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Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation

Fig 4

Single- and double-knockdown of WT1 and GATA4 in XX and XY gonads.

(A-D) XX and XY gonads were dissected from murine embryos (12.5 dpc) and incubated for 72 h with Wt1 and/ or Gata4 vivo-morpholinos. Transfection with appropriate mismatch vivo-morpholinos served as a negative control. For double-knockdown of WT1 and GATA4, vivo-morpholinos were applied simultaneously. (A) Efficient knockdown of WT1 and/or GATA4 was assessed by immunoblot analysis. (B-D) Relative transcript levels were determined by qRT-PCR and normalized to Sdha transcripts according to the 2ΔΔCt method [34]. Results are shown as fold differences between cultures treated with mismatch vs. Wt1- (B) and mismatch vs. Gata4-morpholino (C), respectively. Panel D represents the effect of the double-knockdown of WT1 and GATA4. Error bars represent S.E.M. from independent biological replicates (n ≥ 5). scale = log2. Statistical differences are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0005, paired t-test. n.d., not detectable.

Fig 4