Journal: The Journal of Clinical Investigation
Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice
Figure Lengend Snippet: Confirmation of Lpcat1 GT in ES cells and generation of mice. ( A ) Top: Diagram of Lpcat1 locus. Boxes denote exons, and intervening lines denote introns. The β-geo selection cassette encoding a β-galactosidase/neomycin fusion protein was inserted in intron 9, approximately 400 bp 3′ of exon 9. f1, f2, and r2 denote primers used to confirm the presence of GT cassette in the Lpcat1 locus by qPCR analysis. Bottom: PCR analysis of the Lpcat1 locus in GT ES cells or parental ES cells (129J/Ola) using 2 separate primer sets. The presence of amplicons in GT ES cells with the absence of signal in parental cells demonstrates trapping of the Lpcat1 locus at exon 9. Vertical line indicates discontinuous lanes in the same gel. ( B ) qPCR analysis of GT ES cells. cDNA from GT ES cells (GT) and parental cells (WT) was subjected to qPCR analysis using 3 separate exon-spanning TaqMan primer/probe sets for Lpcat1 as depicted in the bottom panel: 2/3, 9/10, and 12/13. Data were normalized to Actb and represent 3 independent experiments, each performed in triplicate. Note the difference in signal between 2/3 probe and 9/10 probe in GT cells. † P
Article Snippet: Total RNA for qPCR was isolated and reverse transcribed into cDNA by standard methods. qPCR was performed using TaqMan primer/probe sets (Applied Biosystems) specific for Sftpc (assay ID: Mm00488144_m1), Sftpb (assay ID: Mm00455681_m1), Abca3 (assay ID: Mm00550501_m1), Lpcat1 exons 2/3 (assay ID: Mm00628177_m1), Lpcat1 exons 9/10 (assay ID: Mm00461015_m1), Lpcat1 exons 12/13 (assay ID: Mm01340302_g1), Hspa5 (assay ID: Mm00517691), Fasn (assay ID: Mm01253300_g1), and Scd1 (assay ID: Mm00772290_m1) and Actb (part number: 4352933E) as an internal control.
Techniques: Mouse Assay, Selection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction