text:
- Research article
- Open Access
Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing
- Linda Sharp†1,
- Thomas Pratt†1,
- Gillian E. MacKay2, 3,
- Margaret A. Keighren2, 4,
- Jean H. Flockhart2,
- Emma J. Chandler2,
- David J. Price1,
- John O. Mason1 and
- John D. West2Email author
© The Author(s). 2017
- Received: 28 February 2017
- Accepted: 1 June 2017
- Published: 29 June 2017
The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3.
Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex.
Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.
- Mosaic transgene expression
- Green fluorescent protein
- tauGFP
- Adrenal cortex lineages
- Preimplantation embryo
- Time-lapse imaging
Two lines of transgenic mice, TP6.3 and TP6.4, express CAG-tauGFP transgenes TgTP6.3 and TgTP6.4 respectively [1]. Both lines TP6.3 and TP6.4 were produced by electroporation of the same construct into embryonic stem (ES) cells, which were then used to generate ES cell chimaeras and founder transgenic mice. Line TP6.3 expresses a tau-green fluorescent protein (tauGFP) fusion protein almost ubiquitously. Its localisation to microtubules has some advantages over soluble GFP as a lineage marker and it is well suited for labelling axons, blood vessels and preimplantation embryos [1, 2]. This TgTP6.3 tauGFP marker has been used to identify cell fusion between ES cells and other cell types [3] and as a lineage marker for macrophages [4], ovarian cells [5], preimplantation embryos [6] and, in particular, neural tissues [7–12].
TgTP6.4 transgene expression was reported to be strong in many tissues yet only weak in much of the central nervous system but it was not fully characterised [1]. As TgTP6.3 Tg/Tg homozygotes are lethal and TgTP6.3 Tg/− hemizygotes are smaller than wild-type (WT) siblings we evaluated the second tauGFP transgenic line to determine whether TgTP6.4 had any advantages over the TgTP6.3 marker transgene. We investigated growth of TgTP6.4 Tg/− hemizygotes and the viability of TgTP6.4 Tg/Tg homozygotes and we characterised expression of the TgTP6.4 transgene in embryos and adults by confocal microscopy of tauGFP fluorescence. This revealed that TgTP6.4 Tg/− mice showed widespread mosaic expression. As mosaicism can be useful for analysis of clonal lineages that are established during development or by adult stem cells, we also compared mosaic expression in the adrenal cortices of TgTP6.3 Tg/− and TgTP6.4 Tg/− hemizygotes.
C57BL/6 and outbred CD-1 mice were purchased from Bantin and Kingman, Hull, UK and A/J/Ola/Hsd mice were purchased from Harlan Olac Ltd., Bicester, UK. BALB/c/Eumm, CBA/Ca, C57BL/OlaWs (a C57BL/OlaHsd sub-colony), (C57BL × CBA/Ca)F1 hybrids, TP6.3 and TP6.4 mice were bred and maintained under conventional conditions at the University of Edinburgh with a light cycle of 14 h light (05:00 h. - 19:00 h.) and 10 h dark or 12 h light (06:00 h. - 18:00 h.) and 12 h dark. Founder TP6.3 and TP6.4 mice were produced from chimaeras made with E14Tg2a ES cells that had been transfected with the CAG-tauGFP vector, pTP6 [1, 2]. Founder mice were on a largely outbred MF1 genetic background and the transgenes were bred onto other strains as required. Transgenic mice were maintained by crossing TgTP6.3 Tg/− and TgTP6.4 Tg/− hemizygotes to non-transgenic, wild-type (WT) mice. Both TP6.3 and TP6.4 mice were initially crossed to inbred C57BL/6 mice to produce the first colony of each transgenic line, and some mice were also crossed to outbred, albino CD-1 mice to facilitate confocal microscopy of tauGFP expression in eye tissues. These colony I mice were used for the initial investigations shown in Fig. 3 and Additional file 1: Table S1. For later studies, TgTP6.3 Tg/− and TgTP6.4 Tg/− mice from the first colonies were crossed to (C57BL × CBA/Ca)F1 hybrid mice to produce a second colony for each line. These colony II mice were maintained by crossing transgenic mice to (C57BL × CBA/Ca)F1 mice at each generation and were used for the investigations shown in Figs. 1, 2, 4, 5, Additional file 1: Figures S2, S3 and S4 and Table 1. In all genetic crosses the female parental genotype is shown first. TauGFP-positive offspring were identified at weaning by the green fluorescence of their ear punch biopsies, using a fluorescence microscope with a fluorescein isothiocyanate (FITC) filter set. Some tauGFP-positive newborn pups were identified as described elsewhere [13].
Analysis of postnatal growth
Newborn pups from crosses between (C57BL × CBA/Ca)F1 females and hemizygous TgTP6.4 Tg/− males were sexed and weighed on postnatal days (P) 1 and 7 and then at weekly intervals thereafter until P84. Individual pups were marked with a marker pen until they were old enough for an ear punch biopsy, which was used both for identification and classification of their tauGFP phenotype.
Analysis of preimplantation embryos
For collection of preimplantation embryos, females were superovulated by intraperitoneal (i.p.) injections of 5 international units (I.U.) of pregnant mare's serum gonadotrophin (PMSG; Folligon, Intervet) at approximately 12 noon followed 48 h later by 5 I.U. of human chorionic gonadotrophin (hCG; Chorulon, Intervet). After hCG injections, each female was caged with a male overnight and mating was verified by the presence of a vaginal copulation plug the following morning, which was designated embryonic day (E) 0.5. Preimplantation embryos were flushed from the reproductive tract with KSOM-H handling medium [14] and their ages in hours post coitum (p.c.) were timed from the mid-point of the dark period (midnight) following the hCG injection. For collection of 1-cell stage embryos, mice were killed at approximately 10:00 h. on the morning after the hCG injection, cumulus masses were released from the oviducts and cumulus cells were dispersed in a solution of 100 I.U. hyaluronidase (Sigma-Aldrich, Gillingham, UK) per ml of phosphate-buffered saline (PBS). Two cell-stage embryos were flushed from the oviducts at approximately 10:00 h. on the following day (34 h.).
For time-lapse monitoring, preimplantation embryos were cultured for 24 h in drops of KSOM culture medium under mineral oil in thin glass-bottomed dishes (WillCo HBSt 3522, Intracel Ltd. Royston, UK). The dishes were placed in an environmental chamber on top of the heated stage (THD 60, Linkam Scientific Instruments Ltd., Tadworth, UK) of an inverted Leica DMIRB/E confocal microscope, and the atmosphere within the chamber was maintained at 37 °C, 5% CO2 in air (Additional file 1: Fig. S1). Images were acquired every 15 or 30 min for approximately 24 h. in both fluorescent (FITC) and transmitted light mode, using the Leica TCS NT confocal system. The time-lapse files contained both FITC and transmitted light channels from each time point. These were merged to provide photographs showing GFP fluorescence overlaid on a transmitted light image for the figures. Each image from the FITC channel is a pseudocoloured greyscale representation of the intensity of the emission signal from the sample, rendered on a 0–255 pixel intensity scale, which represents the strength of fluorescence. For quantitative analysis, files for each time point were opened in the freeware image analysis program, Scion Image (http://downloads.informer.com/scion-image/), each embryo was outlined as a region of interest (ROI) and an average pixel intensity reading obtained. If embryos moved between frames (time points), the ROI was either moved or redrawn for analysis of the next frame. Embryos that were cultured from the 2-cell stage but failed to cleave were excluded from the analysis. The embryos sometimes drifted out of focus so, typically, a short (1–2 h.) culture period was recorded at the beginning of the experiment and examined, so the focus could be adjusted if required. The focus was also checked prior to overnight recording. These interruptions meant that each time-lapse experiment comprised several separate files, which sometimes differed slightly in pixel intensity because of fluctuations in laser power that occurred while scanning was interrupted.
Analysis of transgenic fetuses
Fetuses were produced by natural matings and staged from the date of the copulation plug, as described above. Females were killed by cervical dislocation, and the numbers of implantation sites, moles (resorbing conceptuses), dead fetuses and live fetuses were noted. Conceptuses were removed from the uterus and the fetuses dissected free of their placentas and extraembryonic membranes with fine forceps. Fetuses were classified for tauGFP expression with a Leica M2FLIII, fluorescence dissecting microscope using a GFP filter set. For comparison of fetal sizes, E14.5 fetuses were blotted dry, weighed and their crown-rump lengths were measured.
