Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Transcriptional Regulation of Lung Cytidylyltransferase in Developing Transgenic Mice

doi: 10.1165/rcmb.2005-0401oc

Figure Lengend Snippet: Figure 6. CCT transcriptional activity in vitro. (A and B) Fetal lung explants isolated from CCT promoter–reporter transgenic mice were cultured in chemically defined medium. After 24 or 48 h, tissues were harvested for analysis of CCT and -galactosidase activities (A) or CCT, - galactosidase, and -actin content by immunoblotting (B). In A, CCT and -galactosidase activities are expressed as pmoles/min/mg protein and RLU/mg protein, respectively. In B, the far left lanes in the top and middle rows represent -galactosidase and CCT standards, respectively. In C, rat fetal (Day 18), newborn, and adult type II alveolar epithelial cells were isolated, and nucleofection was performed on 2 106 freshly isolated cells by electroporation. Suspended cells were co-transfected with CCT promoter fragments (1,867/71 or 169/71) coupled to luciferase (1.5 g), and a pSV–-galactosidase (0.5 g) plasmid to normalize for transfection efficiency. After electroporation, cells were cultured for an additional 24 h before analysis for luciferase and -galactosidase activities. Promoter activities in primary type II cells are expressed as relative light units calculated as ratios of luciferase to -galactosidase and additionally corrected for transfectional efficiencies as determined by GFP labeling. The data are from type II cells isolated from 20 fetal pups, 22 neonates, and two adult rats. (D) Immunoreactive CCT levels and CCT activity was assayed in freshly isolated rat primary type II cells during development. *P 0.05 versus other groups. In A, †P 0.08 or *P 0.05 versus 24 h samples by t test. In C, **P 0.01 versus all other groups and *P 0.05 versus fetal (Day 18) or adult promoter activity by ANOVA.

Article Snippet: The Basic Nucleofector Solution for primary mammalian epithelial cells, a plasmid construct encoding green fluorescence protein (GFP) driven by CMV (pmaxGFP), and electroporation apparatus were provided by Amaxa (Gaithersburg, MD).

Techniques: Activity Assay, In Vitro, Isolation, Transgenic Assay, Cell Culture, Western Blot, Electroporation, Transfection, Luciferase, Plasmid Preparation, Labeling