Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides.
Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down-regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an alpha-catenin specific MO, to down-regulate gene expression during murine preimplantation development. Experiments applied to both two- and eight-cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 microM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two-cell embryos with Lipofectin and 20 microM alpha-catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in alpha-catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development.
Siddall
Laura S
LS
Department of Obstetrics and Gynaecology, The University of Western Ontario, Rm 238, Medical Science Building, London, Ontario, Canada.
Barcroft
Lisa C
LC
Watson
Andrew J
AJ
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051178:alpha Catenin
C495272:Ctnna1 protein, mouse; D003598:Cytoskeletal Proteins; D008244:Lysophosphatidylcholines; D009025:Morpholines; D016376:Oligonucleotides, Antisense; D010714:Phosphatidylethanolamines; D012333:RNA, Messenger; D051178:alpha Catenin; C020888:1,2-dielaidoylphosphatidylethanolamine