FIG. 3.

Knockdown of the Ppp1cc gene in differentiating spermatogonia/early spermatocytes by the Cre-Lox method. a) Genomic PCR showing detection of the floxed allele. Lane 1: absence of the floxed allele is identified by a single 600-bp band in wild-type mice; lane 2: the presence of the homozygous floxed allele is detected as a single 750-bp band; lane 3: mice heterozygous for the floxed allele (the center band is a nonspecific PCR amplification product). The schematic exon-intron organization of the Ppp1cc wild-type allele and the floxed allele are denoted by arrows corresponding to their respective PCR bands. b) RT-PCR confirming the specificity of expression of the Cre transgene driven by the Str8-Cre promoter. For all the tissues included in the study, Cre message could only be detected in testis. Br, brain; Spl, spleen; Kid, kidney; Liv, liver; Ts, testis. c) Northern blot analysis confirming the targeted deletion of the Ppp1cc gene in the testis is shown. Ppp1cc2 mRNA was drastically reduced in Ppp1cc^Δflx/Δflx; Tg^Str8-Cre mice compared to control (single-headed black arrow). The double-headed arrow shows the presence of a ∼1.0-kb truncated message produced as a result of splicing occurring between exon 1 and exon 4 (the putative splicing event is shown below the panel as schematic). It is to be noted that Ppp1cc1 mRNA remained almost unaltered in both GcKO and control mice.