Cloning and characterization of cat POU5F1 and NANOG for identification of embryonic stem-like cells.
POU5F1 and NANOG play important roles in the maintenance of embryonic stem cell pluripotency. Recently, we isolated cat embryonic stem (ES)-like cells from cat blastocysts generated in vivo. In an effort to identify genetic markers for the characterization of cat ES-like cells, we have determined the coding sequences (CDSs) of cat POU5F1 (cPOU5F1) and NANOG (cNANOG). The sequence identities of cPOU5F1 with orthologous genes of the human and mouse were 92 and 82%, respectively, at the nucleotide level and 94 and 83%, respectively, at the amino acid level. We identified POU-specific and POU homeodomain sequences in the CDS of cPOU5F1. The sequence identities of cNANOG with its human and mouse orthologs were 69 and 68%, respectively, at the nucleotide level and 69 and 58%, respectively, at the amino acid level. We identified a homeodomain, SMAD4 domain and tryptophan repeat domain (W/QXXXX) in the CDS of cNANOG. We examined the expression of cPOU5F1 and cNANOG mRNA in ES-like cells and fibroblast feeder cells by RT-PCR. Transcripts of cPOU5F1 and cNANOG were detected at a high level in ES-like cells. However, these two genes were undetectable in cat fibroblast feeder cells and 6 adult tissues. We also examined ES-like cells by immunocytochemistry and demonstrated that cPOU5F1 and cNANOG are present at high levels in cat ES-like cells and are undetectable in cat fibroblast feeder cells. These results confirmed that cat ES-like cells can be successfully isolated from in vivo-produced blastocysts and that the expression of cPOU5F1 and cNANOG can be used as a biomarker for characterization of cat ES-like cells.
Yu
Xian-Feng
XF
Division of Applied Life Science (BK21 program), Graduate School of Gyeongsang National University, Jinju, Korea.
Kim
Jae-Hwan
JH
Jung
Eun-Ji
EJ
Jeon
Jin-Tae
JT
Kong
Il-Keun
IK
D000595:Amino Acid Sequence; D000818:Animals; D002415:Cats; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000166:cytology*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000502:physiology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger