Using GFP video to track 3D movement and conditional gene expression in free-moving flies

Background

In vivo imaging and quantification of fluorescent reporter molecules is increasingly useful in biomedical research. For example, tracking animal movement in 3D with simultaneous quantification of fluorescent transgenic reporters allows for correlations between behavior, aging and gene expression. However implementation has been hindered in the past by the complexity of operating the systems.

Results

We report significant technical improvements and user-friendly software (called FluoreScore) that enables tracking of 3D movement and the dynamics of gene expression in adult Drosophila, using two cameras and recorded GFP videos. Expression of a transgenic construct encoding eGFP was induced in free-moving adult flies using the Gene-Switch system and RU486 drug feeding. The time course of induction of eGFP expression was readily quantified from internal tissues including central nervous tissue.

Conclusions

FluoreScore should facilitate a variety of future studies involving quantification of movement behaviors and fluorescent molecules in free-moving animals.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.     

Imaging and tracking of single GFP molecules in solution

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.     

Ubiquitous GFP expression in transgenic chickens using a lentiviral vector

We report the first ubiquitous green fluorescent protein expression in chicks using a lentiviral vector approach, with eGFP under the control of the phosphoglycerol kinase promoter. Several demonstrations of germline transmission in chicks have been reported previously, using markers that produce tissue-specific, but not ubiquitous, expression. Using embryos sired by a heterozygous male, we demonstrate germline transmission in the embryonic tissue that expresses eGFP uniformly, and that can be used in tissue transplants and processed by in situ hybridization and immunocytochemistry. Transgenic tissue is identifiable by both fluorescence microscopy and immunolabeling, resulting in a permanent marker identifying transgenic cells following processing of the tissue. Stable integration of the transgene has allowed breeding of homozygous males and females that will be used to produce transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These results demonstrate that a transgenic approach in the chick model system is viable and useful even though a relatively long generation time is required. The transgenic chick model will benefit studies on embryonic development, as well as providing the pharmaceutical industry with an economical bioreactor.     

Protein expression from synthetic genes: selection of clones using GFP

Construction of synthetic genes is today the most elegant way to optimize the heterologous expression of a recombinant protein. However, the selection of positive clones that incorporate the correct synthetic DNA fragments is a bottleneck as current methods of gene synthesis introduce 3.5 nucleotide deletions per kb. Furthermore, even when all predictable optimizations for protein production have been introduced into the synthetic gene, production of the protein is often disappointing: protein is produced in too low amounts or end up in inclusion bodies. We propose a strategy to overcome these two problems simultaneously by cloning the synthetic gene upstream of a reporter gene. This permits the selection of clones devoid of frame-shift mutations. In addition, beside nucleotide deletion, an average of three non-neutral mutations per kb are introduced during gene synthesis. Using a reporter protein downstream of the synthetic gene, allows the selection of clones with random mutations improving the expression or the folding of the protein of interest. The problem of errors found in synthetic genes is then turned into an advantage since it provides polymorphism useful for molecular evolution. The use of synthetic genes appears as an alternative to the error-prone PCR strategy to generate the variations necessary in protein engineering experiments.     

A zebrafish gene trap line expresses GFP recapturing expression pattern of foxjlb

Foxj1 has been found to play an important role in cilia formation and function in vertebrates.The zebrafish or Xenopus genome expresses two Foxj1 genes, foxjla/FoxJ1 and foxj1b/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZ10 by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxjl1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxjlb. Although normal expression offoxjlb is dramatically reduced,T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxj1b expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

A zebrafish gene trap line expresses GFP recapturing expression pattern of foxjlb

Foxj1 has been found to play an important role in cilia formation and function in vertebrates.The zebrafish or Xenopus genome expresses two Foxj1 genes, foxjla/FoxJ1 and foxj1b/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZ10 by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxjl1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxjlb. Although normal expression offoxjlb is dramatically reduced,T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxj1b expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

Background  

Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice.     

Automatic recognition and annotation of gene expression patterns of fly embryos

Vol. 23, No.     

Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression

Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.     

