I-SceI-Mediated Transgenesis in Xenopus.

Transgenic frogs can be very efficiently generated using I-SceI meganuclease, a nuclease with an 18-bp recognition site. The desired transgene must be flanked by I-SceI sites, in either a plasmid or a polymerase chain reaction (PCR) product. After a short in vitro digestion with the meganuclease, the complete reaction is injected into fertilized eggs, where the enzyme mediates genomic integration by an unknown mechanism. Posttransgenesis development is typically normal, and up to 70% of the embryos integrate the transgene.

Confirming Antibody Specificity in Xenopus.

Verifying that a new antibody recognizes its target can be difficult. In this protocol, expression of a target protein in Xenopus embryos is either knocked down using CRISPR-Cas9 technology (for zygotic proteins) or enhanced by microinjection of a synthetic mRNA (for maternal proteins). Western blotting analysis is then performed. If the antibody recognizes the target protein, the western blot will show a relatively weak band for CRISPR-injected embryos and a relatively strong band for RNA-injected embryos. This represents a straightforward, powerful strategy for confirming antibody specificity in Xenopus.

Assessing the Immune Response When Raising Antibodies for Use in Xenopus.

Frog-specific antibodies usually must be raised for work in Xenopus Selecting a host animal whose immune system will respond to a target antigen with an antibody response is essential to obtaining high-quality antibodies. To determine whether an immunized animal has produced antibodies against an antigen, western blotting using Xenopus embryo or egg extract as the protein source can be performed as described here. When a protein of the expected size is detected by western blotting in the immune sera but not the preimmune sera, the antibody has been successfully raised.

Purifying Antibodies Raised against Xenopus Peptides.

Antibody production for work in Xenopus involves the immunization of a host with an antigen, usually a Xenopus protein or peptide alien to the host. The antibody-containing serum, normally returned to the investigator by the company/bioresource unit where it was raised, is comprised of all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances, such as drugs and microorganisms, that were in the blood. It is often necessary to separate the target antibody from the rest of the serum components to minimize nonspecific protein-antibody interactions in downstream applications (e.g., when performing western blotting). Most antibody production companies provide a column containing the peptide coupled to glass beads. A purification procedure for using this type of column (i.e., one that is based on controlled-pore glass beads) is described here.

Raising Antibodies for Use in Xenopus.

For work in Xenopus, frog-specific antibodies must usually be raised, although a few antibodies against mammalian proteins cross-react. To produce an immunogen for antibody production, human embryonic kidney (HEK) expression systems can be used as described here. For most laboratories, the actual method of raising the antibody is determined by local ethical regulations controlling the adjuvant and injection protocols used. Because these steps are often outsourced, they are not included in this protocol.

Chytrid fungus infections in laboratory and introduced Xenopus laevis populations: assessing the risks for U.K. native amphibians.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is notorious amongst current conservation biology challenges, responsible for mass mortality and extinction of amphibian species. World trade in amphibians is implicated in global dissemination. Exports of South African Xenopus laevis have led to establishment of this invasive species on four continents. Bd naturally infects this host in Africa and now occurs in several introduced populations. However, no previous studies have investigated transfer of infection into co-occurring native amphibian faunas. A survey of 27 U.K. institutions maintaining X. laevis for research showed that most laboratories have low-level infection, a risk for native species if animals are released into the wild. RT-PCR assays showed Bd in two introduced U.K. populations of X. laevis, in Wales and Lincolnshire. Laboratory and field studies demonstrated that infection levels increase with stress, especially low temperature. In the U.K., native amphibians may be exposed to intense transmission in spring when they enter ponds to spawn alongside X. laevis that have cold-elevated Bd infections. Exposure to cross-infection has probably been recurrent since the introduction of X. laevis, >20 years in Lincolnshire and 50 years in Wales. These sites provide an important test for assessing the impact of X. laevis on Bd spread. However, RT-PCR assays on 174 native amphibians (Bufo, Rana, Lissotriton and Triturus spp.), sympatric with the Bd-infected introduced populations, showed no foci of self-sustaining Bd transmission associated with X. laevis. The abundance of these native amphibians suggested no significant negative population-level effect after the decades of co-occurrence.

pTransgenesis: a cross-species, modular transgenesis resource.