Analysis of tauGFP expression in tissue sections from fetal and adult transgenic mice
Hemizygous adults and E13.5 and E17.5 fetuses were produced as described above. Fetuses were decapitated and fixed in ice cold 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight at 4 °C. Some small tissue samples from adults were also fixed by this immersion method. Other adult samples were perfusion fixed, while the mouse was deeply anaesthetised with a 0.4 ml i.p. injection of 25% urethane in saline. The vascular system was then flushed with ice-cold 0.9% saline via cardiac puncture, followed by perfusion with ice-cold 4% PFA in PBS. After perfusion, the mouse was killed and various organs, including brain, heart, liver, kidney and eyes, were removed and fixed overnight in fresh ice-cold 4% PFA in PBS.
Fixed samples were washed in two changes of PBS and embedded in 4% agarose (LMP Agarose, Bethesda Research Laboratories, Life Tech inc., USA). Blocks were trimmed, mounted on glass with adhesive and 200 μm sections were cut with a vibratome. Prior to mounting, some vibratome tissue sections were counterstained with propidium iodide (PI) solution (0.1% PI, 0.05% RNase, 0.1% Triton X-100 in PBS) for 5 min then rinsed in PBS, to label nuclear DNA fluorescent red. In some cases RNase was omitted, so cytoplasmic RNA was also counterstained. Sections were washed in PBS, immersed in a mixture of glycerol and PBS (1:1 v/v) at 4 °C until saturated and mounted on glass slides in a 1:1 mixture of glycerol and PBS containing 1% Vectashield antifade reagent (H-100, Vector Labs Inc., USA). The coverslips were sealed with Aquamount. Sections were viewed with an upright Leica TCS NT confocal microscope (Leica Microsystems, Germany). Bright-field images were collected in the transmitted light channel and GFP was detected in the FITC channel. For sections that were counterstained with propidium iodide, optical sections were acquired simultaneously in the FITC (green) and tetramethylrhodamine isothiocyanate (TRITC; red) channels. Single optical sections and stacks of sections were acquired with a minimum of 4 averages per optical section.
Analysis of tauGFP expression by fluorescence activated cell sorting
TauGFP-positive and negative E14.5 fetuses, from crosses to outbred CD-1 strain mice, were dissected in ice-cold Earle's Balanced Salt Solution (EBSS), pre-equilibrated in 5% CO2. Whole brains, hippocampus, cerebral cortex, ventral telencephalon, central and dorsal thalamus, and midbrain and hindbrain tissues were dissected and dissociated using the Worthington Papain Dissociation System, (Worthington Biochemical Corporation, USA). Dissociated cells were sorted for green fluorescence using a Fluorescence Activated Cell Sorter (FACS; Becton Dickinson, Rutherford, NJ, USA) to generate histograms of green fluorescence intensity (FL1 channel) versus cell number for 10,000 cells per sample. Gates identifying cell populations with different fluorescent intensities were defined using a tauGFP-positive TP6.3 brain sample, considered to contain 100% fluorescent cells and the same gates were applied to all samples. After gating to exclude outliers (predominantly dead cells), the percentage of cells in three gated regions, corresponding to different fluorescent intensities, were compared: region M1 included all fluorescent cells, M2 included cells with low fluorescence and M3 included cells with high fluorescence.
Analysis of mosaicism in adrenal glands
In TgTP6.4 Tg/− and many TgTP6.3 Tg/− hemizygous mice, the adrenal cortex was not uniformly tauGFP-positive but, in sections, showed a radial pattern of tauGFP-positive and tauGFP-negative stripes, most of which extended continuously across the full width of the adrenal cortex. This 2-dimensional radial stripe pattern in sections was analysed as a 1-dimensional pattern of stripe widths by measuring around the adrenocortical circumference as described previously [15]. The width of each tauGFP-negative and tauGFP-positive stripe was measured in calibrated images of single sections near the middle of the adrenal gland using UTHSCSA Image Tool software for Microsoft Windows (http://compdent.uthscsa.edu/dig/itdesc.html). Measurements were made around the complete circumference of the adrenal cortex at a similar distance from the outer edge of the adrenal gland (equivalent to 20% of the distance from the capsule to the inner margin of cortex). This provided the numbers of tauGFP-negative and tauGFP-positive stripes, the observed mean stripe widths, the total measured circumference and the proportions of tauGFP-negative and tauGFP-positive cells at the depth of the measurements for each adrenal cortex analysed. To allow for differences in stripe widths at different radial positions, the stripe width was expressed as a proportion of the circumference.
The radial stripes are believed to represent coherent clones of cells derived from stem cells at the periphery of the adrenal cortex [15, 16]. However, it is misleading to compare observed stripe numbers directly because each stripe may comprise several adjacent coherent clones and the number of clones per stripe is likely to vary according to the proportions of tauGFP-positive and tauGFP-negative cells in the tissue. We, therefore, converted the observed number of tauGFP-positive stripes plus tauGFP-negative stripes to a 'corrected stripe number' for each section, which adjusts for the expected number of clones per stripe [15]. First, the observed mean tauGFP-negative stripe width was divided by the correction factor 1/(1-p), where p is the proportion of tauGFP-negative cells around the circumference [15, 17, 18]. As the radial stripes in sections of the adrenal cortex form a complete circle, the calculated 'corrected mean stripe width' will be the same for both the positive and negative stripes, as explained previously [15, 17, 18]. Thus, correction of mean tauGFP-positive stripe widths, using the proportion of tauGFP-positive cells as p, produced identical results. As the 'corrected mean stripe width' applies to both tauGFP-negative and tauGFP-positive stripes, the 'corrected stripe number' for the positive plus negative stripes is the reciprocal of the corrected mean stripe width, expressed as the proportion of the measured circumference. The calculated corrected stripe number adjusts for differences in proportions of tauGFP-positive cells among adrenals and is likely to be proportional to the number of active coherent clones of stem cells, so was used to compare different groups of adrenal glands as described elsewhere [15].
Minimum group sizes were guided by previous experience and power calculations. For comparison of postnatal growth, TP6.4 group sizes were chosen to ensure sufficient power to detect, as significant (P < 0.05), mean body mass differences that were smaller than those previously published for TP6.3 [1, 2]. For quantitative analysis of adrenal mosaicism, results for only one adrenal gland per mouse were included. For mice where both adrenal glands were imaged, the choice of including the left or right adrenal was randomised by tossing a coin. The choice of parametric or non-parametric tests was guided, in part, by normality tests. GraphPad Prism versions 5.0c and 7 (GraphPad Software Inc., La Jolla, CA) were used for most statistical tests including 2-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison post-tests, repeated measures 2-way ANOVA followed by Bonferroni multiple comparison post-tests, 1-way ANOVA, the paired t-test, the Mann–Whitney U-test and the Kruskal-Wallis test followed by Dunn's multiple comparison post-tests. An online statistical calculator (http://vassarstats.net/index.html) was used for the chi-square goodness of fit test and Fisher's exact test. Error bars shown are 95% confidence intervals (CI).
Postnatal growth and viability of hemizygous TgTP6.4 Tg/− transgenic mice
Viability of TgTP6.4 Tg/Tg homozygotes
We were unable to identify any adult TgTP6.4 Tg/Tg homozygotes by overt differences in GFP fluorescence and, as the TgTP6.4 insertion site is unknown, we had no simple PCR method for distinguishing TgTP6.4 Tg/Tg homozygotes from TgTP6.4 Tg/− hemizygotes. In the absence of an easy method for identifying homozygotes, we used a statistical analysis of offspring phenotype frequencies from a genetic test cross to determine whether homozygotes were viable. Thirty-nine tauGFP-positive progeny, from TgTP6.4 Tg/− × TgTP6.4 Tg/− crosses, were crossed to non-transgenic mice to determine whether any only produced tauGFP-positive offspring and so were likely to be homozygotes. Thirty-six mice were classified as hemizygotes because they produced both tauGFP-positive and tauGFP-negative offspring in either their first viable litter (35 cases) or their second litter (one case). One other mouse produced three litters with 9/9, 7/8 and 9/12 tauGFP-positive offspring, respectively. As the overall frequency (25/29) was significantly higher than the expected 50% tauGFP-positive offspring (P = 0.0002), this mouse might have been a germline homozygous/hemizygous mosaic, rather than a hemizygote but it died before further investigations could be performed. Five of the other 36 mice produced equal numbers of tauGFP-positive and tauGFP-negative offspring, 16 produced more tauGFP-positive and 15 produced more tauGFP-negative offspring. As the 16:15 ratio did not differ significantly from the 1:1 ratio expected if all 36 mice were TgTP6.4 Tg/− hemizygotes (P = 1.0000), there was no evidence that any of these were homozygous/hemizygous mosaics. This is also supported by the overall frequency of tauGFP-positive offspring from these 36 mice (177/345; 51.3%), which was not significantly different from the expected 50% (P = 0.6714) for hemizygotes. The remaining two males failed to sire any offspring in three-months and we cannot exclude the possibility that these were infertile homozygotes. One third of the tauGFP-positive offspring from TgTP6.4 Tg/− × TgTP6.4 Tg/− crosses are expected to be homozygous but none of 37 fertile offspring bred as homozygotes. The probability of this occurring by chance is only (2/3)37 (P = 0.0000003), if homozygotes are actually viable and fertile. These results imply that homozygous TgTP6.4 Tg/Tg mice do not survive to reproduce.
