Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.     

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.     

Colony variation in Sinorhizobium meliloti inoculant strain U 45

A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red. The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium. All colonies were stable except G, which segregated into G and W2-like types. Immune diffusion patterns of all colony types were identical. The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar. The parent cultures and their variants exhibited a range of N₂-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa. Variant R2 from the FD culture was ineffective on both cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies. Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth. When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79. Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants. The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.     

Chromosome instability and tumor predisposition inversely correlate with BLM protein levels

Independent mouse models for Bloom syndrome (BS) exist, each thought to disrupt Blm gene function. However, animals bearing these alleles exhibit distinct phenotypes. Blm(tm1Ches) and Blm(tm1Grdn) homozygous mutant animals exhibit embryonic lethality while in another, Blm(tm3Brd), homozygosity yields viable, fertile animals with a cancer predisposition. Further characterization reveals the Blm(tm3Brd) allele to be a hypomorph, producing a diminished quantity of normal mRNA and protein. The Blm(tm3Brd) allele produces sufficient normal protein to rescue Blm(tm1Ches) lethality. Evaluation of viable animals reveals an inverse correlation between the quantity of Blm protein and the level of chromosome instability and a similar genotypic relationship for tumor predisposition indicating that Blm protein is rate limiting for maintaining genomic stability and the avoidance of tumors.     

What tubulin drugs tell us about microtubule structure and dynamics

A wide range of small molecules, including alkaloids, macrolides and peptides, bind to tubulin and disturb microtubule assembly dynamics. Some agents inhibit assembly, others inhibit disassembly. The binding sites of drugs that stabilize microtubules are discussed in relation to the properties of microtubule associated proteins. The activities of assembly inhibitors are discussed in relation to different nucleotide states of tubulin family protein structures.     

Development of hematological respiratory variables in late chicken embryos: the relative importance of incubation time and embryo mass

Oxygen demand increases during embryonic development, requiring an increase in red blood cells (RBCs) containing hemoglobin (Hb) to transport O(2) between the respiratory organ and systemic tissues. A thorough ontogenetic understanding of the onset and maturation of the complex regulatory processes for RBC concentration ([RBC]), Hb concentration ([Hb]), hematocrit (Hct), mean corpuscular indices (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration ([MCHb])) is currently lacking. We hypothesize that during the last half of incubation when the respiratory organ (the chorioallantoic membrane) envelops most of the egg contents, mean corpuscular indices will stabilize. Accordingly, Hct, [RBC] and [Hb] must also all change proportionally across development. Further, we hypothesize that the hematological respiratory variables develop and mature as a function of incubation duration, independently of embryonic growth. As predicted, a similar increase in Hct (from 18.7±0.6% on day 10 (d10) to 34.1±0.5% on d19 of incubation), [RBC] (1.13±0.03×10(6)/μL to 2.50±0.03×10(6)/μL) and [Hb] (6.1±0.2 g% to 11.2±0.1 g%) occurred during d10-19. Both [RBC] and [Hb] demonstrated high linear correlation with Hct, resulting in constant [MCHb] (~33 g% from d10 to d19). The decrease in MCV (from ~165 μ(3) on d10 to ~140 μ(3) on d13) and MCH (~55 pg to ~45 pg) during d10-13, may be attributed to a changeover from larger primary to smaller secondary and adult-type erythrocytes with MCV and MCH remaining constant (~140 μ(3) and ~45 pg respectively) for the rest of the incubation period (d13-19). Hematological respiratory values on a given incubation day were identical between embryos of different masses using either natural mass variation or experimental growth acceleration, indicating that the hematological variables develop as a function of incubation time, irrespective of embryo growth.     

Genomic instability in induced stem cells

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.