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Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

Authors
Publisher
The American Society for Cell Biology
Publication Date
Source
PMC
Keywords
  • Article
Disciplines
  • Biology
  • Medicine

Abstract

Fig. 2. (A) Genetic diagnosis of PITPα −/− clones. Alb J G et al. Mol. Biol. Cell 2002;13:739-754 Copyright © 2002 The American Society for Cell Biology (A) Genetic diagnosis of PITPα −/− clones. R1- and AB-1–derived PITPα −/+ ES cells were challenged with 2 mg/ml G418 and surviving clones were isolated. Genomic DNA was prepared from −/+ ES cell controls and high G418-resistant ES cell clones and analyzed as detailed in Figure 1B. Diagnostic signatures of the wild-type (+) and targeted (−) PITPα alleles are indicated at right. Approximately 5% of total R1- and AB1-derived −/+ ES cells that passed the high G418 selection exhibited physical signatures consistent with two targeted PITPα alleles as shown. (B) Immunoblot analysis of candidate PITPα −/− clones. ES cell strains were grown to confluence on gelatin-coated tissue culture dishes, harvested, and lysed by repeated trituration through a tuberculin syringe (Pierce Chemical). A sample load of 150 μg of total cell lysate was resolved by SDS-PAGE, and displayed proteins were transferred to nitrocellulose membranes. Membranes were decorated with PITPα-specific antibodies and developed with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham Biosciences). PITPα genotypes are indicated at top. Chicken antibodies specific for PITPα were raised against a peptide corresponding to the C-terminal 15 PITPα amino acids. (C) Measurements of PITPβ levels. Top, an immunoblot experiment performed as described in A with 150 μg of total cell lysate per lane and PITPβ-specific antibodies as probe. The PITPβ-specific antibodies were rabbit polyclonal antibodies directed against the C-terminal 18 residues of this protein and were generously provided by Bruce Hamilton (University of California, San Diego, CA). PITPα genotypes are indicated at top. Bottom, quantitative ELISA assay (see MATERIALS AND METHODS) was used to measure levels of PITPβ in isogenic PITPα +/+, −/+, and −/− ES cell lines (left, PITPα genotype indica

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