A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae

Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.     

New tools for gene manipulation in chicken embryos

Genomics has changed the pace by which genes are analyzed. Rather than looking at genes one by one, gene expression today is studied at the genome level. Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes. Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates. With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo has become an efficient in vivo system to study gene function during development. A major advantage of in ovo RNAi is the fact that the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes. Thus, this new approach is a valuable tool for functional genomics.     

Regulating gene expression with light-activated oligonucleotides

Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.     

Genomically integrated transgenes are stably and conditionally expressed in neural crest cell-specific lineages

Neural crest cells (NCCs) are a transient embryonic structure that gives rise to a variety of cells including peripheral nervous system, melanocytes, and Schwann cells. To understand the molecular mechanisms underlying NCC development, a gene manipulation of NCCs by in ovo electroporation technique is a powerful tool, particularly in chicken embryos, the model animal that has long been used for the NCC research. However, since expression of introduced genes by the conventional electroporation method is transient, the mechanisms of late development of NCCs remain unexplored. We here report novel methods by which late-developing NCCs are successfully manipulated with electroporated genes. Introduced genes can be stably and/or conditionally expressed in a NCC-specific manner by combining 4 different techniques: Tol2 transposon-mediated genomic integration (Sato et al., 2007), a NCC-specific enhancer of the Sox10 gene (identified in this study), Cre/loxP system, and tet-on inducible expression (Watanabe et al., 2007). This is the first demonstration that late-developing NCCs in chickens are gene-manipulated specifically and conditionally. These methods have further allowed us to obtain ex vivo live-images of individual Schwann cells that are associated in axon bundles in peripheral tissues. Cellular activity and morphology dynamically change as development proceeds. This study has opened a new way to understand at the molecular and cellular levels how late NCCs develop in association with other tissues during embryogenesis.     

逆转录病毒与转基因鸡

摘要：随着生物制药业的迅速发展,转基因鸡输卵管生物反应器正逐渐成为人们关注的焦点,转基因鸡以其卓越的优势必将成为科学研究的热点和生物制药领域的新兴产业之一。目前,转基因鸡最成功的制备方法即逆转录病毒介导法,并已成功制备出表达标记基因的转基因鸡。本文主要综述了逆转录病毒载体制备转基因鸡的优势,逆转录病毒制备转基因鸡的方法和转基因鸡的研究进展,同时也讨论了转基因鸡的意义和存在问题。     

Cloning and sequencing of the CRABP-I locus from chicken and pufferfish: analysis of the promoter regions in transgenic mice

Retinoic acid (RA), a derivative of vitamin A, is an important molecule for development and homeostasis of vertebrate organisms. The intracellular retinoic acid binding protein CRABP-I has a high affinity for RA, and is thought to be involved in the mechanism of RA signalling. CRABP-I is well conserved in evolution and shows a specific expression pattern during development, but mice made deficient for the protein by gene targeting appear normal. However, the high degree of homology with CRABP-I from other species indicates that the protein has been subject to strong selective conservation, indicative of an important biological function. In this paper we have compared the conservation in the expression pattern of the mouse, chicken and pufferfish CRABP-I genes to substantiate this argument further. First we cloned and sequenced genes and promoter regions of the CRABP-I genes from chicken and the Japanese pufferfish, Fugu rubripes. Sequence comparison with the mouse gene did not show any large blocks of homology in the promoter regions. Nevertheless, the promoter of the chicken gene directed expression to a subset of the tissues that show expression with the promoter from the mouse gene. The pattern observed with the pufferfish promoter is even more restricted, essentially to rhombomere 4 only, indicating that this region may be functionally the most important for CRABP-I expression in the developing embryo     

Identification and biochemical characterization of an avian sulfatase homologous to the human ARSE, the gene for X-linked chondrodysplasia punctata

Despite many efforts, the mouse homolog of ARSE, the gene implicated in X-linked recessive chondrodysplasia punctata, has not yet been identified. This absence has so far impaired a deep study of the role of this gene. For this reason, we searched the avian homolog and here report the identification of a chicken sulfatase, cARS, that shares high degree of homology with the cluster of sulfatases located on the short arm of the human X chromosome. cARS activity against a sulfated artificial substrate is heat labile and inhibited by warfarin, features that are characteristic of ARSE. The expression in pharyngeal arches, somites, and leg buds during chick development is consistent with cARS being the functional ortholog of ARSE, matching the tissues affected in this genetic disorder. The identification of the ARSE chicken gene is an important step for the study of its natural substrate and its role during development.     

