Generation of stable Xenopuslaevis transgenic lines expressing a transgene controlled by weak promoters

• Published in: Transgenic research Vol. 18; no. 5; pp. 815 - 827
• Main Authors: L’hostis-Guidet, Anne, Recher, Gaëlle, Guillet, Brigitte, Al-Mohammad, Abdulrahim, Coumailleau, Pascal, Tiaho, François, Boujard, Daniel, Madigou, Thierry
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 2009
• Subjects: Animal Genetics and Genomics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Disease transmission; Eggs; Enzymes; Genetic Engineering; Integration; Life Sciences; Molecular Medicine; Nuclei; Plant Genetics and Genomics; Post-transcription; Promoters; Regulatory sequences; Sperm; Transgenes; Transgenic animals; Transgenics; Woodchuck hepatitis virus; Xenopus; Posttranscriptional regulatory element (WPRE); Transgenesis; I-SceI meganuclease
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-009-9273-0

Combining two existing protocols of trangenesis, namely the REMI and the
I-SceI
meganuclease methods, we generated
Xenopus
leavis
expressing a transgene under the control of a promoter that presented a restricted pattern of activity and a low level of expression. This was realized by co-incubating sperm nuclei, the I-SceI enzyme and the transgene prior to transplantation into unfertilized eggs. The addition of the woodchuck hepatitis virus posttranscriptional regulatory element in our constructs further enhanced the expression of the transgene without affecting the tissue-specificity of the promoter activity. Using this combination of methods we produced high rates of fully transgenic animals that stably transmitted the transgene to the next generations with a transmission rate of 50% indicating a single integration event.