Chromosomal localization of a proinsulin transgene inserted with a transposon-based vector into Japanese quail, Coturnix coturnix

The overall goals of this research were to develop a reproducible method of detecting stable DNA insertion into Japanese quail and provide a method for gene location on avian chromosomes. This research resulted in the development of a different method of obtaining chromosome spreads in Japanese quail, the establishment of primed in situ hybridization as a method for the chromosomal gene detection in birds, development of Teflon-coated coverslip slides to facilitate laser microdissection of 0.5 ƒÝm samples, and chromosomal identification of proinsulin transgene insertions by laser microdissection and nucleotide sequence from G2 Japanese quail. The 28S rDNA was found on a macrochromosome and a microchromosome pair by primed in situ hybridization, fluorescent in situ hybridization, and silver staining. Teflon-coated coverslip slides were created to facilitate laser microdissection of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail produced in Dr. Richard Cooper¡¦s laboratory were identified by laser microdissection and found to have 2-5 chromosomal insertions of the proinsulin transgene

The mammalian centrosome and its functional significance

Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome’s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome’s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome’s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease

The Formation of the First Stars in the Universe

In this review, I survey our current understanding of how the very first stars in the universe formed, with a focus on three main areas of interest: the formation of the first protogalaxies and the cooling of gas within them, the nature and extent of fragmentation within the cool gas, and the physics -- in particular the interplay between protostellar accretion and protostellar feedback -- that serves to determine the final stellar mass. In each of these areas, I have attempted to show how our thinking has developed over recent years, aided in large part by the increasing ease with which we can now perform detailed numerical simulations of primordial star formation. I have also tried to indicate the areas where our understanding remains incomplete, and to identify some of the most important unsolved problems.Comment: 74 pages, 4 figures. Accepted for publication in Space Science Review

On the issue of transparency and reproducibility in nanomedicine.

Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio-nano papers, the community responds

Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting

Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2-5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host\u27s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production. © Springer Science+Business Media B.V. 2006

Small Molecule Optoacoustic Contrast Agents: An Unexplored Avenue for Enhancing In Vivo Imaging

Almost every variety of medical imaging technique relies heavily on exogenous contrast agents to generate high-resolution images of biological structures. Organic small molecule contrast agents, in particular, are well suited for biomedical imaging applications due to their favorable biocompatibility and amenability to structural modification. PET/SPECT, MRI, and fluorescence imaging all have a large host of small molecule contrast agents developed for them, and there exists an academic understanding of how these compounds can be developed. Optoacoustic imaging is a relatively newer imaging technique and, as such, lacks well-established small molecule contrast agents; many of the contrast agents used are the same ones which have found use in fluorescence imaging applications. Many commonly-used fluorescent dyes have found successful application in optoacoustic imaging, but others generate no detectable signal. Moreover, the structural features that either enable a molecule to generate a detectable optoacoustic signal or prevent it from doing so are poorly understood, so design of new contrast agents lacks direction. This review aims to compile the small molecule optoacoustic contrast agents that have been successfully employed in the literature to bridge the information gap between molecular design and optoacoustic signal generation. The information contained within will help to provide direction for the future synthesis of optoacoustic contrast agents

Noninvasive Monitoring of mRFP1- and mCherry-Labeled Oncolytic Adenoviruses in an Orthotopic Breast Cancer Model by Spectral Imaging

Genetic capsid labeling of conditionally replicative adenoviruses (CRAds) with fluorescent tags offers a potentially more accurate monitoring of those virotherapy agents in vivo. The capsid of an infectivity-enhanced CRAd, Ad5/3, delta 24, was genetically labeled with monomeric red fluorescent protein 1 (mRFP1) or its advanced derivative, “mCherry,” to evaluate the utility of each red fluorescent reporter and the benefit of CRAd capsid labeling for noninvasive virus tracking in animal tumor models by a new spectral imaging approach. Either reporter was incorporated into the CRAd particles by genetic fusion to the viral capsid protein IX. Following intratumoral injection, localization and replication of each virus in orthotopic breast cancer xenografts were analyzed by spectral imaging and verified by quantitative polymerase chain reaction. Fluorescence in tumors increased up to 2,000-fold by day 4 and persisted for 5 to 7 weeks, showing oscillatory dynamics reflective of CRAd replication cycles. Capsid labeling in conjunction with spectral imaging thus enables direct visualization and quantification of CRAd particles in tumors prior to the reporter transgene expression. This allows for noninvasive control of CRAd delivery and distribution in tumors and facilitates quantitative assessment of viral replication. Although mCherry appeared to be superior to mRFP1 as an imaging tag, both reporters showed utility for CRAd imaging applications

Noninvasive Monitoring of mRFP1- and mCherry-Labeled Oncolytic Adenoviruses in an Orthotopic Breast Cancer Model by Spectral Imaging

Genetic capsid labeling of conditionally replicative adenoviruses (CRAds) with fluorescent tags offers a potentially more accurate monitoring of those virotherapy agents in vivo. The capsid of an infectivity-enhanced CRAd, Ad5/3, delta 24, was genetically labeled with monomeric red fluorescent protein 1 (mRFP1) or its advanced derivative, “mCherry,” to evaluate the utility of each red fluorescent reporter and the benefit of CRAd capsid labeling for noninvasive virus tracking in animal tumor models by a new spectral imaging approach. Either reporter was incorporated into the CRAd particles by genetic fusion to the viral capsid protein IX. Following intratumoral injection, localization and replication of each virus in orthotopic breast cancer xenografts were analyzed by spectral imaging and verified by quantitative polymerase chain reaction. Fluorescence in tumors increased up to 2,000-fold by day 4 and persisted for 5 to 7 weeks, showing oscillatory dynamics reflective of CRAd replication cycles. Capsid labeling in conjunction with spectral imaging thus enables direct visualization and quantification of CRAd particles in tumors prior to the reporter transgene expression. This allows for noninvasive control of CRAd delivery and distribution in tumors and facilitates quantitative assessment of viral replication. Although mCherry appeared to be superior to mRFP1 as an imaging tag, both reporters showed utility for CRAd imaging applications