PQBP1 Affects Lipid Storage Figure 6. Morphological analysis of lipid storage in intestinal cells. A) Worms were stained by culturing from the L1 stage to adult stage on a Nile Red-containing plate. Control worms (N2) show strong stains in intestinal cells. In the T21D12.3 mutants, Nile Red stains intestinal cells weakly and the stain signals in body cavity outside of eggs seem higher than that of N2. Nile Red stains in the T07A9.1 mutants were not changed so 2 remarkable. B) Nile Red staining signals in intestinal cells were analyzed quantitatively by collecting signals from 6 worms (25 mm ). Asterisks indicate p,0.001 in Student’s t-test. Quantitative analysis showed a significant difference between N2 and T21D12.3 mutants but not between N2 and T07A9.1 mutants. doi:10.1371/journal.pone.0004104.g006 disorders. Although the phenotype we observed in this study might be a downstream consequence of essential molecular functions of PQBP1, still this work shows a novel finding to suggest a possible involvement of PQBP1 in the lipid metabolism. Materials and Methods Plasmid construction for transgenic C. elegans expressing venus under the control of T21D12.3 enhancer/promoter An about 6.7 kb DNA fragment containing an operon of T21D12.2 (pat-6) and T21D12.3 gene was amplified from N2 genome DNA with primers 59-GCCTATCAATATCAGGCACA and 59-CTTCGTCCCACCTACATAAC (Fig. 1) with KOD plus (Toyobo). One Adenine wad added to the end of the amplified DNA fragment during reaction. The genome fragment was subcloned into pGEMT-T Easy vector (Promega) and the resultant plasmid (pGEMT-T-T21D12.3) was digested with Pml I (BioLabs) which is unique in the exon 4 of the T21D12.3 gene. Venus gene cDNA was amplified from pFX-Venus vector [13] with primers 59-ATGAT- GATAAGCAATCACGTGGAATGGTGAGCAAGGGCG and 59-TTTCAGCATTATTGCCACGTGTCTTGTACAGCTCGT- CCATGC, then the fragment was subcloned into the Pml I site of pGEMT-T-T21D12.3 with recombinase in-Fusion kit (Clontech). The Venus Open Reading Frame was adjusted to that of T21D12.3 by inserting ‘‘GA’’ to 59 and ‘‘T’’ to 39 end of Venus cDNA, giving rise to the plasmid was named pGEMT-T-T21D12.3-Venus. The junctions between T21D12.3 and Venus were confirmed by sequencing with primers AGAAATTTCCACAACAAGAA and TCTGGACCCTGTGACGTCAT. Generation of transgenic C. elegans pGEMT-T-T21D12.3-Venus (20 mg/ml) and pFX-RXT-myo-2 (20 mg/ml), a marker of pharyngeal muscle to distinguish transgenic worms, were mixed and injected into the germ line of adult worm, according to the method by Mello et al. [27]. After 3 days, fluorescence-positive F1 worms were picked up, and transferred to a new dish. After additional culture for 3–4 days, fluorescence-positive F2 worms were selected and named tmEx1642. Screening of mutant C. elegans of T21D12.3 and T07A9.1 The deletion mutations used in this work were isolated as previously described [15]. Primers used in nested PCR screening for detecting T21D12.3 (tm3004) are as follows; first round: 59- CCGGCGGCGATACCCATTAA-39, 59-CCTAATCAAACGA- CACCGTC-39, second round: 59-GCGATACCCATTAAG- TACGA-39, 59-GTCTGGCTCAACAACTATGG-39. Primers for screening T07A9.1 (tm3045) are as follows; first round: 59- TCGCAAGTCCCTCATCATAG-39, 59- CGAAATAAC- TGTGTGCGCCT-39, second round: 59- ACAGTGAT- GACGTCGTCTAG-39, 59- CTATTGCGATTGAGATGCTC- 39. Sequence analysis of the deleted regions was performed as previously described. These deletion alleles were outcrossed at least twice. PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4104