The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo.
For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo.
Ochiai
Hiroshi
H
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan.
Sakamoto
Naoaki
N
Suzuki
Kenichi
K
Akasaka
Koji
K
Yamamoto
Takashi
T
D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D038101:Insulator Elements / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012617:Sea Urchins / Q000196:embryology / Q000235:genetics*
D049452:Green Fluorescent Proteins; D015252:Deoxyribonucleases, Type II Site-Specific