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Published on: September 29, 2013
Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis
Donald A Yergeau1, Clair M Kelley, Haiqing Zhu
1Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 8, 2012
Summary
Class II DNA transposons facilitate Xenopus genome modification for transgenesis. A breeding strategy using double-transgenic frogs (hoppers) enables efficient transposon remobilization and large-scale insertional mutagenesis screens.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Class II DNA "cut-and-paste" transposons are tools for Xenopus genome modification.
- Transposons can be mobilized (excised and re-integrated) by supplying transposase enzyme in trans.
Purpose of the Study:
- To evaluate two methods for Xenopus transposon remobilization.
- To establish a breeding-mediated strategy for efficient insertional mutagenesis screens.
Main Methods:
- Remobilization via micro-injection of transposase mRNA.
- Remobilization via germline expression of transposase from a transgene in double-transgenic "hopper" frogs.
- Outcrossing hopper frogs to generate progeny with new transposon integration events.
Main Results:
- Both micro-injection and breeding strategies effectively remobilize transposons.
- Breeding-mediated strategy bypasses time-consuming micro-injection.
- Outcrossing hopper frogs generates novel integration events in large progeny numbers.
Conclusions:
- Breeding-mediated transposon remobilization is an efficient method for Xenopus transgenesis.
- This strategy facilitates large-scale insertional mutagenesis screens in Xenopus tropicalis.
- Xenopus tropicalis serves as a tractable model system for genetic screens.
Related Concept Videos
02:46Genetic Screens
Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes...
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which result in visible changes...
01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.