Lin - Sca-1 + c-Kit + (LSK) cells shows increased self-renewal of Tet2 -/- HSPCs upon MSI2 upregulation. (D) Venn diagram showing common and unique differentially expressed genes across different genotypes identified by RNA-Seq (FDR <0.05 and |log2FC| >1.5). “WT MSI2” indicates wild-type HSPCs transduced with MSI2 overexpressing vector; “Tet2 -/- MSI2” indicates Tet2 -/- HSPCs transduced with MSI2 overexpressing vector; “Tet2 -/- HMD” indicates Tet2 -/- HSPCs transduced with empty HMD vector. All groups are compared against WT HSPCs transduced with empty HMD vector, “WT HMD” (n=2 replicates each group). (E) Heatmap showing relative expression of Wnt polarity genes across all groups. (F) Heatmap showing relative expression of Cell cycle genes across groups. (G) Gene set enrichment analysis of MSI2 HyperTRIBE binding targets in Tet2 -/- MSI2 vs. WT HMD. All data are means ± SDs. *p < 0.05, **p < 0.01.

Article Snippet: Bulk RNA-seq was performed in two biological replicates using primary HSPCs (LSK cells) isolated from a well-established TET2 mouse model. Total RNA was extracted using the RNeasy Mini Kit, and poly(A) enrichment and library preparation were carried out with the NEBNext Ultra II Directional RNA Library Prep Kit.

Techniques: Over Expression, Construct, Western Blot, RNA Sequencing, Transduction, Plasmid Preparation, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Systematic functional dissection of germline noncoding risk variants impacting clonal hematopoiesis

doi: 10.64898/2026.01.21.700897

Figure Lengend Snippet: (A) CFU-C replating assay shows the colonies number from sorted murine GFP+ TET2+/- HSPCs. (B-C) Colony images of 2 nd plating from TET2+/- and TET2-/-HSPCs, respectively. (D) Heatmap showing expression of genes across all genotypes highlighting genes uniquely up- and down-regulated in the KO HMD group (E) Heatmap represents mitochondrial electron transport chain genes expression in different experimental groups. (F) Volcano plots show DEGs expression compared WT MSI2 vs WT HMD, Tet2-/- HMD vs WT HMD, Tet2-/- MSI2 vs Tet2-/- HMD, and Tet2-/- MSI2 vs WT HMD. (G) Gene ontology analysis of downregulated and upregulated pathways between Tet2 -/- MSI2 vs. WT HMD.

Article Snippet: Bulk RNA-seq was performed in two biological replicates using primary HSPCs (LSK cells) isolated from a well-established TET2 mouse model. Total RNA was extracted using the RNeasy Mini Kit, and poly(A) enrichment and library preparation were carried out with the NEBNext Ultra II Directional RNA Library Prep Kit.

Techniques: Expressing

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency impairs memory B‐cell and plasma cell differentiation of IgG1+ germinal center B‐cells . (A) Schematic diagram of the protocol of SRBC primary and secondary immunizations. (B) Representative cytometry plot and quantification of IgM+ and IgG1+ GC B‐cells (CD19+CD95+GL7+) from Tet2 ‐WT and Tet2 ‐KO mice upon primary ( n = 8) and secondary immunization ( n = 18) with SRBC. (C) Representative cytometry plot and quantification of IgM+ and IgG1+ MBCs, (Lin (CD4, CD8, Gr1)‐IgD‐GL7‐CD19+CD38+) from Tet2 ‐WT and Tet2 ‐KO mice upon primary and secondary immunization with SRBC (primary immunization: Tet2 ‐WT [ n = 7] and Tet2 ‐KO [ n = 9]; secondary immunization: Tet2 ‐WT [ n = 9] and Tet2 ‐KO [ n = 10]). (D) Representative cytometry histograms and quantification of the expression of CD80 on the cell surface of IgM+ and IgG1+ MBCs from Tet2 ‐WT ( n = 7) and Tet2 ‐KO ( n = 9) mice upon primary immunization with SRBC. (E) Representative cytometry plot of PBs (CD19+CD138+CD43+) from Tet2 ‐WT ( n = 4) and Tet2 ‐KO ( n = 5) mice upon secondary immunization with SRBC. (F) Representative cytometry plot and quantification of IgM+ and IgG1+ PBs (CD19+CD138+CD43+) from Tet2 ‐WT ( n = 4) and Tet2 ‐KO ( n = 5) mice upon secondary immunization with SRBC. All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. GC, germinal center; MBC, memory B cell; PBs, plasmablasts; SRBC, sheep red blood cells.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Clinical Proteomics, Cell Differentiation, Cytometry, Expressing, Two Tailed Test