Viability of TgTP6.4 hemizygotes and homozygotes assessed by frequencies of tauGFP-positive mice produced in different crosses
Genetic cross (female × male)
Cross 1
(experimental cross)
Cross 2
(control cross)
Cross 3
Tg/− × Tg/−
Tg/− × −/−
−/− × Tg/−
A. Mice classified for tauGFP at ~3 weeks
Total born
Dead before weaning
52 (15.5%)a
3 (2.1%)
8 (2.3%)
Dead after weaning (most unclassified)
5 (1.5%)
3 (0.9%)
Viable at weaning
TauGFP not classified
TauGFP classified
133 (55.9%)b,c
57 (43.5%)d
79 (39.3%)e
105 (44.1%)
74 (56.5%)
122 (60.7%)
Pre-weaning death attributable to crossj
B. Fetuses classified for tauGFP at E14.5
Total conceptuses
Moles (early deaths)
16 (8.3%)f
9 (4.7%)
Dead fetuses
2 (1.0%)f
2 (1.0%)
Total live fetuses
129 (73.7%)g,h
85 (47.0%)i
46 (26.3%)
96 (53.0%)
Death attributable to crossk
To investigate whether homozygous, TgTP6.4 Tg/Tg E14.5 fetuses were viable, we compared observed and expected frequencies of tauGFP-positive fetuses in two crosses (Table 1). The control and experimental crosses produced close to 50 and 75% tauGFP positive E14.5 fetuses respectively, as expected if all hemizygotes and homozygotes survived to E14.5. Survival of most homozygotes to E14.5 is also suggested by the relatively low frequency of dead embryos, which was not significantly elevated in the experimental cross. However, there was some evidence that homozygous tauGFP fetuses were slightly smaller than normal by E14.5. After allowing for variation among litters, tauGFP-positive fetuses in the experimental cross were significantly lighter than tauGFP-negative fetuses (P < 0.0001 by a 2-way ANOVA) and had shorter crown-rump lengths (P < 0.0001; Additional file 1: Figure S2). In the control cross, fetal mass differences were less pronounced (P = 0.0343) and crown-rump length differences were not significant (P = 0.0597), implying that hemizygous TgTP6.4 Tg/− fetuses were more normal in size at E14.5. In summary, the breeding experiments indicate that most TgTP6.4 Tg/Tg homozygotes survive to E14.5 but some may already be retarded and they probably all die between E14.5 and weaning.
TauGFP expression in TgTP6.4 Tg/− preimplantation embryos
Preimplantation embryos from the reciprocal cross between TgTP6.4 Tg/− females and (C57BL × CBA/Ca)F1 males, initially all retained oocyte-encoded tauGFP but TgTP6.4 Tg/− and TgTP6.4 −/− embryos could be distinguished by their tauGFP levels by the 4-cell stage (Fig. 2d-f). There was no overlap in pixel intensity from 45 h. p.c. for the experiment shown in Fig. 2h and a repeated measures 2-way ANOVA with Bonferroni multiple comparison post-tests showed that pixel intensity differed significantly between the two genotypes from 42.5 h., consistent with the onset of embryo-coded TgTP6.4 expression by the late 2-cell stage. Comparisons of pixel intensity between TgTP6.4 −/− embryos, with some residual tauGFP, and control WT embryos, with no tauGFP, showed no overlap in pixel intensity at any ages analysed (34.6 to 57.6 h.) and a repeated measures 2-way ANOVA with Bonferroni multiple comparison post-tests confirmed that pixel intensities remained significantly different at 57.6 h. This indicates that residual oocyte-encoded tauGFP remained at least until the 4-cell stage.
TauGFP expression in fetal and adult TgTP6.4 Tg/− tissues
Fluorescence activated cell sorting (FACS) analysis of cell suspensions prepared from whole E14.5 fetal brains confirmed that tauGFP fluorescence was present in fewer TgTP6.4 Tg/− cells than TgTP6.3 Tg/− cells (Fig. 3q-t). The analysis using three gated regions (Fig. 3q) is described in the Methods. Fig 3r shows that 95% of all the cells from whole TgTP6.3 Tg/− brains were fluorescent (M1 gate in Fig. 3q), of which 87% were highly fluorescent (Fig. 3r). In contrast, only 25% of cells from whole TgTP6.4 Tg/− brains were fluorescent, 10% of which were highly fluorescent (Fig. 3s). Nevertheless, this profile shows more fluorescence than the TgTP6.4 −/− negative controls (Fig. 3t). Similar results were obtained for cells from separate brain regions although the hippocampus showed more fluorescence than other TgTP6.4 Tg/− brain regions (Additional file 1: Table S1).
Comparison of mosaic tauGFP expression in TgTP6.3 Tg/− and TgTP6.4 Tg/− adrenal cortices
The pattern of radial stripes seen in the adrenal cortex of some TgTP6.3 Tg/− and TgTP6.4 Tg/− mice (Fig. 4m-p,t) has been reported for several other experimental systems and is believed to represent coherent clones of cells, which are derived from stem cells at the periphery of the adrenal cortex, and move towards the medulla (see Discussion). After correcting for the expected number of multiple adjacent clones in a single stripe, the corrected stripe number can provide an indirect estimate of the number of clones of active stem cells as described elsewhere [15, 17, 18] so this can be used to compare stem cell function in different groups. The uncorrected mean stripe width varies with the percentage of tauGFP-positive cells in the adrenal cortex, which will affect the number of clones per stripe, but the corrected mean stripe width adjusts for this, as shown in Additional file 1: Figure S3. The corrected mean stripe width was used to calculate a corrected stripe number, as explained in the Methods, to provide a quantitative comparison of TgTP6.3 Tg/− and TgTP6.4 Tg/− stripe patterns.
The aim of the present study was to determine whether the TgTP6.4 marker transgene offered any advantages over TgTP6.3. Although tauGFP expression in TgTP6.3 Tg/− cells provides an excellent marker, TgTP6.3 Tg/Tg homozygotes do not survive and TgTP6.3 Tg/− hemizygotes are slightly smaller than normal. TgTP6.4 Tg/− growth was less affected than TgTP6.3 Tg/− hemizygotes but TgTP6.4 Tg/Tg homozygotes died between E14.5 and weaning age, as reported for TgTP6.3 Tg/Tg homozygotes [2] and the widespread mosaicism means that TgTP6.4 is unsuitable as a conventional cell lineage marker. The reason why both TgTP6.3 Tg/Tg and TgTP6.4 Tg/Tg homozygotes die was not investigated but high levels of GFP or tau protein are neurotoxic [19]. Other tauGFP transgenic mice have been produced by inserting a tauGFP transgene into a specific locus [20–22] or randomly inserting a CAG-tauGFP construct into the genome to produce another CAG-tauGFP transgenic line [23], but no mosaic phenotypes were described for these mice. While mosaicism in TgTP6.3 Tg/− mice was only identified in the cornea and adrenal cortex, mosaicism became increasingly common in TgTP6.4 Tg/− mice during our investigation and eventually affected all tissues examined and all TgTP6.4 Tg/− mice.
Mosaic patterns produced by mouse chimaeras and X-inactivation mosaics have been useful for visualising clonal lineages and for analysing the extent of cell mixing and movement during development, growth and maintenance of adult tissues [24–29]. Mosaic transgene expression can provide a useful alternative for these types of investigations [15], particularly when mosaicism is widespread, as in TgTP6.4 Tg/− mice. We used this approach to investigate whether there were any overt differences in maintenance of the adrenal cortex in TgTP6.3 Tg/− and TgTP6.4 Tg/− adults.
