Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs.
Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan.
Shota
S
Nakade
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan.
Tetsushi
T
Sakuma
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan.
Yuto
Y
Sakane
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan.
Yoshihiro
Y
Hara
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Atsushi
A
Kurabayashi
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Keiko
K
Kashiwagi
Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Akihiko
A
Kashiwagi
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
Takashi
T
Yamamoto
Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. msobara@hiroshima-u.ac.jp.
Masanobu
M
Obara
D000818:Animals; D001483:Base Sequence; D000078842:Cytoglobin; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D020440:Gene Duplication; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010802:Phylogeny; D012689:Sequence Homology, Nucleic Acid; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics*
D000078842:Cytoglobin; D029867:Xenopus Proteins; D005914:Globins; D003851:Deoxyribonucleases
Homeolog; TALEN; Targeted mutagenesis; Xenopus laevis