Automatic recognition and annotation of gene expression patterns of fly embryos

MOTIVATION: Gene expression patterns obtained by in situ mRNA hybridization provide important information about different genes during Drosophila embryogenesis. So far, annotations of these images are done by manually assigning a subset of anatomy ontology terms to an image. This time-consuming process depends heavily on the consistency of experts. RESULTS: We develop a system to automatically annotate a fruitfly's embryonic tissue in which a gene has expression. We formulate the task as an image pattern recognition problem. For a new fly embryo image, our system answers two questions: (1) Which stage range does an image belong to? (2) Which annotations should be assigned to an image? We propose to identify the wavelet embryo features by multi-resolution 2D wavelet discrete transform, followed by min-redundancy max-relevance feature selection, which yields optimal distinguishing features for an annotation. We then construct a series of parallel bi-class predictors to solve the multi-objective annotation problem since each image may correspond to multiple annotations. SUPPLEMENTARY INFORMATION: The complete annotation prediction results are available at: http://www.cs.niu.edu/~jzhou/papers/fruitfly and http://research.janelia.org/peng/proj/fly_embryo_annotation/. The datasets used in experiments will be available upon request to the correspondence author.     

红系特异的GFP基因在转基因小鼠中的整合和表达

应用荧光定量PCR技术对由位点控制区LCR的HS2元件和 β 珠蛋白基因启动子指导的红系特异表达绿色荧光蛋白 (GFP)基因的转基因小鼠中外源基因拷贝数进行测定 ,使用荧光显微镜和流式细胞仪检测小鼠外周血中GFP的表达水平 ,并运用荧光原位杂交技术 (FISH)确定了其中两只转基因小鼠中外源基因的整合位点 ,结果表明 :在转基因小鼠中外源基因的拷贝数各不相同且相差较大 ,而且拷贝数与GFP基因的表达量之间未呈现出相关性 ;FISH分析确定出两只转基因小鼠的外源基因整合于不同的染色体上 ;杂交信号的强弱与拷贝数的多少相一致     

Monitoring phase-specific gene expression in Histoplasma capsulatum with telomeric GFP fusion plasmids

Dimorphism is an essential feature of Histoplasma capsulatum pathogenesis, and much attention has been focused on characteristics that are unique to the saprophytic mycelial phase or the parasitic yeast phase. Recently, we identified a secreted calcium-binding protein, CBP, that is produced in large amounts by yeast cells but is undetectable in mycelial cultures. In this study, the green fluorescent protein (GFP) was established as a reporter in H. capsulatum to study regulation of CBP1 expression in cultures and in single cells grown under different conditions and inside macrophages. One GFP version that was optimized for human codon usage yielded highly fluorescent Histoplasma yeast cells. By monitoring GFP fluorescence during the transition from mycelia to yeast, we demonstrated that the CBP1 promoter is only fully active after complete morphological conversion to the yeast form, indicating for the first time that CBP1 is developmentally regulated rather than simply temperature regulated. Continuous activity of the CBP1 promoter during infection of macrophages supports the hypothesis that CBP secretion plays an important role for Histoplasma survival within the phagolysosome. Broth cultures of Histoplasma yeasts carrying a CBP–GFP protein fusion construct were able to secrete a full-length fluorescent fusion protein that remained localized within the phagolysosomes of infected macrophages. Additionally, a comparison of two Histoplasma strains carrying the CBP1 promoter fusion construct either epichromosomally or integrated into the chromosome revealed cell-to-cell variation in plasmid copy number due to uneven plasmid partitioning into daughter cells.     

Systematic optimization of active protein expression using GFP as a folding reporter

Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.     

Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

An elaborated model of fly small-target tracking

Flies have the capability to visually track small moving targets, even across cluttered backgrounds. Previous computational models, based on figure detection (FD) cells identified in the fly, have suggested how this may be accomplished at a neuronal level based on information about relative motion between the target and the background. We experimented with the use of this small-field system model for the tracking of small moving targets by a simulated fly in a cluttered environment and discovered some functional limitations. As a result of these experiments, we propose elaborations of the original small-field system model to support stronger effects of background motion on small-field responses, proper accounting for more complex optical flow fields, and more direct guidance toward the target. We show that the elaborated model achieves much better tracking performance than the original model in complex visual environments and discuss the biological implications of our elaborations. The elaborated model may help to explain recent electrophysiological data on FD cells that seem to contradict the original model.Acknowledgement This work was supported by the US Office of Naval Research under agreement number N68936-00-2-0002.