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Leaflet - Newmarket Saleyards, Newmarket, pre 1987 Reg. No: HT 27693
- Summary:
- Supply list with descriptions of products, codes and sizes. Items include turpentine, cement and floor polish. Affixed to masonite notice board hung in Administration Offices at Newmarket Saleyards.
Maintenance of the Newmarket yards was carried out by the Melbourne City Council. It mainly involved replacing rotten or broken timber fence rails and posts; repair of fittings on gates and rails; re-laying bluestone pitchers; and cleaning and maintenance of the administration buildings.The yard rails were made of hardwood, some of the older posts were jarrah but most were red gum. Sometimes damage to the yard would be caused by animals pushing against fences or becoming stuck between fence rails. Emergency maintenance and repairs had to be made during the sales, with the work being done quickly around the stock.
- Description:
- Rectangular piece of brown cardboard. Card has lines ruled in red ink. Text is written in blue ink.
- Acquisition Information:
- Donation from State Government of Victoria Major Projects Unit, 1990
|16.80 cm (Width), 23.70 cm (Length)
More information
|Tagged with:
|working life, livestock, rural victoria, livestock sale yards, making history - newmarket saleyards
|Themes this item is part of:
|Municipality of Melbourne, Victoria, Sustainable Futures Collection, Working Life & Trades Collection, Newmarket Collection
|Primary Classification:
|Secondary Classification:
|Livestock Markets
|Tertiary Classification:
|reference materials
|Text: 'MOP HEADS 10834 OXY SIZE 120-240' [extensive text]
|Place Used:
|Newmarket Saleyards, Newmarket, Victoria, Australia, pre 1987
|Melbourne City Council (MCC), Newmarket, Victoria, Australia, pre 1987
|Vincent, K 1992, "On the fall of the hammer : a personal history of Newmarket saleyards", Lee White ed, State Library of Victoria, Melbourne.
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New device helps police gather online evidence
By Benjamin J. Ramano
The Seattle Times
SEATTLE — Microsoft has developed a small plug-in device that investigators can use to quickly extract forensic data from computers that may have been used in crimes.
The COFEE, which stands for Computer Online Forensic Evidence Extractor, is a USB "thumb drive" that was quietly distributed to a handful of law-enforcement agencies last June. Microsoft General Counsel Brad Smith described its use to the 350 law-enforcement experts attending a company conference Monday.
The device contains 150 commands that can dramatically cut the time it takes to gather digital evidence, which is becoming more important in real-world crime, as well as cybercrime. It can decrypt passwords and analyze a computer's Internet activity, as well as data stored in the computer.
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New device helps police gather online evidence
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Ex-police officer indicted for excessive use of ECD at traffic stop
Calif. woman talking to police after lawyer says kidnapping wasn't hoax
Mo. police to limit tear gas use in settlement with Ferguson protesters


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Page 1
B The Author(s), 2009
Published Online: 24 June 2009DOI: 10.1007/s11307-009-0236-x
Mol Imaging Biol (2010) 12:15Y24
Comparison of Gene-Transfer Efficiency
in Human Embryonic Stem Cells
Feng Cao,1,2,3Xiaoyan Xie,1Timothy Gollan,1Li Zhao,3Kazim Narsinh,2Randall J. Lee,4
Joseph C. Wu1,2
1Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Edwards Building R354, Stanford, CA, 94305-
2Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University School of Medicine,
Stanford, CA, USA
3Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaxi, China
4University of California San Francisco, Institute for Regenerative Medicine, San Francisco, CA, USA
Technologies designed to allow manipulation and modification of human embryonic stem (hES)
cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety.
The most commonly studied and practiced of these methods include electroporation, lipofection,
nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers
the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion
construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the
firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro
and in vivo studies. In vitro studies examined the transfection efficiency and viability of each
technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction
demonstrated the highest efficiency (H1: 25.3±4.8%; H9: 22.4±6.5%) with >95% cell viability.
Nucleofection demonstrated transfection efficiency of 16.1±3.6% (H1) and 5.8±3.2% (H9).
However, minimal transfection efficiency was observed with electroporation (2.1±0.4% (H1) and
1.9±0.3% (H9)) and lipofection (1.5±0.5% (H1) and 1.3±0.2% (H9); PG0.05 vs. lentiviral
transduction). Electroporation also demonstrated the highest cell death (62±11% (H1) and 42±
10% (H9)) followed by nucleofection (25±9% (H1) and 30±15 (H9)). Importantly, lentiviral
transduction generated a greater number of hES cell lines stably expressing the double-fusion
reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcuta-
neous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust
proliferation as determined by longitudinal bioluminescence imaging. In summary, this study
demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe
techniques for reliable gene transfer in hES cells. The double-fusion construct provides an
attractive approach for generating stable hES cell lines and monitoring engraftment and
proliferation in vitro and in vivo.
Key Words: Human embryonic stem cell, Molecular imaging, Gene transfer, Plasmid
transfection, Lentivirus transduction
yield important discoveries in the areas of cell differen-
he study of embryonic stem (ES) cells will continue to
Correspondence to: Joseph C. Wu; e-mail: firstname.lastname@example.com
Page 2
tiation and growth, tissue engineering, and regenerative
medicine. The ES cells are derived from the inner cell mass
of blastocyst embryos, have unlimited proliferation, and are
the precursor to the three embryonic germ layers [1, 2]. To
efficiently study ES cells and successfully expand their
potential clinical applications, a rapid, reliable, and simple
technique is needed to deliver genes to stem cells. Typically,
gene delivery techniques have been divided into two
categories: viral and non-viral gene delivery. Viral delivery
uses the backbone of a viral genome, such as retrovirus,
lentivirus, adenovirus, and adeno-associated virus (AAV),
with insertion of the gene(s) of interest to be expressed in the
stem cell. The more commonly used non-viral plasmid
delivery systems include liposome, electroporation, and
nucleofection-based, though newer techniques such as
ultrasound [3, 4], microinjection [5], nuclear transfer [6, 7],
and molecular-vibration-mediated delivery [8] have also
been examined, among other techniques.
The advantages of using non-viral delivery compared to
viral vector delivery include decreased immunogenicity and
less insertional mutagenesis. However, the disadvantages of
non-viral delivery may include decreased transfection
efficiency and a shorter period of transgene expression
which may limit long-term studies. For example, the
transfection efficiency and viability of hES cells following
electroporation has been shown to vary depending on the
specific voltage and capacity settings [9, 10]. Cell survival
rates following electroporation are typically low at 24 to 48 h
and often not recorded, though range from >60% at 1 to 2 h
[9], 73% at 2 to 3 h [11], to a median rate of 19% at 48 h [12].
In addition, the efficiency of gene transfer with cationic
liposomes varies significantly based on the commercial
preparation [13]. Liposome preparations may have different
aliphatic chain lengths and structural compositions, as well as
various associated anions. Nucleofection, a liposomal-based
electroporation system that can be adapted with appropriate
buffers for different cell lines, has demonstrated a significantly
higher level of transfection efficiency, with results of 32% to
66% ofviableH9.2cells at24h[11] and20% to22% ofviable
H1 cells at 72 h [14]. The survival rate following nucleofection
has been demonstrated at 74% with a control transfection at
48 h and 50% with transfection of GFP at 48 h [15]. Lastly,
despite the disadvantages associated with the use of viral
vectors for gene transfer, the lentiviral systems have reliably
demonstrated efficient transduction into hES cells. Lentiviral
transduction has demonstrated efficiencies of 20% at low viral
titers to >80% at high viral titer [16, 17].
Although various studies have examined the gene-transfer
efficiency to hES cells in vitro as highlighted above, few have
focused on a direct comparison of these techniques. In this
study, we examine lipofection, electroporation, nucleofection,
and lentiviral transduction of H1 and H9 hES cell lines to
identify the most efficient, reliable, and simple gene-transfer
system for in vitro and in vivo applications. Using a double-
fusion plasmid construct expressing eGFP and Fluc, the
transfection efficiency was carefully demonstrated for each
protocol. Stable cell lines expressing the bi-fusion construct
were generated by lentiviral transduction and nucleofection.
to express the appropriate markers following treatment.
Finally, bioluminescence imaging was used to facilitate non-
invasive monitoring of transplanted hES cells in vivo.
Materials and Methods
Culture of Undifferentiated hES Cells
H1 and H9 hES cells were obtained from Wicell (Madison, WI).
hES cells were maintained on irradiated mouse embryonic
fibroblasts (MEFs) at 5% CO2in medium containing Knockout-
Dulbecco's modified Eagle medium (KO-DMEM), 20% serum
replacement, 1% nonessential amino acids, 1 mM L-glutamine,
0.1 mM β-mercaptoethanol, and 4 ng/ml fibroblast growth factor-2
(FGF-2; GIBCO BRL). The cells were passaged in a 4-day cycle by
incubating with 1 mg/ml collagenase IV at 37°C for 45 min,
maintaining the cells in small clumps. To avoid contamination by
MEFs in the transfection (liposome, electroporation, nucleofection)
analysis, the hES cells were cultured on Matrigel in MEF-
conditioned medium for one passage prior to transfection. Approx-
imately, 2×104/cm2cells were plated uniformly in six-well plates
prior to transfection. Samples of the cells were immunostained with
anti-SSEA-4 (Chemicon, Temecula, CA) and anti-Oct-4 (Santa Cruz
Biotechnology, Santa Cruz, CA) antibodies. Immunofluorescent
labeling was analyzed using a Zeiss LSM 510 Confocal Laser
Scanning Microscope equipped with a Coherent Mira 900 tunable Ti:
sapphire laser for two-photon excitation (Zeiss, Minneapolis, MN).
Construction of pUb-eGFP-Fluc and pUb-eGFP-
The pUb-eGFP-Fluc construct used for lentiviral transduction has
been described previously [18]. The pUb-eGFP-Fluc-SV40-
neomycin was generated by cleaving pUb-GFP-Fluc at the BamH1
restriction enzymes and blunt-end ligating the SV40-neomycin
construct. This plasmid was used for stable transfection of hES
cells using neomycin as a selection marker.
Plasmid Transfection Methods
hES cells (4–5×105/well) onto Matrigel at a confluency of 60–70%.
Qiagen lipofection 2000 was used according to the manufacturer's
protocol. Briefly, 5μg of plasmid DNA (pUb-eGFP-Fluc) was diluted in
The mixture was incubated for 15–20 min at room temperature. The
mixture was then added to 1,000μl of pre-warmed conditioned medium
and transferred to one well of a six-well plates. (b) Electroporation: 5μg
of plasmid DNA (pUb-eGFP-Fluc) was diluted in 500μl of phosphate-
buffered saline (PBS) and added to 4–5×105hES H1 and H9 cells
suspended in 500μl of hES culture medium. Electroporation was
performed in a 4-mm gap cuvette using a Gene Pulser (BioRad,
After pulsing,the cellswere incubatedin the cuvette at room temperature
for 10 min. The cells were transferred to new medium and plated onto
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 3
Matrigel-coated six-well plates at a density of 2×105cells/cm2.
Each plate was analyzed for eGFP expression under a fluorescence
microscope at 24, 48, and 72 h post-electroporation. (c) Nucleofection:
4°C. The culture medium was removed and cells were resuspended in
100μl of the nucleofector solution (Amaxa Biosystems, Cologne,
Germany). DNA (5μg) was added, cells and DNA were gently mixed,
and cells were electroporated using the Nucleofector™ device (Amaxa
Biosystems). Transfections were performed with a program (A23) that
was selected in a series of pilot transfection experiments with the eGFP
reporter gene and proven to result in the highest transfection efficiency.
Lentiviral Vector Preparation and Transduction
A self-inactivating lentivirus was prepared by transient transfection
of 293 T cells as described previously [16, 19]. Briefly, 12µg of the
HIV-1 packaging plasmid (peGFP-Fluc/pRRE; delta-8.9) contain-
ing the eGFP-Fluc reporter gene were cotransfected into 293 T cells
with 9µg of vesicular stomatitis virus G glycoprotein-pseudotyped
envelope vector (pVSV-G), and 6.25µg of REV plasmid (pRSV-
REV). Lentivirus supernatant was collected after 72 h and
concentrated by sediment centrifugation with SW29 rotor at
50,000×g for 2 hours. Concentrated virus was titrated on 293 T
cells. hES H1 and H9 cells (4–5×105) were incubated with virus
particles at MOI of 10 and 8µg/ml polybrene (Sigma). The media
was changed after 12 h by fresh medium.
