Inducible gene trapping with drug-selectable markers and Cre/loxP to identify developmentally regulated genes

Gene trapping in mouse embryonic stem cells is an important genetic approach that allows simultaneous mutation of genes and generation of corresponding mutant mice. We designed a selection scheme with drug selection markers and Cre/loxP technology which allows screening of gene trap events that responded to a signaling molecule in a 96-well format. Nine hundred twenty gene trap clones were assayed, and 258 were classified as gene traps induced by in vitro differentiation. Sixty-five of the in vitro differentiation-inducible gene traps were also responsive to retinoic acid treatment. In vivo analysis revealed that 85% of the retinoic acid-inducible gene traps trapped developmentally regulated genes, consistent with the observation that genes induced by retinoic acid treatment are likely to be developmentally regulated. Our results demonstrate that the inducible gene trapping system described here can be used to enrich in vitro for traps in genes of interest. Furthermore, we demonstrate that the cre reporter is extremely sensitive and can be used to explore chromosomal regions that are not detectable with neo as a selection cassette.     

Selection for retroviral insertions into regulated genes.

Gene traps can be used to monitor faithfully the changes in gene expression accompanying several cellular processes. Here, we present a strategy that combines retroviral gene trap vectors, efficient selection schemes based on fluorescence-activated cell sorting or dominant positive and negative drug selection, and appropriately responsive cell lines in order to enrich for retroviral insertions into regulated genes (i.e., genes participating in cellular differentiation processes and genes induced by growth factors, drugs, or neurotransmitters, etc.). As an example, we applied this approach to the identification of insertions into genes activated by a MyoD protein, using a MyoD-responsive fibroblast line. In a single experiment designed to demonstrate the feasibility of this approach, we have been able to screen thousands of gene trap integrations and to select those that represent direct or indirect targets of MyoD. Distinct patterns of regulation were observed during myogenic determination. Sequences flanking the integrations can be rescued with several approaches, and they can be used to isolate the host genes or can serve as entry points for genome-wide sequencing projects.     

Secretion trap tagging of secreted and membrane-spanning proteins using Arabidopsis gene traps

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a beta-glucuronidase reporter enzyme that is inhibited by N-linked glycosylation specific to the secretory pathway. Treatment of seedlings with tunicamycin inhibits glycosylation, resulting in increased activity of secreted beta-glucuronidase fusions that result from gene trap integration downstream of exons encoding signal peptides. In the 2,059 gene trap lines that we screened, 32 secretion trap expression patterns were identified in a wide variety of tissues including embryos, meristems, and the developing vasculature. Genes disrupted by the secretion traps encode putative extracellular signaling proteins, membrane transport proteins, and novel secreted proteins of unknown function missed by conventional mutagenesis and gene prediction. Secretion traps provide a unique reagent for gene expression studies and can guide the genetic combination of loss of function alleles in related genes.     

基因捕捉及其在植物基因分离和功能基因组学上的应用

基因捕捉是一种报告基因的随机整合技术。基因捕捉系统已成为分离基因、鉴定基因功能的重要手段。基因捕捉(gene traps)包括增强子捕捉(enhancer trap)、启动子捕捉(promoter trap)和基因捕捉(gene trap),通称为基因捕捉(gcne traps)。在增强子捕捉中,报告基因与一个基本启动子融合,这个启动子不能使报告基因表达,但可被临近的增强子激活。在启动子捕捉和基因捕捉中,报告基因的启动子被去除,融合基因只有以正确的方向插入到转录单元内才能表达。对基因捕捉系统的结构特征、构建方法、应用范围、研究现状和应用前景等作了系统论述,并对有关问题进行了讨论。     

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.     

