Journal: PLoS ONE
Article Title: Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant
Figure Lengend Snippet: Molecular analysis of the Plcd3 mNab mutation. A. Schematic representation of the genomic context of the Plcd3 mNab mutation. The Plcd3 mNab mutation is caused by the insertion of 5.4 kbp of IAP sequences in intron 2 of Plcd3 . Hybridisation with a probe (P) derived from intron 1 using primers 572 and 573 ( Table S1 ) reveals a restriction fragment length polymorphism in BamHI digested genomic DNA of wild-type (+/+), oltSH (+/−) and oltNH (−/−) mice, respectively. The wild-type allele shows a 4.1 kbp fragment and the mutant allele a fragment of 4.5 kbp. The increased length of the mutant fragment is caused by the BamHI site (B) within the inserted IAP. The Plcd3 locus is shown in reverse orientation with respect to the chromosomal DNA, with Plcd3 exon 1 to the left. The intracisternal A particle (IAP) has no env sequences and is inserted in intron 2 in reverse orientation with respect to the transcription of the Plcd3 gene. B. Location of the primers used to analyse the Plcd3 mNab mutant and the wild-type Plcd3 locus (f, forward; r, reverse). On the left, electrophoresis of amplified genomic DNA fragments that are indicative of the wild-type Plcd3 and the Plcd3 mNab allele using the primers indicated. On the right, the PCR products from genomic DNA around Plcd3 intron 2 obtained from wild-type and oltNH mice using primers f1046 and r1049 ( Table S1 ) are shown. The 5.4 kbp long fragment in the oltNH mutant contains the inserted IAP sequence. C. Northern blot analysis of mutant dorsal skin. A DIG-labelled probe derived from the 5′ region of Plcd1 hybridises to transcripts only in phenotypically wild-type animals. A DIG-labelled RNA probe derived from the 3′ region of Plcd3 hybridises to two transcripts of 3 kb and 2.6 kb, respectively (marked by arrowheads). oltSH mice express both transcripts, while oltNH mice show only the 2.6 kb transcript. Note that the wild-type mouse in lane 2 shows the mutant Plcd3 transcript and expresses Plcd1 . Hybridisation with a Gapdh -specific probe as loading control is also shown. D. Western blot analysis of mutant dorsal skin using antibody IF12–15 directed against the catalytic region of PLCD3 protein and 4H 5–9 directed against the PH domain of PLCD3. The phenotypes of the mice (Wt, oltNH , oltSH ), from which the lysates were obtained, are given on top of each lane. The 2.6 kb mutant Plcd3 transcript is translated to a truncated protein of 75 kDa, which is detected by the antibody IF12–15, but not the PH domain-specific antibody 4H 5–9 (lanes 2 and 3). This antibody also bound to an unknown protein of 110 kDa in all samples. Arrowheads indicate the wild-type protein. An immunoblot using antibody against actin is given below as a control. Note that the phenotypically wild-type in lane 2 is the same as the one in lane 2 of the Northern blot in C.
Article Snippet: DIG labelled probes for DNA blot hybridisations were synthesised by amplifying genomic fragments using the PCR DIG probe synthesis kit (Roche).
Techniques: Mutagenesis, Hybridization, Derivative Assay, Mouse Assay, Electrophoresis, Amplification, Polymerase Chain Reaction, Sequencing, Northern Blot, Western Blot