Journal: bioRxiv

Article Title: HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo

doi: 10.1101/2024.06.10.598329

Figure Lengend Snippet: (A) Mice were injected with 3 μg of the internal control plasmid encoding Renilla luciferase and 15 μg of reporter plasmid (pGYL1 or pGYL17, see Table S1E) encoding Firefly luciferase by HDI. Livers were collected and assayed for firefly (FF) and Renilla (Rluc) luciferase activity at two time points: 20 h and 7 d after HDI. Data represent mean +/- SEM. Shown are the changes in each activity over time for mice injected with either a mixture of pGYL1 + pGYL18 plasmid DNA, or pGYL17+ pGYL18 plasmid DNA. Luciferase activity consistently decreased from 20 h to 7 d post-injection. The firefly luciferase activity from pGYL1 showed a greater decrease from 20 h to 7 d (212-fold) than that of pGYL18 (30-fold), while Renilla luciferase activity showed a similar decrease (∼9- fold). Each data point represents one mouse liver. See detailed data in Table S2B. (B) Of 36 mice, twenty-three mice were successfully injected with ∼3 ml of injection solution. 4 mice (marked as hollow circles) experienced slight resistance at the very end of the 3 ml injection, which often resulted in lower luciferase activity, as shown. Based on these luciferase activity data, we excluded from further analysis three of the lowest liver samples (marked as pink dots; XE86-C6, XE86-D5 and XE86-F5), whose Renilla luciferase activity ranged from 1,358 to 2,605; these could represent mice with poor HDI injections. Background signal, based on liver extract luciferase activity from mice without HDI transfection is marked by gray circles (background activity ∼100).

Article Snippet: Plasmids hSTARR-seq_SCP1 vector (Addgene plasmid # 99292, RRID:Addgene_99292; listed as pTYC6 in Table S1E) and hSTARR-seq_ORI vector (Addgene plasmid # 99296, RRID:Addgene_99296; pTYC7 in Table S1E) were gifts from Alexander Stark [ ]. hSTARR-seq_SCP1 was modified to generate plasmid pTYC10 by removing the SCP1 promoter sequence by BanII digestion and replacing it with a minimal mouse Alb promoter sequence, comprised of 318 nt obtained from pLIVE plasmid (cat. #MIR 5420, Mirus Inc, Madison, WI), where the Alb promoter HNF1 TF binding site was changed from GATC to AATC to prevent bacterial methylation and to enable expression of the full intrinsic activity of the Alb promoter in HDI-transfected livers [ ].

Techniques: Injection, Plasmid Preparation, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo

doi: 10.1101/2024.06.10.598329

Figure Lengend Snippet: (A) Mouse study design. Reporter plasmid encoding firefly luciferase driven by Alb minimal promoter, or by 1 of the 4 other individual DHS sequences indicated along the X-axis of panel B, was mixed with a normalization control plasmid encoding Renilla luciferase, then delivered to mouse liver by HDI on day 0, followed by TCPOBOP injection on day 6. (B) Normalized luciferase reporter activity for the DHS sequences shown along the X-axis, with TCPOBOP or corn oil (vehicle control) treatment (mean +/- SEM for n=3-7 individual livers per group; each symbol represents a single mouse liver). TCPOBOP-induced reporter activity was assessed by t-test: **, p <0.01; ***, p<0.001 for mean of corn oil control vs TCPOBOP group activity. The mean luciferase activity of the empty vector control group was set = 1 for normalization. “+”, TCPOBOP stimulates chromatin opening at the DHS . (C) Impact of time after HDI (20 h vs. 7 d) on signal-to-noise ratio (S/N) for liver luciferase reporter activity. S/N was calculated as the ratio of normalized activity for Alb enhancer plasmid (pGYL17) vs empty vector plasmid (pGYL17) at each time point. Data shown are mean +/- SEM values, with the empty vector at 20 h group mean set = 1. t -test: **, p <0.01; n=3-7 per group. (D) qPCR analysis of interferon-related response genes showing the mean (+/- SEM) expression level of each gene in livers from mice treated by HDI to deliver plasmid DNA or vehicle control. Plasmids delivered by HDI (see Table S1E): HDI control = pGYL18 only; HDI plasmid = pGYL18 + pGYL17 or pGYL18 + pGYL1. Significance: t -test comparing livers with HDI delivery of reporter plasmid DNA and collected 20 h vs. 7 d later: *, p <0.05; **, p <0.01 for n=3-7 livers/group. See Table S1A for qPCR primers.

Article Snippet: Plasmids hSTARR-seq_SCP1 vector (Addgene plasmid # 99292, RRID:Addgene_99292; listed as pTYC6 in Table S1E) and hSTARR-seq_ORI vector (Addgene plasmid # 99296, RRID:Addgene_99296; pTYC7 in Table S1E) were gifts from Alexander Stark [ ]. hSTARR-seq_SCP1 was modified to generate plasmid pTYC10 by removing the SCP1 promoter sequence by BanII digestion and replacing it with a minimal mouse Alb promoter sequence, comprised of 318 nt obtained from pLIVE plasmid (cat. #MIR 5420, Mirus Inc, Madison, WI), where the Alb promoter HNF1 TF binding site was changed from GATC to AATC to prevent bacterial methylation and to enable expression of the full intrinsic activity of the Alb promoter in HDI-transfected livers [ ].

Techniques: Plasmid Preparation, Luciferase, Injection, Activity Assay, Expressing

Journal: bioRxiv

Article Title: HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo

doi: 10.1101/2024.06.10.598329

Figure Lengend Snippet: Mice were injected with vehicle control (No STARR-seq reporter, plasmid pGYL18 only), STARR-seq vector with ORI promoter (STARR_proORI; STARR-TYC5 +pGYL18), STARR-seq vector using SCP1 as a promoter (STARR_proSCP1; STARR-TYC4 + pGYL18), and STARR-seq vector using Alb minimal promoter (STARR_proALB; STARR-TYC6 + pGYL18). See Table s1E for plasmid details. Shown are average gene expression for the indicated interferon-related genes, as determined by qPCR of extracted liver RNA. No significant difference was found in comparisons to the No STARR-seq reporter control ( t -test, n=3-4). Livers were harvested 7 d after HDI, at which time hepatocytes recover from initial damage induced by HDI . Data represent mean +/- SEM. The level of gene expression in the No STARR-seq reporter group (HDI vehicle-injected) was set = 1 for normalization. 1

Article Snippet: Plasmids hSTARR-seq_SCP1 vector (Addgene plasmid # 99292, RRID:Addgene_99292; listed as pTYC6 in Table S1E) and hSTARR-seq_ORI vector (Addgene plasmid # 99296, RRID:Addgene_99296; pTYC7 in Table S1E) were gifts from Alexander Stark [ ]. hSTARR-seq_SCP1 was modified to generate plasmid pTYC10 by removing the SCP1 promoter sequence by BanII digestion and replacing it with a minimal mouse Alb promoter sequence, comprised of 318 nt obtained from pLIVE plasmid (cat. #MIR 5420, Mirus Inc, Madison, WI), where the Alb promoter HNF1 TF binding site was changed from GATC to AATC to prevent bacterial methylation and to enable expression of the full intrinsic activity of the Alb promoter in HDI-transfected livers [ ].

Techniques: Injection, Plasmid Preparation, Expressing