Generation of Stable Cell Line
Stable hES cell lines were generated by fluorescence-activated cell
sorting (FACS) in the lentiviral transduction group and by G418
selection (50–100µg/ml) over 2 weeks in the nucleofection group.
Colonies were isolated, propagated, and analyzed for eGFP
expression and Fluc activity.
Cell Viability Analysis
Cell viability was determined using dissociated cells prior to
plating, as well as at 24 and 48 h after transfection. Viability was
measured using trypan blue exclusion and expressed as a
percentage of the initial number of cells used for each transfection.
The H1 and H9 hES cells treated with the various transfection
protocols were plated onto cover slips within six-well plates, fixed
in 5% paraformaldehyde for 1 min, and blocked with 1% bovine
serum albumin in PBS. They were incubated with the primary
antibody in PBS at 37°C for 1 h, followed by several PBS washes,
and incubation with a secondary antibody at 37°C for 1 h, with
final PBS washes. Slides were mounted with Vectorshield (Vector
Laboratories, Inc., Burlingame, CA, USA).
Fluorescence-activated Cell Sorter Analysis
Twenty-four hours after transfection, the cells were washed twice
with KO-DMEM and dissociated with Accutase II. After centrifu-
gation, the cells were resuspended at 1×106cells/ml in PBS and
stored on ice for a maximum of 1 h before analysis. Acquisition
was performed on a FACS Calibur system (BD Biosciences,
Heidelberg, Germany) and samples were analyzed using FlowJo
software (Tree Star Inc., Ashland, OR). Analysis-gating criteria for
eGFP-expressing cells were set according to the level of auto-
fluorescence of a non-transfected control.
Transplantation of hES Cells into Nude Mice
Harvested control hES and stable hES-DF cells (transduced with
lentivirus) were kept on ice for G30 min for optimal viability. Adult
immunodeficient nu/nu mice (n=5) were injected subcutaneously
with 1×106hES-DF cells in 50µL of PBS. Control animals (n=3)
received 1×106non-transduced hES cells. All animals had an
uneventful recovery and underwent bioluminescence imaging at
various time points. Study protocols were approved by the Stanford
Animal Research Committee.
Optical Bioluminescence Imaging of hES Cell
Bioluminescence imaging was performed with the Xenogen In Vivo
Imaging System (IVIS, Alameda, CA). Following intraperitoneal
injection of the reporter probe D-luciferin (375 mg/kg body weight),
animals were imaged for 30 min with 1-min acquisition intervals. The
same mice were scanned at various time points for a 6-week period
according to the specific study design. Bioluminescence was
quantified in units of maximum photons per second per centimeter
squared per steradian (P s−1cm−2sr−1), as described previously [18].
Statistical Analysis
Quantitative data were expressed as mean±SD. Means were
compared using one-way ANOVA and the Student t test. P values
of less than 0.05 were considered statistically significant.
hES Cells Maintained on a Feeder-free Culture
System Express Stem Cell Markers
Previous studies have detailed the need to separate hES cells
from contaminant feeder layer cells prior to the initiation of
studies [20]. For our studies, the H9 and H1 cells were
reseeded onto Matrigel-coated plates to eliminate any
possible contaminating effects of the MEF feeder layer.
Additional studies were performed on these hES cells to
confirm that separation from the feeder layer had not
changed the basic characteristics of the cells. The cell
doubling time was tested by cell counting, which revealed
similar results between the hES cells on feeder layer and
feeder-free plates (37±3.4 h vs. 36±2.1 h; P=NS). The two
cell lines showed the same morphology under brightfield
microscopy (Fig. 1). Immunohistochemistry staining also
showed similar expression pattern of Oct-4 and SSEA-4
under fluorescence microscopy for H9 and H1 hES cells.
These results confirm that H9 and H1 hES cells grown in a
feeder-free cell culture system maintained similar growth
and surface marker expression characteristics compared to
hES cells grown on conventional feeder layers.
