Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs
- 264 Downloads
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.
KeywordsHomeolog TALEN Targeted mutagenesis Xenopus laevis
We thank Dr. Ken-Ichi T. Suzuki (Hiroshima University, Japan) for his participation in helpful discussions. The authors wish to express their thanks to Dr. Daniel Voytas (University of Minnesota, St. Paul, MN) for supplying the Golden Gate TALEN and TAL Effector Kit. This study was supported by KAKENHI (25890014) to T.S. from the Japan Society for the Promotion of Science.
- Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148CrossRefPubMedGoogle Scholar
- Sakuma T, Hosoi S, Woltjen K, Suzuki K, Kashiwagi K, Wada H, Ochiai H, Miyamoto T, Kawai N, Sasakura Y, Matsuura S, Okada Y, Kawahara A, Hayashi S, Yamamoto T (2013a) Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18:315–326CrossRefPubMedGoogle Scholar
- Xi Y, Obara M, Ishida Y, Ikeda S, Yoshizato K (2007) Gene expression and tissue distribution of cytoglobin and myoglobin in the Amphibia and Reptilia: possible compensation of myoglobin with cytoglobin in skeletal muscle cells of anurans that lack the myoglobin gene. Gene 398:94–102CrossRefPubMedGoogle Scholar