Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos.
To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.
Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine,  Hiroshima University, Minami-ku, Hiroshima 734-8553,  Japan.
Hiroshi
H
Ochiai
Naoaki
N
Sakamoto
Kazumasa
K
Fujita
Masatoshi
M
Nishikawa
Ken-ichi
K
Suzuki
Shinya
S
Matsuura
Tatsuo
T
Miyamoto
Tetsushi
T
Sakuma
Tatsuo
T
Shibata
Takashi
T
Yamamoto
D000595:Amino Acid Sequence; D000818:Animals; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008648:Mesoderm / Q000166:cytology / Q000502:physiology; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D010641:Phenotype; D061251:Primary Cell Culture; D012617:Sea Urchins / Q000196:embryology / Q000235:genetics*; D019076:Transgenes / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
D049452:Green Fluorescent Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases; D000072481:DNA Ligase ATP