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

Abstract

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.

A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles

“…The degree of penetrance and tissue-specificity from injecting these plasmids was consistently higher than what has been reported with other plasmids and with what we observed directly with injecting plasmids that lacked the attB and insulator elements (Table 1). They were also comparable with what has been reported in F 0 animals with several bona fide transgenesis methods [e.g., (Sparrow et al, 2000;Ogino et al, 2006;Hamlet et al, 2006;L'hostis-Guidet et al, 2009), and other literature referenced in Results] as well as with injections of bacterial artificial chromosomes [50-60% (Fish et al, 2012)]. The one possible exception was with REMI combined with insulator sequences (Sekkali et al, 2008), where frequencies of animals exhibiting the most highly penetrant expression pattern of the CarA promoter were higher than ours (64% vs. 34%).…”

“…By st. 32-33/34, patterns of expression that were characteristic of each of the two neuronal promoters (categories 1 and 2) became evident in the majority of embryos (NβT, 60%; NF-M, 59%), and these numbers improved through st. 39-40 (Table 2) and st. 43-46 (Table 1), when expression in live animals became easier to observe as they became more transparent. The expression patterns and steadily growing frequency of promotertypic expression that we observed here at these earlier stages were similar to what has been reported for these same promoters in F 0 animals with transgenesis methods (Kroll and Amaya, 1996;Roosa et al, 2000;Sparrow et al, 2000;Ryffel and Lingott, 2000;Ogino et al, 2006;Sekkali et al, 2008;L'hostis-Guidet et al, 2009). …”

“…2A1 and category 1, Table 1). Such expression at equivalent developmental stages has been observed by others working with this promoter using transgenesis procedures such as REMI and the I-SceI meganuclease methods (Ryffel and Lingott, 2000;L'hostis-Guidet et al, 2009); 45% showed some weak expression in tail muscle in addition to strong expression in CNS ( Fig. 2A2; category 2, Table 1); 21% showed high degrees of ectopic expression outside of CNS ( Fig.…”

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons

“…Previous reports have usually used transgenic constructs to label and observe more superficially located cells in the skin, heart, eye, muscle, or tail, where the opacity of X. laevis embryos presents less of a problem. (e.g., Moritz et al, 1999;Jansen et al, 2002;Lim et al, 2004;Smith et al, 2005;Scheenen et al, 2009;Yokoyama et al, 2011;Vivien et al, 2012;Haeri et al, 2013;Loots et al, 2013;Tam et al, 2013;Zhuo et al, 2013) [although also see (Love et al, 2011) and(L'Hostis-Guidet et al, 2009) as these authors used a few widely expressed neuronal promoters to test different types of transgenesis].…”

A guide to the productive poking, prodding and injection of cells

“…Finally, the method for generating transgenic X. laevis embryos described by Bryan Allen and Daniel Weeks (Chapter 9) is clearly exciting, but it is worth noting that since it was first described in 2005 (Allen andWeeks, 2005) no other lab has (apparently) published using the technique. In this light, a general review of other transgenic methods (Ogino et al, 2006;Waldner et al, 2006;L'Hostis-Guidet et al, 2009), as well as a discussion of the application of these methods to X. tropicalis, a diploid relative with a shorter generation time and a sequenced genome, would seem to be both appropriate and useful.…”