As 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.

Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes.

The digestion of lignocellulose is attracting attention both in terms of basic research into its metabolism by microorganisms and animals, and also as a means of converting plant biomass into biofuels. Limnoriid wood borers are unusual because, unlike other wood-feeding animals, they do not rely on symbiotic microbes to help digest lignocellulose. The absence of microbes in the digestive tract suggests that limnoriid wood borers produce all the enzymes necessary for lignocellulose digestion themselves. In this study we report that analysis of ESTs from the digestive system of Limnoria quadripunctata reveals a transcriptome dominated by glycosyl hydrolase genes. Indeed, > 20% of all ESTs represent genes encoding putative cellulases, including glycosyl hydrolase family 7 (GH7) cellobiohydrolases. These have not previously been reported in animal genomes, but are key digestive enzymes produced by wood-degrading fungi and symbiotic protists in termite guts. We propose that limnoriid GH7 genes are important for the efficient digestion of lignocellulose in the absence of gut microbes. Hemocyanin transcripts were highly abundant in the hepatopancreas transcriptome. Based on recent studies indicating that these proteins may function as phenoloxidases in isopods, we discuss a possible role for hemocyanins in lignin decomposition.

A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos.

We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.

Zygotic nucleosome assembly protein-like 1 has a specific, non-cell autonomous role in hematopoiesis.

Nucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates alpha-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non-cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues alpha-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

Intact RNA-binding domains are necessary for structure-specific DNA binding and transcription control by CBTF122 during Xenopus development.

CBTF122 is a subunit of the Xenopus CCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122 containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (an in vivo target of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF122 fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo.

Linker length modulates DNA cross-linking reactivity and cytotoxic potency of C8/C8' ether-linked C2-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers.

A C2/C2'-exo-unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 4b (DRG-16) with a C8-O(CH2)nO-C8' diether linkage (n = 5) has been synthesized that shows markedly superior in vitro cytotoxic potency (e.g., >3400-fold in IGROV1 ovarian cells) and interstrand DNA cross-linking reactivity (>10-fold) compared to the shorter homologue 4a (SJG-136; n = 3). In contrast, for the C-ring unsubstituted series, the corresponding n = 5 dimer (3c) is generally less cytotoxic and has a lower interstrand cross-linking reactivity compared to its shorter n = 3 homologue (3a). Dimer 4b cross-links DNA with >10-fold efficiency compared to 4a, and also inhibits the activity of the restriction endonuclease BamH1 more efficiently than either 3a or 4a. The C2-exo-unsaturated PBD dimers 4a,b are not only more effective than their C-ring saturated counterparts in terms of induced DeltaTm shift, but they also exert this effect more rapidly. Thus, while 3a and 3c exert 68 and 35% of their maximum effect immediately upon interaction with DNA, this level increases to 76 and 97% for 4a and 4b, respectively. Molecular modeling shows a rank order of 4b (n = 5) > 4a (n = 3) > 3a (n = 3) > 3c (n = 5) in terms of binding energy toward duplexes containing embedded target 5'-GAT(1-2)C cross-link sequences, reflecting the superior fit of the C2-exo-unsaturated rather than saturated C-rings of the PBD dimers. A novel synthesis of core synthetic building blocks for PBD dimers via stepwise Mitsunobu reaction and nitration with Cu(NO3)2 is also reported.

Xenopus nucleosome assembly protein becomes tissue-restricted during development and can alter the expression of specific genes.

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development we have isolated the cDNA encoding Xenopus NAP1L, raised an antibody against recombinant xNAP1L and examined the expression pattern of this mRNA and protein. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation we overexpressed this protein in animal pole explants and tested the effect on expression of a series of potential target genes. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation.