Inhibition of Oct4 expression in mouse preimplantation embryos using morpholino antisense oligonucleotides.
Morpholino oligonucleotides (MO) can induce gene silencing by binding to a target mRNA and inhibiting its translation, and this technique has been especially successful in studies of embryonic development in various vertebrates. But in mice MO-induced downregulation of target genes has not been widely reported. In this study, we examined whether MO delivery using ethoxylated polyethylenimine (EPEI) delivery reagent is useful for silencing gene expression in the mouse preimplantation embryo, by targeting endogenous gene Oct4. To optimize the conditions for MO delivery, we examined the MO concentration, the EPEI concentration, the treatment time, and the number of MO treatments. The MO treatment was performed at the 2-cell, the morula, the blastocyst, and the hatched blastocyst stage. We first determined the optimal conditions for MO delivery into the nucleus using fluorescein isothiocyanate (FITC)-labeled MO, and demonstrated that treatment with a combination of 20 microM MO and 0.56 microM EPEI for 3 hrs produced effective MO delivery. MO-induced downregulation of Oct4 was then examined. Two-step MO treatment at the 2-cell and blastocyst stages successfully suppressed Oct4 expression. This MO treatment resulted in marked reduction of Oct4 protein at the blastocyst stage. After cultivation of blastocysts for further 4 days, derivatives of embryos either differentiated to trophoblastic cells or showed developmental arrest at the blastocyst. This phenocopy is similar to Oct4-deficient embryos. Overall, our results indicate that MO delivery with EPEI is an effective tool for analyzing gene function in mouse preimplantation embryos.
Tsuji
Isao
I
Department of Obstetrics and Gynecology, Kinki University of Medicine, Osaka-Sayama, Osaka, Japan. Issa.and.ryoma@m2.dion.ne.jp
Mitani
Tasuku
T
Mitsuhashi
Akiyo
A
Watanabe
Yo
Y
Hosoi
Yoshihiko
Y
Hoshiai
Hiroshi
H
D000818:Animals; D001755:Blastocyst / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D009025:Morpholines / Q000737:chemistry; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism
D009025:Morpholines; D050814:Octamer Transcription Factor-3; D016376:Oligonucleotides, Antisense; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger