266 IDENTIFICATION OF Oct-4 AND Nanog, THE TWO PLURIPOTENCY MARKER GENES IN RABBIT PRE-IMPLANTATION-STAGE EMBRYOS

Abstract

Several genes, including Oct-4 and Nanog, coordinate the embryogenesis of mammalian embryos. Whereas Oct-4 has an activator effect, the Nanog protein blocks the transcription of several genes in early stages; however, the product of these two genes appears parallel and directs early embryogenesis. The goal of this work was to isolate the Oct-4 and Nanog genes from rabbit, based on the sequences of other species published so far. The sequence of known genes has been analyzed, and primers have been designed based on similarity of sequences. Oocyte-to-blastocyt-stage embryos were collected from superovulated rabbits in RNase-free water. Embryonic mRNA was isolated by using the Dynal mRNA isolation KIT (Dynal, Biotech, Oslo, Norway). Real-time PCR was performed in a ABI PRISM^® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction mixture consisted SYBR^® Green PCR Master Mix (Applied Biosystems), 300 nM of each primer, and an 1/8 aliquot of the embryo cDNA in a 25-¼L final volume. The cDNA template was denatured by heating to 95°C for 10 min and amplified by 45 cycles of 95°C for 15 s and 60°C for 1 min, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. The reaction mix was electrophoretically separated to facilitate fragment isolation. In the case of Nanog, a 131-bp fragment was cloned. Sequence analysis revealed significant homology with the mouse sequence. Based on this finding, new primers were designed in order to isolate a larger fragment as well as the genomic copy of the gene. In the case of Oct-4, several combinations of primers were tested, because of the rather conservative sequence of the Oct-4 transcription factor family. As a result, an optimal primer pair was found that yielded a 450 bp fragment, expected according to known sequences. After direct sequencing, there was a high similarity to Oct-4 genes, indicating that the isolated cDNA is probably part of the rabbit Oct-4 cDNA. For further analysis, the cDNA fragments of both genes were isolated (MinElute Gel Extraction Kit; Qiagen, Valencia, CA, USA) and cloned into bacterial plasmids (TOP 10 Cloning KIT; Invitrogen, Carlsbad, CA, USA). At this point, it can be stated that: (1) both genes have been successfully identified in rabbit genome, (2) a method has been developed to detect expression of genes, and (3) gene-specific primers have been produced. Our further goal is to clone the whole coding region of the genes and to identify the sequence. The cloned fragments will be used for in situ hybridization in implanted stage embryo sections.

This research was supported by EU-FP6-MEXT-CT-2003-59582, Wellcome Trust (Grant No. 070246), OTKA T046171, and NKTH BIO-00017/2002.