Figure 2. Expression of Oct4 isoforms in Oct4A-knockout oocytes and embryos.

(a) Real-time RT-PCR Ct values of Oct4 together with the internal control Hprt1. The single-oocyte samples were run with preamplification for 18 cycles and tested with the Fluidigm system. The rest of the groups were tested with the ABI PRISM Sequence Detection System 7900 after preamplification for 10 cycles. The Oct4 Taqman probe-primer set from ABI (Mm00658129_gH, spanning exon 2 and 3) consistently detected background signals in all Oct4A-knockout embryos of all preimplantation stages. (b) Further testing using equally efficient Oct4A specific primers (spanning exon 1 and exon 2) eliminated such background signals in all 3 biological replicates of Oct4A-knockout oocytes (pool of 10 per sample) and 2 replicates of single blastocyst samples, indicating expression of truncated Oct4 transcripts. (c) Gel image of RT-PCR using the same Exon2–3 Oct4 Taqman probe–primer set as a and using the same samples as b. The amplicons from the bright bands were cut out and sequenced. They were found to match the Oct4 reference sequence as showed in d. (e) The same samples were tested by PCR using different primers spanning exons 3 and 4, and again the amplicons were found to match the Oct4 reference sequence as showed in f, further confirming the presence of Oct4 isoforms in Oct4-knockout oocytes and embryos. Values represent the means of 2–5 biological replicates, with error bars representing S.D. in a and the means from 3 technical replicates, a result representative of one biological replicates in b. Uncropped versions of c and e is shown in Supplementary Fig. 5.