Identification Of Nanog And Oct4 Gene In Mylopharyngodon Piceus And Establishment Of A Muscle Cell Line From Oryzias Latipes

As the first species of Four Major Chinese Carps,Mylopharyngodon piceus is one of the dominant varieties of freshwater aquaculture with great significance in economics and science in China.Currently,most of the researches on M.piceus have been mainly focused on artificial breeding,nutrition and feedstuff,disease control and prevention but lack of some basic data on molecular level.Induced pluripotent stem cell possesses unlimited self-renewal capacity and could differentiate into different kinds of cell types under a certain condition.Nanog and Oct4 gene are important homeodomain transcription factors responsible for pluripotency and self-renewal and participate in the induction of iPS as vital roles in mammals.In order to induce fish iPS,we have isolated and characterized the nanog,oct4 gene and nanog promoter sequence in M.piceus,analysed their gene structures,expression patterns in diverse adult tissues and early developmental embryos and proved their maternal inheritance which was distinguished from mammals.In addition,we have established 1 somatic cell line from Oryzias latipes,studied its basic biological characteristics and examined the activities of nanog promoter with 3 different length in O.latipes in muscle cell line by Luciferase Reporter Assay.Here are the results:1)The 3295 bp gDNA sequence and 1550 bp cDNA of Mpnanog were isolated which was composed of 4 exons encoding a protein of 390 amino acid residues.A multiple protein sequence alignment exposed that MpNanog possessed a conserved 63residues homeodomain and the similarity between MpNanog and Nanog from Megalobrama amblycephala was up to 95%.A phylogenetic tree showed that MpNanog protein was clustered together with other fish and separated from tetrapods.2)RT-PCR indicated that the expression of Mpnanog was detected from unfertilized egg to blastula stage with a high-level signal while a sharp drop of the signal occurred from gastrula stage.It also showed a specific expression in ovary.The fluorescence in situ hybridization?FISH?further confirmed the Mpnanog transcripts were diffused in the cytoplasm of oogonia?stage I?and primary oocytes?stage II?and the whole mount in situ hybridization?WISH?demonstrated that Mpnanog positive signals were present in all blastomeres of these early embryos.3)The 1.8 kb 5'transcriptional regulatory sequence of Mpnanog was isolated and dual-luciferase plasmid cassettes of 3 different length of Mpnanog promoter were constructed,termed as psi-CHECK-2-Mpnanog-1775?1070?550.And then the activities of 3 dual-luciferase plasmids in somatic cell line were examined by Luciferase Reporter Assay.4)The 3412 bp cDNA of Mpoct4 was isolated which was composed of 5 exons encoding a protein of 472 amino acid residues;A multiple protein sequence alignment exposed that MpOct4 possessed a conserved POU_S domain and a POU_HDD homeodomain.The similarity between MpOct4 and Oct4 from Megalobrama amblycephala was up to 98.31%.A phylogenetic tree showed that MpOct4 protein was clustered together with other fish and separated from tetrapods.5)RT-PCR indicated that the expression of Mpoct4 was detected from unfertilized egg to gastrula stage with a high-level signal while a sharp drop of the signal occurred from neurula stage.It also showed a specific expression in ovary.The fluorescence in situ hybridization?FISH?further confirmed the Mpoct4 transcripts were diffused in the cytoplasm of oogonia?stage I?and primary oocytes?stage II?and the whole mount in situ hybridization?WISH?demonstrated that Mpoct4 positive signals were present in all blastomeres of these early embryos.6)The muscle cell line developed from O.latipes termed as OLM was established.The cryopreservation and viability,growth curve,species identification,chromosomal analysis and transfection by liposome of this cell line were characterized and analysed.The results indicated OLM was indeed derived from O.latipes,of which the predominant chromosomal number was 48.Moreover,OLM exhibited a typical�S�shape and the transfection efficiency of OLM was up to 40%.In conclusion,we have isolated and characterized the homologous pluripotent gene Nanog and Oct4 of mammals in M.piceus and validated their maternal inheritance;Meanwhile,we have successfully developed 1 somatic cell line from O.latipes with high transfection efficiency and tested the activities of Mpnanog promoter by Luciferase Reporter Assay,which could provide a useful tool for fish iPS research.