Analysis of the chicken TBP-like protein(tlp) gene: evidence for a striking conservation of vertebrate TLPs and for a close relationship between vertebrate tbp and tlp genes.

TLP (TBP-like protein), which is a new protein dis-covered by us, has a structure similar to that of the C-terminal conserved domain (CCD) of TBP, although its function has not yet been elucidated. We isolated cDNA and genomic DNA that encode chicken TLP (cTLP) and determined their structures. The predicted amino acid sequence of cTLP was 98 and 91% identical to that of its mammalian and Xenopus counterparts, respectively, and its translation product was ubiquitously observed in chicken tissues. FISH detection showed that chicken tlp and tbp genes were mapped at 3q2.6-2.8 and 3q2.4-2.6 of the same chromosome, respectively. Genome analysis revealed that the chicken tlp gene was spliced with five introns. Interestingly, the vertebrate tbp genes were also found to be split by five introns when we focused on the CCDs, and their splicing points were similar to those of tlp. On the contrary, another TBP-resembling gene of Drosophila, trf1, is split by only one intron, as is the Drosophila 's tbp gene. These results support our earlier assumption that vertebrate TLPs did not directly descend from Drosophila TRF1. On the basis of these results together with phylogenetical exam-ination, we speculate that tlp diverged from an ancestral tbp gene through a process of gene duplication and point mutations.     

Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector,RCASBP

Functional genomics in avian models has lagged behind that of mammals, and the production of transgenic birds has proven to be challenging and time-consuming. All current methods rely upon breeding chimeric birds through at least one generation. Here, we report a rapid method for the ubiquitous expression of GFP in chicken embryos in a single generation (G-0), using the avian retroviral vector, Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP). High-titre RCASBP retrovirus carrying eGFP was injected into unincubated (stage X) blastoderms in ovo. This resulted in stable and widespread expression of eGFP throughout development in a very high proportion of embryos. Transgenic tissues were identified by fluorescence and immunohistochemistry. These results indicate that chicken blastodermal cells are permissive for infection by the RCASBP virus. This system represents a rapid and efficient method of producing global gene expression in the chicken embryo. The method can be used to generate avian cells with a stable genetic marker, or to induce global expression of a gene of choice. Interestingly, in day 8.5 embryos, somatic cells the embryonic gonads were predominantly GFP positive but primordial germ cells were GFP negative, indicating viral silencing in the embryonic germline. This dichotomy in the gonads allows the isolation or enrichment of the germ cells through negative selection during embryonic stages. This transgenic chicken model is of value in developmental studies, and for the isolation and study of avian primordial germ cells.     

Gene transfer into chicken embryos as an effective system of analysis in developmental biology

Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus-mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.     

In vivo gene transfer by electroporation allows expression of a fluorescent transgene in hamster testis and epididymal sperm and has no adverse effects upon testicular integrity or sperm quality

The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.     

New technology for an old favorite: lentiviral transgenesis and RNAi in rats

The ability to produce targeted deletions in the mouse genome via homologous recombination has been a hallmark of mouse genetics, and has lead to the production of thousands of gene knockouts. New technologies are making it possible to disrupt gene function in many other species. This article reviews some of these methods, highlighting the powerful combination of lentiviral vectors with RNA interference (RNAi), which allows one to produce transgenic animals expressing short hairpin RNA (shRNA) to “knock down” specific gene expression. Lentiviral transduction of embryos has been shown to be a highly efficient means of transgenesis, and is particularly promising for animals that are considered difficult to genetically modify by DNA pronuclear injection. This technique has been popular for introducing transgenes for shRNA expression into rodents and its utility for creating new genetic models has already been demonstrated. One of the purported advantages of in vivo RNAi is that shRNA expressing transgenes would be expected to act in a dominant nature, resulting in a phenotype in founder animals. However, one possible concern with lentiviral-mediated transgenesis is the potential for mosaicism in founders, and the data for this phenomenon and the potential causes and solutions are discussed. Emphasis is placed on the application of in vivo RNAi, and other reverse genetic methods, for creating new genetic models in the rat.