Mosaic TgTP6.3 Tg/− and TgTP6.4 Tg/− adrenals showed qualitatively similar radial stripes across the adrenal cortex. This pattern has previously been reported for other experimental systems, including mouse and rat chimaeras [24, 28, 30–33], mouse X-inactivation mosaics [33] and some other mouse lines that show mosaic transgene expression [15, 34–36]. The stripes are believed to represent coherent clones of cells that are derived from stem cells at the periphery of the adrenal cortex and they maintain the tissue as they move slowly across the cortex before undergoing apoptosis [15, 16, 37–40]. The corrected stripe numbers were similar in the two genotypes and were comparable to results reported previously for mouse chimaeras, X-inactivation mosaics and other mosaic transgenic mice [15, 33], suggesting that maintenance of the adrenal cortex involved a similar number of active clones of stem cells in all these groups.
Mosaic transgene expression is thought to involve position effect variegation caused by stochastic transgene silencing during development [34, 41–43]. Indirect evidence suggested that mosaic expression of a different transgene in the mouse adrenal cortex involved stochastic silencing at the level of the chromosome or transgene, rather than at the cellular level [44] but this may not apply to all mosaic transgenic lines. Transgene silencing can occur if the transgene integrates near an endogenous heterochromatic site [41], which may prevent the transcriptional machinery from accessing the transgene, or if it triggers the formation of heterochromatin by integrating as a series of repeated copies [45, 46]. Repeat-copy transgenes are also associated with methylation changes that could mediate stochastic transgene silencing if transgene methylation levels vary among cells [47]. Moreover, several genes that alter the extent of transgene silencing have been identified in a mutagenesis screen and some have been characterised [48, 49]. However, the copy number is not known for either of the transgenic lines that we studied and the mechanism(s) that caused widespread mosaic silencing of TgTP6.4 and more restricted silencing of TgTP6.3 is unknown.
TauGFP expression in TgTP6.3 Tg/− preimplantation embryos was characterised previously [1, 2] and was similar in TgTP6.4 Tg/− embryos. TauGFP fluorescence was ubiquitous in preimplantation embryos of both transgenic lines, so mosaic expression presumably involves transgene silencing in some cells during development. As there was no evidence for an age-effect on the percentage of cells expressing tauGFP in either TgTP6.3 Tg/− or TgTP6.4 Tg/− adrenals, we have no evidence that silencing is a continuous process. The simplest model assumes that transgene silencing involves a single stochastic event that occurs during development and the silent or active state is then stably inherited by daughter cells. As the frequency of TgTP6.4 Tg/− mosaic expression changed over a number of generations of breeding and ultimately affected all tissues examined and the entire TP6.4 colony, it seems likely that the genetic background somehow affects the probability of transgene silencing but, again, the mechanism remains unknown.
In contrast to TgTP6.4 Tg/− mice, mosaic expression did not occur in all TgTP6.3 Tg/− mice, nor did it occur in so many tissues. Of the TgTP6.3 Tg/− tissues examined, mosaicism was only identified in the adrenal cortex and cornea. Moreover, when mosaicism occurred in the adrenal cortex of TgTP6.3 Tg/− mice, there were significantly more tauGFP-positive cells than in TgTP6.4 Tg/− adrenals. This implies that silencing of the TgTP6.3 transgene affects fewer cells per tissue as well as fewer tissues and fewer mice. In both TgTP6.3 Tg/− and TgTP6.4 Tg/− mice, the mosaic pattern in the cornea was in the form of radial stripes, strongly suggesting it was in the corneal epithelium layer, as described for studies of the corneal epithelium using other mosaic systems [17, 18, 50–57].
Mosaicism in TgTP6.3 Tg/− mice is clearly more restricted than in TgTP6.4 Tg/− hemizygotes so it might arise later in development but this was not tested directly. Investigation of why the extent of mosaicism differs between these two transgenic lines would probably require breeding both transgenes onto the same inbred genetic background, as well as knowledge of the insertion sites, copy numbers and epigenetic modifications around the insertion sites. Stem cells at the tissue's periphery are thought to maintain both the adult corneal epithelium [55–60] and the adrenal cortex [15, 16, 38–40]. It may also, therefore, be worth investigating whether transgene silencing can occur in tissue-specific stem cells as well as earlier in development.
Although widespread mosaic expression means that the TgTP6.4 transgene is not a suitable alternative to TgTP6.3 as a standard lineage marker, it provides a useful system for identifying and analysing clonal lineages. We exploited this mosaicism to demonstrate that similar clonal lineages maintained the adult adrenal cortex in different groups of mice. Differences, in the frequency and tissue distribution of mosaicism, between related transgenic lines, such as TP6.3 and TP6.4, may also provide a useful experimental system for investigating transgene silencing.
We thank William Seldon for technical help during his undergraduate summer project, Andrew Sanderson for assistance with the FACS analysis, Katy Gillies, Denis Doogan, Maureen Ross and Jim Macdonald for other technical help and Steven Morley for helpful discussion.
This work was supported by the Wellcome Trust (research grant 059904/JS to JDW JOM and DJP and equipment grant 052578/Z/97/Z to JBL Bard, JA Davies, DJP, N Spears and JDW), the University of Edinburgh Moray Endowment Fund and PhD studentship funding for GEM from the College of Medicine and Veterinary Medicine, University of Edinburgh. The funding bodies played no role in the design of the study, in the collection, analysis and interpretation of data or in writing the manuscript.
Availability of data and materials
Raw numerical data have been deposited in Edinburgh DataShare, the University of Edinburgh's data repository (http://dx.doi.org/10.7488/ds/2049). TP6.3 and TP6.4 embryos have been frozen by the College of Medicine and Veterinary Medicine Cryopreservation Service, University of Edinburgh. For availability, email firstname.lastname@example.com or JOM
Authors' contributions
TP produced the transgenic mice and participated in lab work, LS & GEM carried out lab work, participated in data analysis, produced some figures and helped draft the manuscript, MAK, JHF and EJC carried out lab work, DJP and JOM designed and supervised parts of the study, JDW designed and supervised part of the study, analysed the data, produced some figures and drafted the manuscript. LS and TP contributed equally to this work. All authors contributed to the preparation of the final manuscript and gave final approval for publication.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval
Animal work was performed in accordance with institutional guidelines and UK regulations and was authorised by the UK Home Office (licences PPL 60/1989, 60/2374 and 60/2887).
Publisher's Note
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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
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Have you ever looked around your home and thought to yourself that it looks pretty dark and depressing? If the answer is yes, you've come to the right place. The focus of today's blog post is to explore the different ways that you can brighten up your home.
Once you try these amazing ideas, you'll be happy with how you've managed to transform your home into a bright and airy place! Keep reading to find out how.
Flickr / vickimoore
Stick with light color schemes
The golden rule to making your home bright and airy is to stick with light color schemes. Choosing bright green or terracotta red might have been a good idea at the time. But now that you have to live with it, it's not!
However, light color schemes will make any home feel fresh and bright. They are also perfect for homes with small rooms, as light colors give the illusion rooms are bigger than they are! Your homeowner's association management will also approve of you using light colors in your home.
Some folks opt to paint their walls white. Not only is this a neutral and light color, but white paint is also the cheapest color to buy too!
Less is more
Are you someone that has a lot of junk without even knowing it? If so, it's time to have a spring clean and clear the clutter away from your home!
People often feel better about their homes when they know everything within them is organized and "has a place." And when you store things away, it also means you have to spend less time cleaning your home.
Most of us lead busy lives, and so some folks decide to hire a cleaner for their homes. The trouble is; quite a few people are reluctant to do so if their home is messy! Keeping things in your home to a minimum resolves this problem and won't make you feel guilty about hiring a cleaner.
Install some mirrors in your home
It doesn't matter whether you love or loathe your personal appearance. Mirrors are great for reflecting light into any home, and therefore can make rooms brighter. Ensure that you position your mirrors for optimal light.
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This year, Student Ministries is launching a new curriculum called "The Story." This curriculum is designed to interactively engage students in God's story that is told throughout Scriptures and challenges them to think through how their individual stories are involved in God's overarching story for mankind.
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Well we went to 6 flags and to my sister's house over the weekend. Chris got off work early on Thursday so we headed to 6 flags to enjoy a few hours. Then we got a hotel room and spent a full day at 6flags on Friday. We spent most of the day in the water park, and had a wonderful time.
