Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Induction of SLE autoantibodies occurs in the presence of LPS or Poly I:C, but not other TLR ligands. Wild type (WT) C57BL/6 mice received five immunizations with β2GPI in combination with one of the following TLR ligands: TLR2 : PAM2, FSL-1 (lipoprotein); TLR3 : Poly I:C (dsRNA); TLR4 : LPS; TLR7: IMQ (imiquimod); or TLR9 : CpG-ODN (ssDNA). Serum autoantibodies were detected by ELISA. Bars indicate the mean value +/standard error (S.E.) per group (n = 5 mice/group), and dots indicate values for individual mice. Autoantibody levels for control mice immunized with PBS and TLR ligand were OD 405 < 0.1. The OD 405 + 3 S.D. for unimmunized mice was ≤0.1 for all autoantibodies assayed. (A) Anti-β2GPI and anti-CL antibodies, and (B) hallmark SLE autoantibodies (anti-DNA, anti-Ro/SS-A, anti-La/SS-B, and anti-Sm) were analyzed statistically in separate groupings. For each of the two autoantibody categories, each different TLR ligand (immunized with β2GPI) was compared to the other TLR ligands by 2-way ANOVA, followed by a Tukey's multiple comparison test. ns = not significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: TLR ligands Pam2CSK4 (PAM2), FSL-1, Pam3CSK4 (PAM3), Poly I:C (HMW), imiquimod-R837 (IMQ), and CpG-ODN-1555 + 1466 (CpG-ODN) were from InvivoGen (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Schematic of TLR-2 reporter allele structure and outcomes following editing. The allele includes two open reading frames encoding different fluorescent proteins; an upstream mVenus (green) in the +1 frame and downstream TagRFP (red) in the +3 frame linked by a P2A sequence. The mVenus cassette is interrupted by a 108 bp polylinker sequence that includes several guide target sequences and a stop codon (blue square). Thus in its native configuration, neither fluorescent protein is expressed. Following CRISPR/Cas9 editing, repair via NHEJ will result in expression of TagRFP if the frame shift results in a -2 deletion, or multiple thereof. Alternatively, HDR can be detected with the inclusion of a plasmid donor designed to repair the mVenus gap. b) Mouse embryo reporter validation workflow. Fertilized zygotes from TLR-2 reporter mice (typically male homozygous to WT female) are either microinjected or electroporated with Cas9 RNP with or without a plasmid donor for HDR. The embryos are then cultured to the blastocyst stage where the outcome can be scored by fluorescent imaging, and followed up by PCR-Sanger sequencing to confirm the identify of specific edits.

Article Snippet: TLR-2 mice (B6.129S6- Gt(ROSA)26Sor tm 12 (CAG–Venus*,–TagRFP*) Rkuhn /MurrJ (JAXStrain # 034033; RRID:IMSR_JAX:034033) were generated with a R26-GFP_KI-TLR2 (“traffic light reporter”) targeting vector was designed to insert the TLR reporter construct (“TLR-2”) into the Gt(ROSA)26Sor locus: the TLR reporter construct (“TLR-2”) containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) .

Techniques: Sequencing, CRISPR, Expressing, Plasmid Preparation, Biomarker Discovery, Cell Culture, Imaging

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Position of guides tested for the TLR-2 allele relative to the 108bp polylinker (gray) and TGA stop codon (blue). b) Percentage of TLR-2 heterozygous blastocysts showing visible expression of TagRFP following editing. Mouse zygotes electroporated with Cas9 RNPs with the guides listed on the X axis were cultured to the blastocysts stage and scored for expression of TagRFP, with corresponding levels of overall editing presented in (c) . Of note, the low number of blasts showing TagRFP fluorescence relative to the editing rate for R26-1 reflects its cut position 3’ to the stop codon in the polylinker. d) Percentage of TLR-2 homozyogous blastocysts with visible expression of TagRFP following editing and corresponding editing percentages shown in (e) . Data are representative of at least two technical replicates per guide and plots are showing mean and standard deviation of replicates with total number of blasts assessed across all replicates below each bar.

Article Snippet: TLR-2 mice (B6.129S6- Gt(ROSA)26Sor tm 12 (CAG–Venus*,–TagRFP*) Rkuhn /MurrJ (JAXStrain # 034033; RRID:IMSR_JAX:034033) were generated with a R26-GFP_KI-TLR2 (“traffic light reporter”) targeting vector was designed to insert the TLR reporter construct (“TLR-2”) into the Gt(ROSA)26Sor locus: the TLR reporter construct (“TLR-2”) containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) .

