Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos

Yong Lei,Yi Deng,Hui Zhao,Yonglong Chen,Xiaogang Guo

doi:10.1186/2045-3701-3-21

Abstract

Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

Highlights

Xenopus tropicalis is an animal model widely used in studies for both vertebrate development and diseases because of various advantages including its vigorous fecundity, fast embryonic development, short generation period, similar morphogenetic movement as higher vertebrates and feasibility for forward genetics [1,2]
transcription activator-like effector nucleases (TALENs) have been proven to be very effective for gene disruption in Xenopus as well as other animal models [5,6,7,8,9,10,11,12,13,14,15]
Conclusion remarks In our previous studies, TALENs exhibited remarkable efficiency of gene disruption as well as broad targeting spectrum of DNA sequence in genome [5]. Those advantages make TALENs a promising gene disruption approach for specific gene targeting in various animal models including Xenopus

Summary

Introduction

Xenopus tropicalis is an animal model widely used in studies for both vertebrate development and diseases because of various advantages including its vigorous fecundity, fast embryonic development, short generation period, similar morphogenetic movement as higher vertebrates and feasibility for forward genetics [1,2]. It is difficult to perform specific gene targeting in Xenopus tropicalis with conventional reverse genetic methods. Such obstacles have been recently overcome by engineered endonuclease tools, the zinc finger nucleases (ZFNs) [3] and the transcription activator-like effector nucleases (TALENs) [4,5]. TALENs have been proven to be very effective for gene disruption in Xenopus as well as other animal models [5,6,7,8,9,10,11,12,13,14,15]. We will describe how to perform specific gene disruption with TALENs in Xenopus tropicalis embryos, and eventually establish knockout Xenopus tropicalis line

Methods

Results

Conclusion