Current Genomics, Vol. 4, No. 8, 2003
Contents
Xenopus
Genomics
Guest
Editor: Gerard J.M. Martens
Construction of a Set of Full-Length Enriched cDNA
Libraries as Genomics Tools for Xenopus Tropicalis Research Pp. 635-644
Jisong Peng, Bridget L. Riggs, Hajime Ogino and Bruce Blumberg
The Xenopus Tropicalis Genome Project Pp. 645-652
Paul M. Richardson
and Jarrod Chapman
Xenopus Informatics Pp. 653-664
Jeff B. Bowes and
Peter D. Vize
Xenopus DNA Microarrays Pp. 665-672
Daniel A. Peiffer, Ken W.Y. Cho and Yongchol Shin
Generation and Characterization of Developmental
Mutations in Xenopus tropicalis Pp. 673-685
Takuya Nakayama and Robert M. Grainger
Sleeping Beauty and Xenopus: Transposons as Genetic Tools
Pp. 687-697
M. R. Johnson Hamlet and P. E. Mead
Functional Genomics in Xenopus laevis: Towards
Transgene-Driven RNA Interference and Cell-Specific Transgene Expression
Pp. 699-711
Ron P.H. Dirks, Gerrit Bouw, Rick Van Huizen, Eric J.R. Jansen and Gerard J.M. Martens
Abstracts
[Back to top]
Construction of a Set of Full-Length Enriched cDNA Libraries as Genomics
Tools for Xenopus Tropicalis Research
Jisong Peng, Bridget L. Riggs, Hajime Ogino and Bruce Blumberg
A large variety of mammalian and non-mammalian animal models have been used in research designed to uncover fundamental mechanisms underlying development and disease. Genomics tools have become increasingly necessary for the molecular genetic analysis of important biological questions. However, there are few genomics resources available for the emerging vertebrate model Xenopus tropicalis. Here we discuss our approach towards making a collection of full-length cDNAs from X. tropicalis that will serve as a resource for EST sequencing, microarray development and large-scale functional genomics analysis of Xenopus development.
[Back to top]
The Xenopus Tropicalis Genome Project
Paul M. Richardson and Jarrod Chapman
The Human Genome Project has resulted in the elucidation of the genomic sequence of a number of model organisms as well as a reference sequence for the human genome. The utility of these available genomes has been demonstrated by researchers throughout the world, and spurred the desire to obtain additional genomic information from a number of sources. The United States Department of Energy’s Joint Genome Institute has undertaken a project to sequence the genome of the amphibian Xenopus (Silurana) tropicalis. The primary goal of the project is to produce a high-quality genome sequence and annotation to meet the needs of the research community. In March of 2002, a number of Xenopus researchers from around the world met at the JGI Production Genomics Facility in Walnut Creek, California to discuss goals and strategies for the project. The project is designed to make use of a whole-genome shotgun approach supplemented with extensive BAC end sequences and shotgun sequence from selected BACs. A high-quality draft genome is desired that will meet minimal criteria for contiguity and long-range linking information. At depths of 6-8X sequence coverage, we expect that a large fraction of features of interest (exons, promoters and regulatory regions) will be covered in large contigs of high sequence quality without gaps. In addition, long-range linking of contigs will be achieved through paired end gap-spanning clones so that contigs are ordered and oriented into large scaffolds with gaps of defined size. These scaffolds typically contain multi megabase-sized regions of the genome. This approach has led to high-quality draft genomes of the pufferfish (Fugu rubripes), Ciona intestinalis and the mouse. Since there will be extensive coverage of large inserts for Xenopus including BAC and Fosmid end sequencing, clones will be readily available for finishing selected regions of the genome.
[Back to top] Xenopus
Informatics
Jeff B. Bowes and
Peter D. Vize
The many advantages of the Xenopus experimental system for investigating vertebrate development are limited by the small number of database resources that exist to support this work. This deficit also affects the usefulness of Xenopus data to researchers using other model organism systems. A number of database resources are available, and the applicability of these resources to Xenopus researchers is described. The power of relational databases is also explored as well as the methods that are being used by Xenbase to build an object-relational database that allows users to integrate data from multiple sources, such as literature, gene sequence, sequence annotation and gene expression.
