pTransgenesis: a cross-species, modular transgenesis resource

Abstract

SUMMARYAs studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.

NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

“…Subcloning into the pCS2+ plasmid was done via the BamHI/XbaI restriction sites using the primers 5′-cgccagatctATGAGAGGATCGCATCACC-3′ and 5′-GCTCCTGAAAAT-CTCGCCAAGC-3′ (pCS2+ XtNADK and pCS2+ ΔNADK). pCS2+ XtNADKha was constructed using PCR and pTransgenesis constructs (27). For synthetic mRNA preparation, pCS2+ XtNADK, pCS2+ ΔNADK, or eYFP constructs were linearized with NotI, and mRNA was prepared using an SP6 mMessage Machine mRNA Kit (Ambion).…”

Assessing Primary Neurogenesis in <em>Xenopus</em> Embryos Using Immunostaining

“…Therefore, to track proliferation, differentiation, and movement of neuronal cells in live animals, it would be desired to establish one or more transgenic lines that harbor fluorescent proteins driven by cell typespecific promoter to allow live observation of such cell populations in early Xenopus embryos. The establishment of a X. laevis line with neurospecific β-tubulin promoter driving tauGFP and its applications have provided a nice example 23,34 . With the full promoter sequences of both sox3 and myt1 characterized in vertebrates [35][36][37] , it is should be relatively easy to establish additional transgenic lines in Xenopus which should contribute extensively to both the Xenopus community and a more general field of primary neurogenesis research.…”

2C-Cas9: a versatile tool for clonal analysis of gene function

“…p5E -3.5ubb:loxP-lacZ-loxP was generated by inserting the result of digesting the iZEG plasmid (Novak et al 2000) with XbaI and XhoI and making blunt ends into the p5E ubi (Mosimann et al 2011) after being linearized with BamHI and blunting the ends. pDest cry:GFP was generated by inserting the result of digesting the p1 cry:GFP (Love et al 2011) with SacII and making blunt ends into the pDestTol2pA2 (Kwan et al 2007) that was linearized with BglII. The -3.5ubb:loxP-lacZ-loxPeGFP; cry:GFP construct was generated combining these four plasmids into a single one using Gateway LR Clonase II, Life Technologies: p5E -3.5ubb:loxP-lacZ-loxP, pME GFP (Kwan et al 2007), p3E pA (Kwan et al 2007), and pDest cry:GFP.…”