A high-throughput induction gene trap approach defines C4ST as a target of BMP signaling

Here we describe a novel gene trap protocol to screen for target genes that are regulated during inductive events in undifferentiated and differentiated mouse embryonic stem cells. This approach integrates several features that allows in vitro screening of large numbers of gene trap clones prior to generating lines of mutant mice. Moreover, targets of spatially and temporally restricted signaling pathways can be analyzed by screening undifferentiated ES cells versus ES cells differentiated into embryoid bodies. We employed this protocol to screen 1920 gene trap lines to identify targets and mediators of signaling through three growth factors of the TGFbeta superfamily--BMP2, activin and nodal. We identified two genes that are induced by BMP2 in a differentiation-dependent manner. One of the genes encodes for Chondroitin-4-sulfotransferase and displays a highly specific temporal and spatial expression pattern during mouse embryogenesis. These results demonstrate the feasibility of a high-throughput gene trap approach as a means to identify mediators and targets of multiple growth factor signaling pathways that function during different stages of development.     

Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the beta-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS expression phenotypes are dominant and are scored in primary transformants, this system does not require rounds of sexual recombination, a typical barrier to developmental genetic studies in trees. Gene and enhancer trap lines defining genes expressed during primary and secondary vascular development were identified and characterized. Collectively, the vascular gene expression patterns revealed that approximately 40% of genes expressed in leaves were expressed exclusively in the veins, indicating that a large set of genes is required for vascular development and function. Also, significant overlap was found between the sets of genes responsible for development and function of secondary vascular tissues of stems and primary vascular tissues in other organs of the plant, likely reflecting the common evolutionary origin of these tissues. Chromosomal DNA flanking insertion sites was amplified by thermal asymmetric interlaced PCR and sequenced and used to identify insertion sites by reference to the nascent Populus trichocarpa genome sequence. Extension of the system was demonstrated through isolation of full-length cDNAs for five genes of interest, including a new class of vascular-expressed gene tagged by enhancer trap line cET-1-pop1-145. Poplar gene and enhancer traps provide a new resource that allows plant biologists to directly reference the poplar genome sequence and identify novel genes of interest in forest biology.     

Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.     

Adaptations of an Emergence Trap for Use in Tropical Streams

An emergence trap based on the MPI Schlitz model was designed for use in the tropics and it was tested over one year in Palawan, the Philippines. Instructions for construction and use are given here. Only commonly available materials were used, except for the collection assembly made of UV‐light permeable acrylic glass. Heavy and bulky assemblies were avoided to enable easy transportation in the field. A special modification allows a fast and easy replacement of the screen when damaged by flooding, as is often required when traps are used downstream of headwaters. This type of trap also allows sampling of a wide littoral strip. Problems concerning the use of emergence traps in the humid tropics are discussed based on experience at different longitudinal stream sections in Palawan. The results presented here suggest that this trap should be used especially for qualitative or semi‐quantitative approaches. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The carnegie protein trap library: a versatile tool for Drosophila developmental studies

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.     

Exploring strategies for protein trapping in Drosophila

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.     

Virus population extinction via ecological traps

Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with high-quality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage Φ6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for viral elimination is frequency dependent in spatially structured environments such that population viability is a nonlinear function of habitat loss in dispersal-limited virus populations. We conclude that the ecological concepts applied to species conservation in altered landscapes can also contribute to the development of trap cell therapies for infectious human viruses.     

Temporal Patterns of Gene Expression in the Antenna of the Adult Drosophila Melanogaster

The time course of gene expression in the adult fruit fly has been partially characterized by using enhancer trap and reporter gene constructs that mark 49 different genes. The relative intensity of the reporter protein in individual cells of the antennae was measured as a function of adult age. Most genes showed a graduated expression, and the intensity of expression had a reproducible and characteristic time course. Different genes displayed different temporal patterns of expression and more often than not the pattern of expression was complex. We found a number of genes having patterns that scaled with life span. In these cases the intensity of gene expression was found to be invariant with respect to biological time, when expressed as a fraction of the life span of the line. The scaling was observed even when life span was varied as much as threefold. Such scaling serves to (1) further demonstrate that deterministic mechanisms such as gene regulation act to generate the temporal patterns of expression seen during adult life, (2) indicate that control of these regulatory mechanisms is linked to life span, and (3) suggest mechanisms by which this control is accomplished. We have concluded that gene expression in the adult fly is often regulated in a fashion that allows for graduated expression over time, and that the regulation itself is changing throughout adult life according to some prescribed program or algorithm.