[Back
to top] Xenopus DNA Microarrays
Daniel A. Peiffer, Ken W.Y. Cho and Yongchol Shin
Recent advances in DNA microarray technology have opened the door for large-scale gene expression screening, functional analysis and genomic profiling. Microarrays offer a new opportunity for genome-wide studies and are quickly revolutionizing biological analyses. This review will focus on several important issues related to Xenopus DNA microarray technology, including the advantages of using this model system for array studies, the current status of Xenopus microarrays, experimental designs, and the future of this method in the Xenopus field.
[Back
to top] Generation and Characterization of Developmental Mutations in
Xenopus tropicalis
Takuya Nakayama and Robert M. Grainger
Amphibians have been favored organisms by experimental embryologists for more than a century. Their large, resilient embryos are ideal for manipulations, such as tissue transplantation, explantation and recombination, methods that have been used to demonstrate the existence and location of inducing centers in developing embryos and to define important embryological principles. Molecular biological approaches developed primarily in Xenopus, including a highly efficient method for transgenesis, have provided another dimension to our understanding of a multitude of cellular and developmental problems. Although there have been many developmental mutants reported in amphibians, mainly in axolotl and Xenopus laevis, amphibians have not been as widely used for genetic approaches as other vertebrates, like the mouse and zebrafish. This has been in part because of the long generation time of these species and their large genome size, especially in the case of axolotl. The duplicated genome of X. laevis also presents potential limitations. But another factor mitigating use of these systems for genetics in past times has been the lack of complementary techniques for studying mutants in detail at the molecular level. A more recently introduced model, Xenopus tropicalis, offers an array of new opportunities for genetic studies. Its short generation time and smaller, diploid genome, together with accumulating genomic resources, make X. tropicalis a very attractive model organism for addressing complex issues in modern cell and developmental biology. Here we will introduce the main features of the X. tropicalis system and briefly discuss possible methodologies for isolating developmental mutants for genetic studies.
[Back
to top] Sleeping Beauty and Xenopus: Transposons as Genetic Tools
M. R. Johnson Hamlet and P. E. Mead
Transposons or transposable elements (TEs) are segments of DNA that are able to mobilize from one region of DNA to another. TEs serve as powerful genetic tools in invertebrates such as Drosophila and in plants such as maize. With the molecular reconstruction of the salmonid transposon, Sleeping Beauty, it is possible to adapt transposon technology to vertebrate systems, and Xenopus is no exception. In this review, we give a brief history of TEs followed by a description of how TEs are classified and their mechanism of action. We then give an overview of the many uses of Tes as experimental tools. We focus on the transposon system Sleeping Beauty, and describe its use as a genetic tool to facilitate insertional strategies in vertebrates in general, and Xenopus tropicalis in particular.
[Back
to top] Functional Genomics in Xenopus laevis: Towards Transgene-Driven
RNA Interference and Cell-Specific Transgene Expression
Ron P.H. Dirks, Gerrit Bouw, Rick Van Huizen, Eric J.R. Jansen and Gerard J.M. Martens
The most direct approach to study the physiological role of a protein of unknown function (Functional Genomics) is to change its expression pattern in an intact organism and analyze the phenotypic consequences of this manipulation. The introduction of a method to generate stably transgenic Xenopus laevis has paved the way to the use of tissue/cell- and developmental stage-specific promoters allowing to study the physiological function of proteins in a defined set of fully differentiated cells. Whereas stable (over)expression of proteins in Xenopus is now within reach, stable inhibition of protein expression can only be accomplished randomly, by gene trap approaches. We here report our efforts to induce stable RNA interference (RNAi) in X. laevis via transgene-driven expression of inverted repeats. Stable, and muscle- and neuron-specific knock-down of expression of exogenous green fluorescent protein (GFP) reporter was achieved via RNA polymerase II promoter-driven expression of long GFP RNA duplexes. Unfortunately, our attempts to induce RNAi directed against various endogenous targets, based on the use of RNA polymerase II and III promoters, and long and short inverted repeats have not resulted in a reliable protocol for stable, transgene-driven RNAi in Xenopus. In the second part, we present an example of the use of a cell-specific promoter for functional studies. Cell-specific transgene overexpression of a GFP-tagged member of the p24 family thought to be involved in intracellular protein transport was achieved and this manipulation of the intermediate pituitary melanotrope cell had a phenotypic consequence at its physiological target, the skin melanophore. Thus, the traditional experimental advantages of X. laevis combined with the recently developed technique of stable, non-mosaic Xenopus transgenesis make this lower vertebrate an attractive model organism for Functional Genomics.