Fig. 4

The morpholinos targeting the eif3ha or eif3hb transcripts demonstrate specificity. A: In vitro transcription-translation assays were performed in the presence of specific morpholinos as follows: translational blocking morpholino for eif3ha and eif3hb are represented as 3ha-TB and 3hb-TB, respectively, while the splice blocking morpholino for each transcript is represented as 3ha-SB or 3hb-SB, respectively. The nonspecific morpholinos (TB morpholino for the other eif3h gene) were also used as indicated. A control reaction without any morpholino (No MO) was also included. The specific DNA constructs used were as indicated. B: Semiquantitative reverse transcriptase-polymerse chain reaction (RT-PCR) assays were carried out using total RNA isolated from splice morphants for eif3ha or eif3hb, as shown. In each case, RT-PCR results for the other eif3h gene was also included to exclude the possibility of cross-targeting by the morpholinos. It should be noted that compared with eif3ha, a very low level of eif3hb transcripts is observed at 24 hours postfertilization (hpf), in accordance with the in situ hybridization data and RT-PCR analyses shown in Fig. 3 and Fig. 2, respectively. Embryos were injected with the indicated amounts of SB MOs specific for either eif3ha or eif3hb. A wild-type control (WT) was also included. β-actin (Ac) was used as an internal control.