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. 2016 Jan 25;6:19480. doi: 10.1038/srep19480

Figure 1. In vitro and in vivo validation of knockdown strategy.

Figure 1

(A) In vitro validation. The specificity and off target effects of pmDMY-AS construct was validated in vitro using COS7 cells via co-transfection of pmDMY-AS construct and the ORF plasmids of either Dmy (specific, solid circles), Dmrt1 (partially specific, solid squared), Sox9a2 (non-specific, grey circles) Gsdf (non specific, open circles) or Sf1 (non specific, open squared). The mean absolute copy numbers of respective gene per 5 ng of RNA are plotted in the graph along with SEM. The significance is indicated by a, b, c, in which different letters indicate the significant difference from other group at p < 0.05. (B,C) In vivo validation. Olvas-eGFP medaka were electroporated with pEGFP-AS plasmid (carries an antisense eGFP sequence) and continuous microscopic visualization (from 3days after fertilization (daf) to 5 days after hatching (dah)) of GFP expression in individual embryos was performed to ascertain the changes of GFP production in control (B, 6 daf) and pEGFP-KD (pEGFP-AS electroporated) groups (C, 6 daf). (D,E) Sharp fall in Gfp mRNA production was assessed through real-time PCR at 20 dah (days after hatching). Non-specific effects of pEGFP-AS construct were analysed by measuring the mRNA amount of Dmy, Dmrt1, Spo11, and ERα. Data are presented as both individual values (with dots and triangles) and mean of 10 individual (white and grey columns for control and knockdown groups, respectively) fish of each sex.