We played a lot in the wave pool because it was warm. The little kid section had FREEZING cold water which I couldn't understand. Anyway, we enjoyed the warm wave pool. This little black girl came up to me when I was teaching Ashlyn how to swim. She smiled and watched us and I smiled back at her. She asked if I was teaching Ash to swim and I said Yes. She asked how, so I told her what to do and she tried it. I was telling both girls what a good job they were doing and the little girl was sooo excited I was teaching her. Then Katey came up and smiled at the girl and the buzzer went off that the waves were going to start again. Katey said come on to the girl and they both went off into the water to jump the waves... When the waves would stop, they would come back and we would practice swimming again. We did this for about an hour and it was time to go. We thanked her for playing with us and left. When we were walking out, Katey said that little girl broke my heart mom. I asked why and she said the little girl told her that white people don't like black people. Katey just looked at her and sighed and said I know, but those people are not nice. But I like everyone. And the little girl smiled and said, I know! This broke my heart, but also made me so proud. Its sad that this little girl at 6 yrs old has been affected by racism, its just terrible. But I was so proud of my Katey, she acknowledged that it does happen, and that she thinks those people are not nice, and made it very clear that she likes everyone. It was the first experience that Kathryn has had with this sort of thing, and I was just so proud of how she handled it.
We went to Cassie's after 6 flags and saw her new house, met her really cool neighbors and had a 4Th of July party and watched fireworks. We had a wonderful weekend.


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Ordering Africa: Anthropology, European Imperialism and the Politics of Knowledge
Ordering Africa: Anthropology, European Imperialism and the Politics of Knowledge [1]
When I was an undergraduate at the School of Oriental and African Studies (SOAS) in the early 1980s, the School had a motto: knowledge is power. Students of a radical inclination would denounce this explicit evocation of the School's imperial origins, and evidently the criticism took its toll. A diligent search of the SOAS website suggests that the phrase has now been entirely suppressed, in favour of a cedar which is presumably supposed to make us think of flourishing and growing strong. Evidently, Francis Bacon's dictum, memorably echoed by Francis Reginald Wingate, former Governor General of the Sudan in his introduction to the first issue of Sudan Notes and Records, is no longer considered to be an appropriate summation of the role of British academe in relation to Africa and Asia.
This sort of embarrassment about the relationship between colonialism and academic study has particularly affected anthropology. Unlike the various other disciplines implicated in the production of knowledge about the subject other, anthropology was, as Kathleen Gough pointed out in the 1960s, very largely born of the colonial relationship in continental Europe, as well as in the UK. Almost as soon as it was established, its practitioners came under attack both for their complicity in a colonial order which was already in decline, and for producing studies of non-European societies which concealed, or at least understated, the dramatic impact of colonialism on the lives of ordinary people. Anthropology has, since the 1960s, been frequently forced into a defensive posture in the face of Bernard Magubane's fierce attack on anthropologists who 'believed not only in the inevitability but also in the rightness of the white conquest of the African', and a series of works have - as the current jargon would have it - interrogated the relationship between academic anthropology and imperialism.
This new collection, the product of a conference in 2000, returns to the debate. From the start, it would seem that the editors of the work, and John Mackenzie in his role as series editor, wish to exonerate anthropology of the more serious charges laid against it. Historical analyses of colonialism, in Africa and elsewhere, have tended in recent years to move away from the vision of a totalising project which crushed all before it. Rather, they have stressed the limited nature of colonial power and the contingent and uncertain nature of colonial policies, as well as the profound role which nominal subjects played in shaping 'the colonial state'. Ordering Africa, despite its title, seems to fit largely into this disaggregating mode, questioning the ability of anthropologists to order themselves, let alone the continent of their study.
This reassessment of the complicity of anthropology pursues several different lines. Several of the contributions explore the metropolitan academic milieu, and here, as elsewhere, the real strength of a work with a broad European focus is apparent. British, French and German academic anthropologists feature not as scheming agents of empire but as bickering egotists, driven by national and professional rivalry and personal ambition. Further chapters remind us of the role of the African auxiliaries of linguistic and anthropological research, the people who gathered and supplied data to European 'experts' who often had relatively little direct experience of Africa. These studies do not support the implication that these academic subalterns shaped the production of knowledge, or subverted European ideas about Africa, but they do show how very limited was the evidential basis of colonial knowledge. Furthermore, they remind the reader that, in any case, the primary concern of these auxiliaries was the 'pursuit of localized personal and family objectives', rather than the promotion or remaking of colonialism. Finally Bruce Berman's and John Lonsdale's study of Kenyatta's venture into anthropology, while showing how it might be used as a political tool, also indicates how ineffectual this could be: before later events propelled Kenyatta into the limelight hardly anybody read Facing Mount Kenya, and of those who did few took it seriously.
If the relationship between European and African producers of anthropological knowledge emerges as one theme a second and more significant division, which comes very clearly out of this collection, was that between different groups of Europeans, notably between academic anthropologists (the emergent professionals of this nascent discipline) and the European administrators, missionaries and settlers who gathered ethnographic data as part of their work, or out of interest, or both. This distinction was an important one. Of course, Europeans tried to gather information about the people whom they were supposed to rule, manage or evangelise; but most of them did so in an uncertain and haphazard way, perhaps pushed along by the occasional questionnaire or the need to add some entries to the Political Record Book. As far as research questions or methods were concerned, they only had their preconceptions and prejudices to guide them. All of this was undoubtedly part of the colonial attempt to order and categorise, but it was rather different from the work of the professional anthropologists who were employed by some colonial governments - Condominium Sudan being probably the most enthusiastic of these - specifically to gather 'knowledge' about their subjects. As Douglas Johnson notes in his contribution to the collection, British officials in Sudan had argued the importance of such information right from the beginning of their rule, and they hired their first anthropologists in 1908. Yet despite this, and some rather half-hearted attempts to train future administrators in the art of anthropological research, the influence of professional anthropologists on the administration of Sudan was minimal. Edward Evans-Pritchard worked with Sudan government support to produce his classic works of anthropology; but as he himself insisted, these were of no real direct value to officials. Harold MacMichael, perhaps the leading administrator-ethnographer of Sudan, was clear in his views on professional anthropologists who 'sink the practical in the recondite'.
There are similar examples from other territories, not discussed in Ordering Africa: when MacMichael left Sudan he moved to Tanganyika, another colonial territory, which experimented repeatedly with professional anthropology and where an administrator and an anthropologist co-operated to produce a book on the uses of anthropology (Hutt and Brown's Anthropology in Action). But there too administrators, preferring the product of their own research, found it hard to make use of the kind of knowledge produced by anthropologists. On the whole, it would seem that, as Helen Tilley puts it here, 'anthropology needed empires far more than empires needed anthropology'.
This will not be surprising to many readers. In our search for research grants many of us, not always disingenuously, have argued the relevance of our proposals to some pressing issue, suggesting that by adding to our understanding we can contribute directly to the shaping of policies which improve the human condition. But as anyone who has been involved in such research will know, the application of research-based knowledge to policies is not always simple, and the relationship with policy-makers can be a fraught one. Academics, encumbered by their pressing sense of the complexity of human societies, tend to be reluctant to offer straightforward policy suggestions; policy-makers have no time to read the cautiously crafted prose of scholars. I have, for the last few years, been involved in running what is, I suppose, a modern equivalent of the colonial summer schools of the 1930s: an intensive course taught by academics, for the employees, mainly, of development agencies working in Sudan. The pupils are a willing lot but, as they freely admit, it is not always possible for them to see how exactly the experience of the course will change the ways that they do their jobs. This is especially true of the parts of the course which discuss the anthropology of Sudan, which most seem to find confusing. This kind of information does not empower them; they are already sufficiently empowered by their knowledge of the international bureaucracy of development, which pays them well and gives them budgets to spend, vehicles to drive, and influence over governments. For these individuals, as for their colonial antecedents, ethnographic knowledge about the people among whom they work is a decidedly uncertain and unwieldy tool. Indeed, it might be argued that in the colonial context and now, real anthropological understanding is as likely to be subversive of power as constitutive of it staying the hand of policy-makers by adding layers of complexity to situations which had appeared simple and giving reason for second thoughts. To offer again the current example of Sudan, the only direct effect on policy which I know to have resulted from the 'knowledge' imparted on the course has been the dissuasion of the UN from a plan to use helicopter gunships to chastise pastoralist raiders; a restraint on power, rather than the creation of it.