Techniques: Expressing, Cell Culture, Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) To confirm the TLR-2 allele can be activated in all cells and tissues, we generated a germline model following targeting with Cas9 RNP and R26-52 guides. Founders harboring a -14 bp frameshift allele were bred to generate N1 mice. b) Imaging of 12 tissues showing widespread and consistent activation of TagRFP expression. Scale bar = 200 microns.

Article Snippet: TLR-2 mice (B6.129S6- Gt(ROSA)26Sor tm 12 (CAG–Venus*,–TagRFP*) Rkuhn /MurrJ (JAXStrain # 034033; RRID:IMSR_JAX:034033) were generated with a R26-GFP_KI-TLR2 (“traffic light reporter”) targeting vector was designed to insert the TLR reporter construct (“TLR-2”) into the Gt(ROSA)26Sor locus: the TLR reporter construct (“TLR-2”) containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) .

Techniques: Generated, Imaging, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: Embryo culture testing of SauCas9 guide efficiency for both fluorescence activation (a) and editing efficiency (b) . Plots are showing mean and standard deviation of replicates with total number of blasts assessed across all replicates below each bar. SaC9-g3 showed the best performance with ∼25% fluorescence and an 80% editing rate. c) AAV9 vectors encoding SauCas9 and guide SaC9-g3 were injected IV into 10 week old adult TLR-2 mice (1 x 10 11 vg/mouse) and tissues were harvested 4 weeks later (n=8, including controls). Genotyping of the liver (d) showed up to 30% editing of the TLR-2 allele of which 1/3 were in the TagRFP frame. Mean editing and Standard Deviation is shown for the AAV9-treated animals. e) Immunofluorescence of the liver of AAV9-treated male and female mice, showing substantial RFP activation versus saline control. Scale bar = 200 microns.

Article Snippet: TLR-2 mice (B6.129S6- Gt(ROSA)26Sor tm 12 (CAG–Venus*,–TagRFP*) Rkuhn /MurrJ (JAXStrain # 034033; RRID:IMSR_JAX:034033) were generated with a R26-GFP_KI-TLR2 (“traffic light reporter”) targeting vector was designed to insert the TLR reporter construct (“TLR-2”) into the Gt(ROSA)26Sor locus: the TLR reporter construct (“TLR-2”) containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) .

Techniques: Embryo Culture, Fluorescence, Activation Assay, Standard Deviation, Injection, Immunofluorescence, Saline, Control

Journal: bioRxiv

Article Title: Novel mouse reporter models for the detection of genome editing events in vivo

doi: 10.64898/2026.04.29.721708

Figure Lengend Snippet: a) Schematic of the TLR-7 allele, showing the shortened polylinker sequence (30 nt) as compared to TLR-2 including a stop codon (blue), that can be repaired with an ssODN. b) Mouse blastocyst validation for NHEJ repair shows high editing rates (>95%) with an expected 1/3 of edits resulting in a shift to the +3 frame to produce the red fluorescent signal. c) Validation of HDR functionality of the allele, showing ∼30% repair indicated by mVenus fluorescence and a similar level of NHEJ activation of Katushka2S. Some blastocysts showed mosaic activation of both cassettes. Plots are showing mean and standard deviation of replicates with total number of blasts assessed across all replicates below each bar. d) Germline indel generation (2 base pair deletion) demonstrates robust, widespread Katushka2S activation in all tissues. Scale bar = 200 microns.

Article Snippet: TLR-2 mice (B6.129S6- Gt(ROSA)26Sor tm 12 (CAG–Venus*,–TagRFP*) Rkuhn /MurrJ (JAXStrain # 034033; RRID:IMSR_JAX:034033) were generated with a R26-GFP_KI-TLR2 (“traffic light reporter”) targeting vector was designed to insert the TLR reporter construct (“TLR-2”) into the Gt(ROSA)26Sor locus: the TLR reporter construct (“TLR-2”) containing a CAG promoter Venus*-P2A-TagRFP-bovine hGH polyA, in a reading frame that is shifted by 2 bp (+3) .

Techniques: Sequencing, Biomarker Discovery, Fluorescence, Activation Assay, Standard Deviation