rbg vector  (Addgene inc)

Journal: bioRxiv

Article Title: SLAMseq reveals transfer of RNA from liver to kidney in the mouse

doi: 10.1101/2024.05.16.594511

Figure Lengend Snippet: A) Genomic DNA (gDNA) recombination assay. gDNA was extracted from liver, kidney, heart and spleen and used as templates in a PCR assay designed to detect Cre-mediated recombination at the flox-stop-UPRT locus. The design of this assay is shown in . Cre-mediated recombination was detected in the liver but not in kidney, spleen or heart. B) TBG promoter expression. The AAV8-Cre vector contains the TBG promoter. In the RNAseq data obtained in the SLAMseq experiments, these sequences were over-represented only in liver samples, following AAV8-Cre injection. C) Fluorescent reporting of AAV8-Cre activity. The AAV8-Cre vector used to induce Cre recombination was injected into the mTmG fluorescent reporter mouse. Green fluorescence, indicative of Cre recombination, was detected only in hepatocytes and not in the kidney. D) UPRT immunoblot. Recombinant UPRT was detected by immunoblot (probing for the HA tag) in liver (2 micrograms of tissue homogenate per lane) and kidney (20 micrograms). β-actin was probed as a loading control. Cre-mediated UPRT expression was detected in liver but not kidney.

Article Snippet: The AAV8.TBG.PI.Cre.rBG vector (Addgene #107787) was diluted to 6.25 × 10 12 genome copies per microlitre in sterile PBS and stored in small aliquots at -70 °C.

Techniques: Recombination Assay, Expressing, Plasmid Preparation, Injection, Activity Assay, Fluorescence, Western Blot, Recombinant

Journal: bioRxiv

Article Title: SLAMseq reveals transfer of RNA from liver to kidney in the mouse

doi: 10.1101/2024.05.16.594511

Figure Lengend Snippet: A) Protocol for small initial experiment. An AAV8 vector was used to deliver Cre recombinase specifically to hepatocytes in floxed-stop-UPRT mice approximately 4 weeks prior to RNA labelling. Mice were dosed with 4-thiouracil (400 mg per kg) at 36, 24 and 12 hrs prior to cull. B) Liver injury protocol. Mice in the injury group were dosed with paracetamol (300 mg per kg) at 36 hrs prior to cull; the first dose of 4-thiouracil was then given 3 hrs after paracetamol (i.e. 33 hrs prior to cull) and then at 24 and 12 hrs prior to cull. C) Metabolism of 4TU and 4sU. The ribonucleoside 4sU, 4-thiouridine, is imported into all metazoan cells by equilibrative nucleoside transporters and incorporated into nascent RNA. 4-thiouracil, 4TU, must first be converted into thiolated uridine monophosphate (sUMP) before it can be incorporated into RNA. In higher eukaryotes, salvage pathways converting 4TU to sUMP operate with very low efficiency. The expression of recombinant protozoan UPRT permits the efficient conversion of 4TU to sUMP and thence incorporation into RNA molecules.

Article Snippet: The AAV8.TBG.PI.Cre.rBG vector (Addgene #107787) was diluted to 6.25 × 10 12 genome copies per microlitre in sterile PBS and stored in small aliquots at -70 °C.

Techniques: Plasmid Preparation, Expressing, Recombinant

Journal: bioRxiv

Article Title: SLAMseq reveals transfer of RNA from liver to kidney in the mouse

doi: 10.1101/2024.05.16.594511

Figure Lengend Snippet: A) Biotinylation dotblot. Flox-stop-UPRT mice were treated with AAV8-Tbg-Cre and then injected with 4-thiouracil as either intraperitoneal or subcutaneous injections (n = 3 biological replicates in each group). Incorporation of the 4TU label into liver RNA was assessed using a biotinylation assay. RNA samples were biotinylated and then spotted in a dilution series next to a positive control (biotinylated oligo-dT) onto a nylon membrane. The membrane was probed with a Streptavidin-HRP conjugate. B) Mutation rates in SLAMseq data. Liver RNA was alkylated and sequenced in a SLAMseq protocol designed to analyse mRNA. Increased rates of T>C conversion were detected on the positive strand; increased rates of A>G conversion were detected on the negative strand (data not shown). C) T>C conversion rates in mRNA SLAMseq data. T>C conversion rate in liver mRNA. D) Labelled RNAs in SLAMseq data. Labelled RNA transcripts were detected by comparing gene-wise T>C conversion rates between Cre-positive (RNA labelling) and Cre-negative (control) groups using the beta-binomial method and setting a false discovery rate of 0.05. Each point represents a single gene mRNA; genes for which there was a significant between-group difference in T>C conversion rate are shown in red. E) T>C conversion rates in known hepatocyte marker genes. T>C conversion rates were determined in a pre-specified set of “marker genes”, known to be specifically expressed in defined cell types in other datasets. The marker genes are listed in supplemental Table S11. F) T>C conversion rates in small RNA SLAMseq data. Liver RNA was alkylated and sequenced in a SLAMseq protocol designed to sequence small RNA. Increased rates of T>C conversion were detected on the positive strand; increased rates of A>G conversion were detected on the negative strand (data not shown). The rates of T>C conversion are shown. G) T>C conversion rates stratified by RNA biotype. Increased rates of T>C conversion int the RNA labelling group were observed in reads mapping to all small RNA biotypes. H) Labelled RNAs in SLAMseq data. Labelled RNA transcripts were detected by comparing gene-wise T>C conversion rates between Cre-positive (RNA labelling) and Cre-negative (control) groups using the beta-binomial method and setting a false discovery rate of 0.05. I) Labelling of small RNA in liver. Stratified by RNA biotype. J) miR-122, miR-126a and miR-23a labelling. T>C conversion rates in the known hepatocyte-enriched miRNA, miR-122, and the known endothelial-enriched miRNAs, miR-126a and miR-23a. Data derived from male and female mice, n = 3 in each experimental group.

Article Snippet: The AAV8.TBG.PI.Cre.rBG vector (Addgene #107787) was diluted to 6.25 × 10 12 genome copies per microlitre in sterile PBS and stored in small aliquots at -70 °C.

Techniques: Injection, Cell Surface Biotinylation Assay, Positive Control, Membrane, Mutagenesis, Negative Control, Marker, Sequencing, Derivative Assay