It is left to the final contributor to Ordering Africa to try to restate the argument for the role of 'ethnology', as he calls it, in sustaining colonialism, and to shift the argument away from the immediate usefulness of any particular piece of 'knowledge' to the idea that knowledge in itself is a project of power. Writing of France's African territories, Gary Wilder describes a world of colonial ethnology in which continuities were much more important than divisions, with 'most colonial administrators' (really?) attending the École Coloniale and diligently absorbing the works of academic anthropologists. His approach centres not so much on the assertion that 'knowledge is power', but rather on the Foucauldian concept of power/knowledge. It is not that the one produces the other, but rather that the processes through which power is made and exercised are intricately involved with the processes through which knowledge is created. This neatly sidesteps the empirical discussion of whether or not officials were able to make practical use of information, emphasising instead that the very act of studying, of gathering knowledge, defined a colonial relationship and legitimised the subordination of the subject. Those who are studied must naturally be ruled and directed by those who study them; the most fundamental ordering comes from this simple divide, which is marked by the process of creating knowledge.
This is a point which Archie Mafeje made many years ago; anthropology offered a service to colonialism not through particular acts of 'collusion', but through the 'ontology of its thought categories'. Seen thus, the partial, jumbled nature of much colonial knowledge about subjects is really irrelevant; anthropological knowledge does not have to be accurate in any empirical sense to bestow power. Nor does it matter if such knowledge is too complicated to be applied in any direct way to administration; that is not really what gathering anthropological information is about. Rather it is the process, through which studied and studier repeatedly rehearse their respective roles, which makes power. The elaboration of anthropology as a distinct discipline was important, not because it created a distinction between professionals and amateurs and divided the discipline, but because it legitimised and rationalised this knowledge project and so effectively empowered colonialism. Administrators may have found professional anthropologists irritating or obscure, but they took comfort and moral strength from the idea that they and the anthropologists were working together to understand and improve those whom - naturally enough - they ruled. The argument here is rather out of step with the rest of the collection and, on the whole, readers will leave this book with the sense that the internal combustion engine and the breech-loading rifle were more useful to colonialism than were anthropological texts; but Wilder offers an effectively discomfiting reminder that anthropological knowledge may, after all, have quite a lot to do with power.


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On Halloween night, while ghouls and goblins did their trick-or-treating, an asteroid that is most likely a dead comet made a close flyby of Earth, with radar images revealing its eerie skull shape.
On Saturday (Oct. 31), the asteroid 2015 TB145 passed by Earth at a range of just over 300,000 miles (480,000 kilometers), placing it just outside the orbit of the moon, where it posed no threat to the planet. The timing of the flyby earned the asteroid – which is about 2,000 feet (600 meters) across – the nickname "Spooky" and "Great Pumpkin."
Unfortunately for skywatching hobbyists, 2015 TB145 was extremely difficult to see from the ground, but the online Slooh Community Observatory hosted a webcast Saturday afternoon that featured updates on the asteroid's path, and discussions about the dangers of near-Earth asteroids. [Related: Boo! Halloween Asteroid Looks Just Like a Skull]
Photos: Asteroids and Near-Earth Objects
NASA observed the asteroid with radar in infrared using its NASA Infrared Telescope Facility (IRTF) in Mauna Kea, Hawaii.
"The IRTF data may indicate that the object might be a dead comet, but in the Arecibo images it appears to have donned a skull costume for its Halloween flyby," Kelly Fast, IRTF program scientist and acting program manager for NASA's NEO Observations Program, said in a NASA statement Friday (Oct. 30).
Slooh used a remotely operated observatory in the Canary Islands to record animations of asteroid 2015 TB145 passing by. The "dead comet" asteroid looks like a bright object speeding across a starry background in those views. It was Slooh who nicknamed the asteroid "Spooky," and the online observatory was thrilled to see the space rock's skull shape in radar views.
NASA Pings A Passing Space Peanut
"We all saw those dramatic images of the skull-like object whirling through space captured by NASA's Arecibo Observatory," Slooh's Tricia Ennis wrote in an email update. "We got a kick out of how appropriate our 'Spooky' moniker ended up being, here at Slooh."
The flyby was a treat for scientists, because it allowed them to see the space rock up close, with a radar resolution of as little as 6.6 feet (2 meters) on the surface. On Saturday, scientists fired radio waves at the passing space rock using a 110-foot-wide (34 m) antenna at NASA's Deep Space Network facility in Goldstone, California. The radio waves that bounced off the asteroid and came back to Earth were then collected using the Green Bank Telescope in West Virginia and Puerto Rico's Arecibo Observatory.
Near-Earth Asteroid 2015 TB145 passes by without a fright!
Two Meteors Hit Ancient Earth at Same Time
Radar images from those observations revealed asteroid 2015 TB145 true size (it's a bit larger than previously thought) and its speed. The asteroid is hurtling through space at a whopping 78,293 mph (126,000 km/h).
"This would generate a 6-mile-wide crater if it were to the Earth, something of this size and speed," asteroid impact expert Mark Boslough, a physicist at Sandia National Laboratories in New Mexico, said in the Slooh webcast. He was also impressed by the early radar images of the 2015 TB145.
"This is beautiful. It's such a high-resolution, nice image. You can see it rotating you can see features on the surface," Boslough said. "This is great science."
The speed of 2015 TBA145′s flyby is considered high for an asteroid flyby, according to a statement from NASA's Jet Propulsion Laboratory (JPL). The Halloween asteroid may actually be a comet, but if that's the case it's a dead comet, meaning it no longer contains the volatile ingredients that sublimate in space and give comets their bright tails.
Russian Meteor Strike Aftermath: Photos
"We found that the object reflects about six percent of the light it receives from the sun," said Vishnu Reddy, a research scientist at the Planetary Science Institute, Tucson, Arizona, in the NASA statement. "That is similar to fresh asphalt, and while here on Earth we think that is pretty dark, it is brighter than a typical comet which reflects only 3 to 5 percent of the light. That suggests it could be cometary in origin – but as there is no coma evident, the conclusion is it is a dead comet."
In addition, its orbit is "very oblong with a high inclination to below the plane of the solar system," Lance Benner, who leads NASA's asteroid radar research program, said in an earlier JPL statement.
Benner said this would be the first time that the Goldstone facility has imaged a comet from such a close distance. Benner also called asteroid 2015 TB145 "one of the best asteroids for radar imaging we'll see for several years."
Asteroid 2015 TB145 was only discovered Oct. 10, less than a month from its closest approach to Earth. And by mapping its trajectory, scientist were able to confirm that it made a similarly close approach to Earth in 1975. The next large asteroid to make a close flyby of Earth (that scientists know of) won't take place until 2027.
Original article on Space.com.
Trick Or Treat? Halloween Asteroid Is 'Sears Tower-Sized' | Orbit Animation Potentially Dangerous Asteroids (Images)
Asteroid Basics: A Space Rock Quiz Copyright 2015 SPACE.com, a Purch company. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
This radar image of asteroid 2015 TB145, which NASA says is likely a dead comet, was captured using the Arecibo Observatory in Puerto Rico on Oct. 30, 2015 with a resolution of 25 feet per pixel.
Doomsday Asteroids?
Asteroids and comets capture the human imagination unlike any other objects in space. They're speedy, craggy, not too far away -- and dangerous. Geologic investigations around the globe guarantee a sizeable space rock is bound to kiss our fragile blue marble within the next few centuries years. And if one may have wiped out the dinosaurs, the thinking goes, why not us? Browse through 10 of our favorite ominous objects that astronomers have plucked from the foreboding skies.
Asteroid 951, also known as Gaspra, is safely tucked away within the Asteroid Belt between Mars and Jupiter. For now. This image is the outcome of the very first robotic rendezvous with an asteroid. The Galileo spacecraft swung by Gaspra at about 1,000 miles away in November 1991 on its way to Jupiter. The intel Galileo brought back on the asteroid was breathtaking; Gaspra's irregular, tooth-like shape and lack of big craters suggest that it was born out of a recent (300 to 500 million-year-old) smashup in the Asteroid Belt.
What asteroid was the first known to have a mini-companion orbiting it? Asteroid 243, better known as Ida. The Galileo spacecraft also scouted out this 1,900-by-2,375 mile space rock during the long trip to Jupiter in August 1993. Ida spins around once about every 4 1/2 hours, and its little "moon" Dactyl is roughly 20 times smaller. Like asteroid Gaspra, Ida is oddly shaped -- yet unlike Gaspra, Ida has remained in one piece for much of the solar system's 4.5 billion year existence.