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: The expansion and differentiation blockade of IgG1+ Tet2 ‐KO GC B‐cell are cell‐autonomous . (A) Schematic diagram of the in vitro differentiation system. (B) Representative cytometry plot and quantification of IgM+ and IgG1+ in vitro iGCB (CD19+ CD95+ GL7+) from Tet2 ‐WT and Tet2 ‐KO mice at D4 ( n = 18) and D8 ( n = 15). (C) Schematic illustration of the in vitro culture system of iGCB expressing a specific Ig isotype. (D) Number of live Tet2 ‐WT and Tet2 ‐KO B‐cells cultured on 40LB with IL21 on Day 8 (D8) ( n = 4). (E) Representative cytometry plots of iPC on Day 8. (F) Quantification of flow cytometry data corresponding to D8 iPC ( n = 4). (G) Number of live iPC (CD19+ CD138+) Tet2 ‐WT and Tet2 ‐KO B‐cells on Day 8 (D8) ( n = 4). All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. D, Day; iGCB, induced germinal center B‐cells, iPC, induced plasma cells; SRBC, sheep red blood cells.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: In Vitro, Cytometry, Expressing, Cell Culture, Flow Cytometry, Two Tailed Test, Clinical Proteomics

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency induces upregulation of IgG1 expression on the cell surface of germinal center B‐cells . (A) Representative cytometry histograms and quantification of the MFI of IgM and IgG1 staining on the cell surface of GC B‐cells (CD19+ CD95+ GL7+) from Tet2 ‐WT ( n = 9) and Tet2 ‐KO ( n = 10) mice upon primary immunization with SRBC. (B) Representative cytometry histograms and quantification of the MFI of IgM and IgG1 staining on the cell surface of GC B‐cells (CD19+ CD95+ GL7+) from Tet2 ‐WT ( n = 9) and Tet2 ‐KO ( n = 9) mice upon secondary immunization with SRBC. (C) Representative cytometry histograms and quantification of the MFI of IgM and IgG1 staining on the cell surface of Tet2 ‐WT ( n = 11 [IgM] and n = 13 [IgG1]) and Tet2 ‐KO ( n = 11 (IgM) and n = 13 (IgG1)) iGCB (CD19+ CD95+ GL7+) at D4. (D) Representative cytometry histograms and quantification of the MFI of IgG1 staining on the cell surface of Tet2 ‐WT ( n = 15) and Tet2 ‐KO ( n = 15) iGCB (CD19+ CD95+ GL7+) at D8. (E) Quantification of the MFI of IgG1 staining on the cell surface of iGCB (CD19+ CD95+ GL7+) from Tet2 ‐WT ( n = 11) and Tet2 ‐KO ( n = 11) mice between D4 and D8. All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. GC, germinal center; iGCB, induced germinal center B‐cells; MFI, mean fluorescence intensity.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Expressing, Cytometry, Staining, Two Tailed Test, Fluorescence

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency induces higher c‐Rel activity in IgG1+ germinal center B‐cells . (A , B) Representative cytometry plots (A) and quantification (B) of intracellular and nuclear localization of c‐Rel and phosphorylated p65 (Ser536) in sorted IgM+ and IgG1+ Tet2 ‐WT and Tet2 ‐KO B cells at D4 of the in vitro GC model ( n = 3). (C , D) Representative cytometry plots (C) and quantification (D) of intracellular and nuclear localization of c‐Rel and phosphorylated p65 (Ser536) in sorted IgG1+ Tet2 ‐WT and Tet2 ‐KO B cells at D8 of the in vitro GC model ( n = 3). (E) Expansion factor compared to empty MSCV condition between D11 and D14 and quantification of MFI of IgG1 in IgG1+ iGCB (GFP+ CD19+ CD138− Fas+ GL7+ IgG1+) at D14 of the iGCB transduction model, after transduction of Tet2 ‐KO B‐cells by an empty MSCV, a MSCV containing shRNA Rel 908, or a MSCV containing shRNA Rel 1869 ( n = 3). For (B , D) , P values were calculated using an unpaired two‐tailed t ‐test. For (E) , P values were calculated using a paired RM one‐way ANOVA test. * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant in all experiments. ANOVA, analysis of variance; GC, germinal center; iGCB, induced germinal center B‐cell; MFI, mean fluorescence intensity; MSCV, murine stem cell virus.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Activity Assay, Cytometry, In Vitro, Transduction, shRNA, Two Tailed Test, Fluorescence, Virus

