Journal: Molecular and Cellular Biology
Article Title: Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor ▿
Figure Lengend Snippet: Generation and analysis of mice expressing A-USF. (A) DNA construct pITRp543f2A-USF4 used to generate transgenic mice expressing dominant-negative USF (A-USF). The A-USF coding region is under the control of the human β-globin gene promoter (β-P) and 3′ enhancer (3′E) as well as human LCR elements HS2 and HS3 plus flanking DNA. The DNA construct is flanked on either site by insulator elements derived from the chicken β-globin gene locus (cHS4). (B) SYBR green stain of the RT-PCR analysis of A-USF expression in transgenic (founders I and III and line II F 1 littermates 1 to 3 [II/1 to II/3]) and wild-type (WT) mice. RNA was isolated from the spleens of phenylhydrazine-treated mice, reverse transcribed, subjected to PCR analysis with primers specific to the A-USF coding region, and electrophoresed in 5% Tris-borate-EDTA polyacrylamide gels. (C) Western blot analysis of A-USF expression in transgenic or wild-type mice. Protein was isolated from the spleen or liver of phenylhydrazine-treated mice and subjected to Western blot analysis using an antibody against USF1, which also detects A-USF. (D) qRT-PCR analysis of β maj -globin gene expression in spleens of A-USF transgenic line II F 1 littermates, A-USF founder mouse I, and wild-type mice. Data from the three line II F 1 littermates were combined and are designated II/1/2/3. GAPDH was used as a loading control, and results from samples were normalized to those of the wild type. (E) ChIP analysis of RNA Pol II and USF2 interactions with the β maj -globin gene promoter control mice (WT) and transgenic mice (A-USF). Spleens taken from two phenylhydrazine-treated F 1 females (derived from line II) or wild-type mice were homogenized and subjected to ChIP analysis using antibodies against IgG, RNA Pol II, or USF2. Error bars reflect standard deviations from two independent experiments.
Article Snippet: RNA was subjected to analysis by quantitative real-time reverse transcription PCR (qRT-PCR) or reverse transcription PCR (RT-PCR) using the MyiQ (Bio-Rad) system, and reactions were carried out using the iQ SYBR green super mix (Bio-Rad). qRT-PCR conditions were the following: 95°C for 5 min, followed by 40 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 1 min (readings were taken after every cycle).
Techniques: Mouse Assay, Expressing, Construct, Transgenic Assay, Dominant Negative Mutation, Derivative Assay, SYBR Green Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation