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Temporal and spatial control of transgene expression using laser induction of the hsp70 promoter
Diane M Ramos, Firdous Kamal, Ernst A Wimmer, Alexander N Cartwright, Antónia Monteiro
BMC Developmental Biology , 2006, DOI: 10.1186/1471-213x-6-55
Abstract: We have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings.This laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms.The use of transgenic animals has helped to produce major advances in the field of functional genetics. Typically, these experiments use only model organisms with known regulatory DNA sequences, i.e. enhancers, that drive gene expression at particular times in development and in particular cells. But, while transposable elements such as piggyBac have enabled transgenic manipulations of increasingly diverse organisms[1], the lack of versatile tools for genetic manipulations in these organisms has hindered their use in functional genetic experiments. Specifically, there is a need to provide temporal and spatial control of transgene expression because it is known that within a developmental context, a gene expressed ubiquitously produces different effects than the same gene expressed in a more restricted p
TAILOR: Transgene Activation and Inactivation Using Lox and Rox in Zebrafish  [PDF]
Joon Tae Park, Steven D. Leach
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0085218
Abstract: The ability to achieve precisely tailored activation and inactivation of gene expression represents a critical utility for vertebrate model organisms. In this regard, Cre and other site-specific DNA recombinases have come to play a central role in achieving temporally regulated and cell type-specific genetic manipulation. In zebrafish, both Cre and Flp recombinases have been applied for inducible activation, inactivation and inversion of inserted genomic elements. Here we describe the addition of Dre, a heterospecific Cre-related site-specific recombinase, to the zebrafish genomic toolbox. Combining Dre-based recombination in zebrafish with established Cre/lox technology, we have established an effective strategy for transgene activation and inactivation using lox and rox (TAILOR). Using stable transgenic lines expressing tamoxifen-inducible CreERT2 and RU486-inducible DrePR fusions, we demonstrate that Cre and Dre retain non-overlapping specificities for their respective lox and rox target sites in larval zebrafish, and that their combinatorial and sequential activation can achieve precisely timed transgene activation and inactivation. In addition to TAILOR, the successful application of Dre/rox technology in zebrafish will facilitate a variety of additional downstream genetic applications, including sequential lineage labeling, complex genomic rearrangements and the precise temporal and spatial control of gene expression through the intersection of partially overlapping promoter activities.
Two Types of Tet-On Transgenic Lines for Doxycycline-Inducible Gene Expression in Zebrafish Rod Photoreceptors and a Gateway-Based Tet-On Toolkit  [PDF]
Leah J. Campbell, John J. Willoughby, Abbie M. Jensen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051270
Abstract: The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA), one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE)-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.
Brain selective transgene expression in zebrafish using an NRSE derived motif  [PDF]
Sadie A. Bergeron,Markus C. Hannan,Kandice Fero,Grace H. Li,Tohei Yokogawa,Harold A. Burgess
Frontiers in Neural Circuits , 2012, DOI: 10.3389/fncir.2012.00110
Abstract: Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae.
Temporal and Spatial Requirements of unplugged/MuSK Function during Zebrafish Neuromuscular Development  [PDF]
Lili Jing,Laura R. Gordon,Elena Shtibin,Michael Granato
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008843
Abstract: One of the earliest events in neuromuscular junction (NMJ) development is the accumulation of acetylcholine receptor (AChR) at the center of muscle cells. The unplugged/MuSK (muscle specific tyrosine kinase) gene is essential to initiate AChR clustering but also to restrict approaching growth cones to the muscle center, thereby coordinating pre- and postsynaptic development. To determine how unplugged/MuSK signaling coordinates these two processes, we examined the temporal and spatial requirements of unplugged/MuSK in zebrafish embryos using heat-shock inducible transgenes. Here, we show that despite its expression in muscle cells from the time they differentiate, unplugged/MuSK activity is first required just prior to the appearance of AChR clusters to simultaneously induce AChR accumulation and to guide motor axons. Furthermore, we demonstrate that ectopic expression of unplugged/MuSK throughout the muscle membrane results in wildtype-like AChR prepattern and neuromuscular synapses in the central region of muscle cells. We propose that AChR prepatterning and axonal guidance are spatio-temporally coordinated through common unplugged/MuSK signals, and that additional factor(s) restrict unplugged/MuSK signaling to a central muscle zone critical for establishing mid-muscle synaptogenesis.
A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish
Mike Placinta, Meng-Chieh Shen, Marc Achermann, Rolf O Karlstrom
BMC Developmental Biology , 2009, DOI: 10.1186/1471-213x-9-73
Abstract: We describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 μm targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle.This versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.The study of gene function has been greatly facilitated by the ability to conditionally regulate gene expression at different locations and times throughout the life cycle. A variety of genetic tools that allow such temporal and/or spatial control of gene expression have been developed for study of gene function in several model organisms. These include site-specific recombination using cre or flp recombinases [1-3], tetracycline inducible systems [4], and the Gal4/UAS system [5,6]. Another system for conditional gene regulation takes advantage of the cellular heat shock response, which leads to the transcription of genes that allow cells to tolerate brief periods of stress, or to activate cell death pathways when these stresses are too extreme [7,8]. One particularly well studied heat shock inducible gene i
Modified Whole-Mount In situ Hybridization Protocol for the Detection of Transgene Expression in Electroporated Chick Embryos  [PDF]
Natacha Arede, Ana T. Tavares
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002638
Abstract: Background In vivo electroporation has been extensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization. Methodology/Principal Findings While analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole-mount in situ hybridization (WISH). Conclusions/Significance Here we describe a modification to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA.
Dynamic expression pattern of zebrafish bfabp (fabp7) during early embryogenesis
Dynamic expression pattern of zebrafish bfabp (fabp7) during early embryogenesis

ZHONG Hanbing,ZHU Zuoyan,FAN Qichang,
ZHONGHanbing
,ZHUZuoyan,FANQichang

科学通报(英文版) , 2004,
Abstract: The expression of bfabp is correlated with the development of nervous system1,2]. Recently, a bfabp homologue (fabp7) was identified in zebrafish3] and its genomic structure, mRNA expression by RT-PCR and linkage mapping data were obtained4]. However, these studies did not provide the information of spatial and temporal expression pattern of fabp7 during zebrafish embryogenesis.
Temporal and Spatial Expression Pattern of Four Laminin Alpha Chains in Xenopus laevis  [PDF]
N. Ahmad
Journal of Biological Sciences , 2009,
Abstract: The spatial and temporal expression of laminin α1, α3, α4 and α5-chain in the developing Xenopus laevis embryos was characterized using RT-PCR and whole-mount in situ hybridization. RT-PCR analysis shows varying developmental timings of expression of the laminin α-chain mRNA. The α1-chain mRNA was detected from stage 6 embryo and levels increase as the embryo develops to stage 41. α3 and α5-chain mRNA was found to be expressed maternally and also in the developing zygote. There was no maternal expression of the α4-chain and in the zygote, α4-chain mRNA was first detected at stage 14. Whole-mount in situ hybridization on embryos at stage 15, 25 and 33 showed that the α1, α3, α4 and α5-chain transcripts produced similar pattern of localisation. Transcripts were observed in the outer epithelia, somites, developing neural tube and pronephros.
Nonparametric identification of regulatory interactions from spatial and temporal gene expression data
Anil Aswani, Soile VE Ker?nen, James Brown, Charless C Fowlkes, David W Knowles, Mark D Biggin, Peter Bickel, Claire J Tomlin
BMC Bioinformatics , 2010, DOI: 10.1186/1471-2105-11-413
Abstract: Here we describe a novel approach which uses nonparametric statistics to generate ordinary differential equation (ODE) models from expression data. Compared to other dynamical methods, our approach requires minimal information about the mathematical structure of the ODE; it does not use qualitative descriptions of interactions within the network; and it employs new statistics to protect against over-fitting. It generates spatio-temporal maps of factor activity, highlighting the times and spatial locations at which different regulators might affect target gene expression levels. We identify an ODE model for eve mRNA pattern formation in the Drosophila melanogaster blastoderm and show that this reproduces the experimental patterns well. Compared to a non-dynamic, spatial-correlation model, our ODE gives 59% better agreement to the experimentally measured pattern. Our model suggests that protein factors frequently have the potential to behave as both an activator and inhibitor for the same cis-regulatory module depending on the factors' concentration, and implies different modes of activation and repression.Our method provides an objective quantification of the regulatory potential of transcription factors in a network, is suitable for both low- and moderate-dimensional gene expression datasets, and includes improvements over existing dynamic and static models.Inferring transcriptional regulatory networks in animals is challenging. For example, the large number of genes, the spatial and temporal complexity of expression patterns, and the presence of many redundant and indirect interactions all make it difficult to learn the network. In the long term, it will be necessary to use multiple data sets--including gene expression, genome wide in vivo DNA binding, and network perturbation data--to accurately represent all interactions. Combining multiple data classes in this way, however, is an open and challenging problem.An alternative, intermediate approach is to use only g
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