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Tet2 deficiency induces downregulation of Nfkbia expression in germinal center B‐cells . (A) Normalized gene expression level of Nfkbia by RNAseq of Tet2 ‐WT and Tet2 ‐KO GC B‐cells, data from. ²⁹ (B) Gene expression levels by RT‐PCR of Nfkbia in Tet2 ‐WT and Tet2 ‐KO GC B‐cells at D8 (IgG1+) and D4 (IgM+ and IgG1+) ( n = 4). (C) Quantification of iPC (GFP+ CD19+ CD138+), iGCB (GFP+ CD19+ CD138− Fas+ GL7+), IgG1+ iGCB (GFP+ CD19+ CD138− Fas+ GL7+ IgG1+), and MFI of IgG1 in IgG1+ iGCB at D11 of the iGCB transduction model, after transduction of Tet2 ‐WT B‐cells by a MSCV containing sh Renilla , a MSCV containing shRNA Nfkbia 1224, or a MSCV containing shRNA Nfkbia 1485 ( n = 4). For A and B, P values were calculated using an unpaired two‐tailed t ‐test. For (C) P values were calculated using a paired RM one‐way ANOVA test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant in all experiments. ANOVA, analysis of variance; iGCB, induced germinal center B‐cells; iPC, induced plasma cells; MFI, mean fluorescence intensity; MSCV, murine stem cell virus.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Transduction, shRNA, Two Tailed Test, Clinical Proteomics, Fluorescence, Virus

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: The effect of Vitamin C on plasma cell differentiation of Tet2 ‐WT and Tet2 ‐KO B‐cells . (A) Schematic illustration of Vitamin C treatment in the in vitro iGCB culture system. (B) Number of live Tet2 ‐WT and Tet2‐KO cells cultured on Day 4 (D4) and Day 8 (D8), in the presence and absence of vitamin C ( n = 5). (C) Graph shows iPC (CD19+ CD138+) percentages at D8 upon treatment of Tet2 ‐WT ( n = 6) and Tet2 ‐KO ( n = 6) B‐cells with Vitamin C (250 µM) or vehicle. (D) Quantification of the MFI of IgG1 staining on the cell surface of D8 iGCB (CD19+ CD95+ GL7+) from Tet2 ‐WT and Tet2 ‐KO mice, in the presence and absence of vitamin C ( n = 9). (E) Gene expression levels by RT‐PCR of Nfkbia in Tet2 ‐WT and Tet2 ‐KO iGCB cells at D8 in the presence and absence of vitamin C ( n = 3). (F) Snapshot from UCSC genome browser showing 5hmC mark distribution at the Nfkbia locus (32,380 bp, mm10, chr12: 55.474.813−55.507.192) in D4 iGCB cells, in the presence and absence of vitamin C. All P values were calculated using an unpaired two‐tailed t ‐test, * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, not significant, in all experiments. 5hmC, 5‐hydroxymethylcytosine; D, Day; iGCB, induced germinal center B‐cells; iPC, induced plasma cells; MFI, mean fluorescence intensity.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Clinical Proteomics, Cell Differentiation, In Vitro, Cell Culture, Staining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Fluorescence

Journal: HemaSphere

Article Title: Tet2 deficiency promotes IgG1+ B‐cell expansion and differentiation blockade through deregulation of the Nfkbia –c‐Rel axis

doi: 10.1002/hem3.70296

Figure Lengend Snippet: Diagram depicting the effects of Tet2 inactivation on IgG1 + GC B cells during primary and secondary immunizations, alongside a proposed model of the underlying molecular mechanisms . At the cellular level, Tet2 ‐deficient mice exhibit germinal center (GC) hyperplasia and impaired plasma cell differentiation following T‐dependent immunization. During primary immunization, Tet2 inactivation reduces the generation of IgG1+ GC B‐cells by impairing isotype switching to IgG1 and hindering their differentiation into plasma cells and memory B‐cells. This deficiency enhances the proliferative response of IgG1+ GC B‐cells to external cues, leading to their accumulation during subsequent immunizations and a persistent differentiation blockade. At the molecular level, Tet2 deficiency directly impacts Prdm1 (encoding Blimp1) expression through hypermethylation of its regulatory elements and induces sustained DNA methylation at the Nfkbia locus. This results in the downregulation of Nfkbia , leading to increased c‐Rel activity, which further represses Blimp1 expression. Elevated IgG1 BCR expression likely sustains c‐Rel activation. Consequently, there is an increased proliferation capacity of IgG1+ GC B‐cells, coupled with a blockade in memory and plasma cell differentiation.

Article Snippet: All TaqMan probes were purchased from Applied Biosystems: Prdm1 (Mm00476128), Nfkbia (Mm00477798_m1), Tet2 (Mm00524395), and Rel (Mm01239661).

Techniques: Clinical Proteomics, Cell Differentiation, Expressing, DNA Methylation Assay, Activity Assay, Activation Assay