Assessment of Artificial MiRNA Architectures for Higher Knockdown Efficiencies without the Undesired Effects in Mice.
RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining -MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both "-MG amiRNA" cells and "amiRNA low-expression" cells but they exhibited growth retardation and cataracts, and they could not transmit the -MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by -MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Hiromi
H
Miura
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Hidetoshi
H
Inoko
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Masafumi
M
Tanaka
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan.
Hirofumi
H
Nakaoka
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Minoru
M
Kimura
Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, Nebraska, 68198, United States of America.
Channabasavaiah B
CB
Gurumurthy
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 1-21-20 Korimoto, Kagoshima, Kagoshima 890-0065, Japan.
Masahiro
M
Sato
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan; The Institute of Medical Sciences, Tokai University, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Masato
M
Ohtsuka
D000818:Animals; D002460:Cell Line; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006720:Homozygote; D008297:Male; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*
D035683:MicroRNAs; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins