title: Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos.
abstract: Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.
authors.affiliation: Department of Nanobiomedical Sciences and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University; Insititute of Tissue Regeneration Engineering, Dankook University.
authors.forename: Jae H
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Animal Biotechnology, Sahmyook University.
authors.forename: Yong Ii
authors.initials: YI
authors.lastname: Cho
authors.affiliation: Department of Pharmacy, Sahmyook University.
authors.forename: Sung S
authors.initials: SS
authors.lastname: Choi
authors.affiliation: Department of Nanobiomedical Sciences and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University; Insititute of Tissue Regeneration Engineering, Dankook University.
authors.forename: Hae-Won
authors.initials: HW
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Ajou University.
authors.forename: Churl K
authors.initials: CK
authors.lastname: Min
authors.affiliation: Department of Animal Biotechnology, Sahmyook University; leesj@syu.ac.kr.
authors.forename: Sang J
authors.initials: SJ
authors.lastname: Lee
mesh_terms: D000818:Animals; D001755:Blastocyst; D001757:Blastomeres; D002454:Cell Differentiation; D019070:Cell Lineage; D018076:DNA, Complementary; D047108:Embryonic Development; D005260:Female; D051379:Mice; D011247:Pregnancy
chemical_list: D018076:DNA, Complementary