NASA's Galileo spacecraft was first to get up close and personal with an asteroid, but the Near-Earth Asteroid Rendezvous (NEAR) mission quickly stole all of the thunder. Designed specifically to study asteroids close to Earth, NEAR's main objective was to spy on 433 Eros. Second only to 1036 Ganymed, Eros is one of the largest asteroids drifting dangerously close to Earth. Measuring 21 by 7 by 7 miles, its size easily rivals the space rock thought to have wiped out the dinosaurs. This false-color image shows where the asteroid's surface is denser (red) and less dense (blue) compared to an average reading.
Itokawa (asteroid 25143) may look like a harmless, peanut-shaped pile of rubble -- but the writing is on the wall. In a few million years, it's probably going to wallop Earth. Hayabusa, Japan's asteroid-snooping spacecraft, was set to land on a smooth patch, scoop up a sample and return it to Earth. Although the plucky robot never did make contact, its payload -- set to arrive at Earth by June 2010 -- probably contains at least a few bits of dust and other material from Itokawa. If it returns safely, Hayabusa will be only the second spacecraft to bring back a sample of the solar system from beyond the moon.
No robot has come out to meet New Jersey-sized asteroid 216 Kleopatra. Rather, this series of images was put together by the expansive Arecibo Observatory in Puerto Rico in mid-2000. As for Kleopatra's dog bone shape? Likely the leftovers of a violent collision within the Asteroid Belt that stuck two similarly sized space rocks together.
When it comes to 253 Mathilde, it's a wonder there's any asteroid left at all. This pockmarked, porous wad of rock is denser on its surface than it is at its core -- a complete puzzle to scientists. The leading explanation? Constant pounding from impacts squashed Mathilde's surface into a compact layer of rock. Clockwise, from top left: Mathilde in true color; Mathilde's color enhanced; Mathilde in black and white, as seen by the NEAR spacecraft.
Tempel 1
Tempel 1 is a comet, not an asteroid, but in fact the difference between the two types of wayward objects is still poorly understood. What appear to be plain ol' asteroids sometimes erupt into comets as they swing by the sun and heat up, sporting beautiful ionic tails. To better understand the difference, NASA smashed a robot called Deep Impact (scientific pun intended) into this wily comet. The probe created some July 4th fireworks in 2005 by deliberately slamming into the 3.1-by-4.3 mile core, directly between the two dark-rimmed craters at far right. Talk about a hole-in-one.
In a few centuries, this ominous-looking asteroid known as 1999 JM8 may impact the Earth, putting it on the "watch out!" list for humans. Astronomers used deep-space radar to create these snapshots of the asteroid in 1999 as it tumbled through space. Clockwise, from top left: July 28; Aug. 1; Aug. 2; Aug. 5.
Wild 2
Also known as comet 81P, Wild 2 used to have a near-perfect circular orbit for billions of years -- that is, however, until Jupiter pulled it into a more eccentric loop around the sun. NASA's Stardust spacecraft sampled bits of the comet's tail and was the first-ever mission to return a sample of material from beyond the moon safely to Earth. Shown here is the comet's 3.1-mile-wide nucleus as seen by Stardust on Jan. 2, 2004.
Asteroid Apophis is last on our list, and certainly not the best. It is, after all, named after the Egyptian god of "uncreation" for a reason. Known in scientific circles as both 2004 MN4 and 99942 Apophis, this 885-foot-long space rock used to have a fairly good chance of impacting the Earth within a quarter century. After making a breathtakingly close pass on Friday the 13th in April 2029 -- less than 10 percent of the distance between Earth and the moon -- Apophis was thought to have a 1 in 45,000 chance of hitting Earth in 2036 when it swings back around. However, recent calculations have reduced this risk to a 1 in 250,000 chance. Phew! Despite Apophis' diminished threat to Earth, some scientists advocate sending a transmitter to the asteroid's surface to better track its movement. And now, Russia wants to send a deflection mission there, 'just in case' the calculations wrong.
Slide show presentation originally compiled by Dave Mosher.


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Yohane Griffin and Craig Ashmann were just a couple of regular, 13-year-old soccer players — good ones, mind you (Yohane is a member of Washington Youth Soccer's Elite Player Development program), but on the surface, not all that different from your typical junior-high athlete.
That is, right up until the moment they walked into South King County elementary schools earlier this year to give a few soccer tips as part of Washington Youth Soccer's after-school Soccer for Success program.
That's when they became celebrities.
"It was crazy, kids were asking for our autographs and stuff," Craig recalls. "They thought that we were superstars. It was cool."
Griffin and Ashmann, who both play for Federal Way Football Club, have spent the spring representing Washington Youth Soccer at elementary schools throughout the region, teaching kids about the physical, social and psychological benefits of soccer. It's all part of the state's Soccer for Success program, an innovative youth development program that uses soccer as a tool to combat childhood obesity and promote healthy lifestyles for children in low-income urban communities.
In a typical visit, Griffin and Ashmann — who arrive in full uniform — play soccer with the kids, mostly aged 6-12, share the ways in which soccer has improved their lives, and answer questions ranging from how much they have to practice, to what kind of food they eat to stay fit — even, "Did you just fly in?
"The kids ask good questions," says Washington Youth Soccer Board member Paula Griffin, who accompanies the boys on their visits. "They love seeing and playing with Yohane and Craig; they're always very excited."
"We talk about our soccer experiences, and how soccer has affected our life," Yohane says. Craig adds, "We encourage them that if they work hard, they can get far in soccer."
In addition to the service they are providing the Soccer for Success programs, Craig notes that he and Yohane have benefitted, too, in the form of increased confidence speaking in front of large groups, and the experience of being a role model for younger players.
"We were excited at first, but nervous, too, to go up in front of so many people," he recalls. "But then we had a bunch of little kids that thought we were celebrities; it made it easier to relax."
And, of course, to sign a few autographs.
To learn more about participating in the Soccer for Success program, either as a coach, school administrator, or Club or Association representative, visit http://www.washingtonyouthsoccer.org/programs/soccer_for_success/ or email Ellie Lipton.
Washington Youth Soccer • 500 S. 336th St. Suite 100 • Federal Way, WA 98003
1-877-424-4318 (Toll Free) • (253) 4-SOCCER (476-2237)
PlayOn! is published by Varsity Communications, Inc.
For advertising information,please email David Stolber at Varsity Communications, Inc.


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Lakers sink Jazz in OT
Salt Lake City, UT (Sports Network) – Sasha Vujacic hit the go-ahead three-
pointer with 20.1 seconds left in overtime, as the Lakers overcame a late exit
for Kobe Bryant to beat the Utah Jazz, 105-101, at the Delta Center.
Bryant made two controversial free throws in the last second of regulation and
finished with 30 points on 11-of-31 shooting for Los Angeles. He also had
seven rebounds before fouling out with 2:07 left in overtime.
Smush Parker added 21 points and Lamar Odom had a solid night with 12 points,
13 rebounds and nine assists, as the Lakers snapped a two-game skid.
Deron Williams had 20 points and Matt Harpring added 16 before fouling out for
the Jazz, losers of three in a row and eight of 10.
The Lakers were ahead by as many as 14 points before the Jazz rallied in the
fourth quarter. Harpring canned two foul shots with 23.8 seconds left to boost
Utah to a 91-90 edge. Bryant then missed a jumper and Harpring came up with a
loose ball after a scramble, and made one more foul shot with 4.3 seconds
remaining in the fourth.
Bryant then missed a jumper from near the three-point line with the ball
caroming off the rim, seemingly at the buzzer. However, Devin Brown was
whistled for a foul, even though he barely made contact with 0.4 seconds left.
Bryant hit both foul shots to force overtime with the game tied at 92.
"As soon as he went up, I just put my hands straight up and he kind of gave a
'oh' and I couldn't believe the ref made that call," Brown said. "It's tough
to play the whole game and come back and fight back like we did and then it
comes down to one call."
Williams missed a potential winning shot at the regulation buzzer.
"He fouled me," Bryant said. "He kicked my legs out from under me. When it's a
questionable call the refs usually won't make the call when the game's on the
line, but that was an easy call."
A Williams jumper gave the Jazz the lead at 101-100 with 1:11 left in
overtime. However, Vujacic, a second-year player from Slovenia canned a shot
from the left side to give the Lakers the edge.
Gordan Giricek missed a shot on Utah's ensuing possession and Laron Profit
made both free throws for his only two points of the night with 6.3 seconds
left to seal LA's victory.
Los Angeles was on top 51-43 at the half and stretched that advantage to 66-52
on an Odom jumper with 7:03 left in the third. The Lakers were ahead 75-68
going into the fourth.
"We got some calls late that could have gone either way, but luckily we stayed
tough," Odom said.
Game Notes
Lakers guard Aaron McKie suffered a sprained left knee…This was the first of
four meetings this season. The Jazz won three of four from the Lakers last
season, their first series win over LA since the 1998-99 campaign…Bryant is
20-of-64 shooting in the last two games…Dee Brown had 13 points and Andre
Owens scored 11 for Utah…Luke Walker had 12 points, Vujacic added 11 and
Chris Mihm scored 10 for the Lakers…The Jazz shot 36 percent from the field,
but made 42-of-53 foul shots.


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Want to bring your pet with you?
The NW Pet Fair welcomes your well-behaved and leashed pets to enjoy the show with you! For each pet owner, we require a Pet Waiver to be filled out and turned in when you get to the show. We will also provide Pet Waivers at the show. To get a head start on your waiver and avoid lines, please print one out and bring it with you! Pet Waiver FormWe want to make sure all pets brought to the show are safe, so before you bring your pet please take a moment to read the guidelines by clicking: Bring Your Pet Guidelines
Here are some useful tips for bringing your pet:
- Pottying your pet before you head to the Expo Center will decrease pet accidents.
- Bring a doggie poop bag.
- We suggest bringing water and treats for your pet.
- Dogs must be kept on non-retractable leashes, cats must be in an enclosed carrier or stroller.
- Locate the Pet Relief Areas on the show floor map.
- Pets will not be allowed without a signed waiver.
- Please take a moment to read the Bring Your Pet Guidelines.
- Have fun!!


text:
Take a look at the card:
A closer look:
This is really adorable. I love the pinks so much. Welcome back and we missed you too.
Hugs Tammy
Free Candy Winner to be chosen 03/10/12 if you haven't joined come on over. Make sure you read the rules to qualify for Drawing.
Wow this is absolutely beautiful!! I love the colours you have used so soft and subtle!! Hope you are back to health!!
Saw this over at MIM!
Nat -x-
Oh my gosh, this is so cute! I'm such a sucker for a banner and yours is fabulous! Thank you so much for joining us at Make it Monday! Remember, to be in the running to win the prize from our sponsor this week just leave a comment for another entry. Be sure to mention you saw them on Make It Monday. The more entries you comment on the more chances you have of winning!
Rene :D
Saw this on MIM - love it! Great colouring and the bunting is fab.
Nicki, x
Love the colours, beautifully done
Hugs Julie P
Hi Ivonne! What a lovely card! I love the colours and those fabby flowers that you used :) Saw this over at Make it Monday and saw your lovely comments over on my blog - Thanks so much! :)ReplyDelete
This is precious Ivonne! Love those SC images and you've colored this one so prettily. The pink and green are a wonderful combination and I love the little banner. :-) Hope you're feeling much better. :-) Hugs!ReplyDelete
Thanks for stopping by my blog from MIM. I had to come over to see what you created and wow! What a great card. I love the colors and all of the details. Perfect!ReplyDelete
Beautiful work! I love the banner and the flowers and the color scheme and well just everything about this design!!!
Awww, so glad you are back hun! :)
This is an outstanding card, your papers, banners and paper flowers are just gorgeous, and your colouring of this cute image is excellent! :)
Terrific card!
Absolutely gorgeous!
kim x
This is so sweet Ivonne! I love how you have decorated the inside too!
I'm so glad you could join us for the Teenage Dream challenge at Pollycraft this week!
What a great card you posted on MIM! Love the image and sentiment! I have a birthday candy on my blog btw! Hugs, HannekeReplyDelete
Hi Ivonne
I love your card, it is just beautiful! Your colouring just looks amazing and I think that banner is the perfect touch.
Thank you for sharing with us at Make It Monday this week.
Michelle :o)


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Why I Broke Up With BlackBerry
Note: This post was originally written for Emerson Social Media.
This is the start of a wonderful relationship
The other day, I broke up with my BlackBerry. To be honest, we just weren't getting along lately. I could barely get through a day without needing the charge it several times, it would always freeze on me, I couldn't get BlackBerry 6, and for some reasons companies now or days don't even bother to develop BlackBerry applications (case and point, SCVNGR). I'd always get the shaft.
So, I decided it was time for a change. Since my father barely can use a computer, let alone his own BlackBerry, he decided to let me use his upgrade as an early birthday present (yes, you can start with the Happy Birthdays. I love it).
When it came time for me to chose a phone I was confused. The Thunderbolt had just come out on Verizon, the Incredible was a good option too, but there's something about the iPhone. After upgrading my computer to a MacBook back in August, I had already become pretty familiar with the Apple OS. The thought of learning a whole new system on an Android phone wasn't that appealing, even though the Android marketing is growing fast. In fact, the Android just beat out the BlackBerry in terms of smartphone market share.
But it's hard to deny the iPhone. It's a powerhouse and it seems like everyone has one (not literally). It's fast, sleek, and with the huge number of apps available for downloading, you can make it your own. There is something for every possible need.
Not to mention the fact that people always develop the iPhone applications first. If there is a new app on the market, you know they're going to have it on the iPhone before anything else (ex: Color).
A friend of mine today actually said, "I never thought I'd see you as an iPhone person, but I'm glad you are". So am I.
Here are some of my initial thoughts on my iPhone:
- Typing on a touchscreen isn't as hard as I thought it was going to be. It's taking some getting used to, but I'm learning quickly
- I'm going app crazy. There are so many different things to chose from. WHICH ONES SHOULD I CHOSE?
- I'm really glad someone told me about the iPhone multitasking and how to stop running apps to conserve battery power.
- Apparently there is a glitch with Gmail and the iPhone 4. My email doesn't update automatically on my phone, I need to tell it to update. Being a freak about checking my email, this poses a problem. Or, if I'm doing something wrong, please let me know.
- I'm terrible at Angry Birds. After waiting forever to actually play it, this makes me very angry.
Since I'm still in the honeymoon phase with my phone, things could change. But for right now I'm happy I broke up with BlackBerry.


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At last, some common sense from the Lib Dems. No, not their decision to shelve their policies on free childcare and free care for the elderly – all said and done, that doesn't matter enough to make it worth writing home about. But the really exciting news from Lib Dem HQ is this: their leader, Nick Clegg, a father of three young children including a 10-month-old, has decided to ram childcare guru Gina Ford's feeding bottle down her gullet.
Ford's advice, says Clegg, is "absolute nonsense". He and his wife Miriam tried out the former maternity nurse's strictures with their first child Antonio, and found themselves – as so many have – locked into a rigid timetable of feeding, waking, bathing and sleeping. According to Ford, babies have to be trained to fit into their parents' world – and the way to train them is to give up on all semblance of a life yourself so you can dedicate yourself to proving to Junior exactly who is the boss.
Ford has her followers – 2 million of them, we're told – but there are many like the Cleggs, who suffered in silence before finally deciding to come clean. "I remember saying, 'OK, we have to got to stop this. I have subcontracted my parental instincts to this book'," says Clegg.
In response, Ford has suggested the Lib Dems ought to think about finding themselves a new leader. Hmmm, an interesting idea, Gina, but maybe Clegg has just upped his popularity ratings because a lot of us parents out here agree with him. Perhaps it's not a new Lib Dem leader we need, it's a new childcare guru. And perhaps we'll get one: because Ford's regime was fine in an era of plenty, when the country teemed with both-working-flat-out parents who had hardly a minute to call their own, and who desperately needed to mould their offspring to fit in with their busy working schedules. In the new world of austerity, with an increasing number of families hit by redundancy and shorter working hours, we're nothing like as desperate as we used to be to squeeze our babies into a strict routine while we race around like headless chickens making our careers work.
We used to think we were in control of all sorts of things, but life has proved us wrong. The financial crisis taught us that you never really know what's going to happen next: and as with banking, so with babies. Ford's words of so-called wisdom appealed to a certain sort of parent, in a certain sort of economic climate: in the recession-hit world of today, there are fewer such parents around, and economic circumstances favour a more relaxed, instinctive, pleasurable approach to childrearing – as Nick Clegg and his wife, and hopefully many others, are realising.