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 4
Lentivirus transduction and Plasmid Nucleofection
Demonstrate Higher Efficiencies in vitro
Following a review of the literature (previously detailed)
[11, 21–23], transfection conditions yielding the highest
efficiencies for hES cells were utilized for each transfec-
tion protocol. Within 24 h, there was a significant
difference in gene-transfer efficiencies in H9 and H1
cells. In particular, lentiviral transduction demonstrated
the highest level of Fluc gene expression, with biolumi-
nescence measurements of 0.95±0.13×106photons/s for
H9 and 1.37±0.16×106for H1. Nucleofection demon-
strated lower transfection efficiency compared to lenti-
viral transduction (3.2±3.9×105photons/s for H9 and
5.6±0.12×105for H1; PG0.05 for both). Importantly,
both electroporation and lipofection had very low
transfection efficiencies compared to lentiviral transduc-
tion and nucleofection (Fig. 2). In our studies, the H1
cell line had higher transfection efficiency than the H9
hES cell line for all protocols tested, suggesting varying
susceptibility to gene transfer in different hES lines.
FACS Analysis of eGFP Expression in H1 and H9
hES Cell Lines
The double-fusion construct allows us to simultaneously
analyze eGFP by FACS and Fluc by bioluminescence
imaging. Consistent with the Fluc bioluminescence activity
shown above, the eGFP expression was highest in lentiviral-
transduced cells followed by nucleofection-transfected cells
(Fig. 3). Lentiviral transduction demonstrated the highest
transduction efficiency, with 25.3±4.8% and 22.4±6.5%
positive eGFP cells for H1 and H9, respectively. Nucleo-
fection demonstrated transfection efficiency of 16.1±3.6%
(H1) and 5.8±3.2% (H9). Low eGFP expression was again
seen in electroporation (2.1±0.4% (H1) and 1.9±0.3% (H9))
and lipofection (1.5±0.5% (H1) and 1.3±0.2% (H9)).
Higher Cell Viability Demonstrated
in lentiviral-transduced Cells
The protocols and expertise to isolate and grow sufficient
quantities of hES cells are complex and technically
challenging. Due to this laborious process, a high hES cell
viability following gene transfer is essential. It is commonly
known that electroporation of hES cells results in significant
cell death, despite efforts to optimize and minimize the
energy delivered [9]. The use of lentivirus, on the other
hand, has previously demonstrated high levels of cell
viability following transduction [16, 17]. To address this
issue, we examined the cell morphology and assessed the
cell viability using trypan blue exclusion at 24 h following
gene transfer with the various protocols. The H1 and H9
cells were examined by overlaying brightfield microscopy
with eGFP fluorescence. H9 cells clearly demonstrate
significantly lower viability in electroporation treated cells
(Fig. 4), followed by nucleofection-treated cells. Similar
results were obtained for H1 cells (data not shown). The
trypan blue assay details the cell viability following electro-
Fig. 1. Immunohistochemistry of stem cell markers on undifferentiated H9 and H1 cell lines grown in feeder-cell free cultures in
vitro. H9 and H1 cells were reseeded onto Matrigel-coated plates. Brightfield and immunohistochemistry staining with
fluorescence microscopy for Oct-4 and SSEA-4 stem cell markers was performed. DAPI staining is used as a nuclear marker
(scale bar=50µm in mid- and right fluorescence staining; 200µm in left brightfield).
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 5
poration at 62±11% (H1) and 42±10% (H9)) followed by
nucleofection at 25±9% (H1) and 30±15% (H9) at 24 h. By
contrast, the other four groups all showed greater than 95%
In vivo Imaging to Monitor hES Cell Survival,
Proliferation, and Teratoma Formation
The ability to monitor transplanted hES cells in vivo without
sacrificing the animals has been made possible by incorpo-
rating newer imaging technologies such as optical biolumi-
nescence or positron-emission tomography [24]. Following
subcutaneous transplantation, bioluminescence imaging was
performed on days 2, 7, 14, 35, 42, and 56 (Fig. 5a). H9-DF
cells (via stable lentiviral transduction) showed persistent
engraftment and survival as measured by bioluminescence
activities: day 2 (55.2±14.3×106), day 7 (21.2±6.1×106), day
14 (17.2±5.0×106), day 35 (19.5±3.2×106), day 42 (28.1±
7.2×106), and day 56 (65.3±14.1×106p/s/cm2/sr). Control H9
cells (not expressing the GFP-Fluc) cannot be detected as
expected. Following imaging at day 56, animals were
sacrificed and the teratoma explanted. Histologic analysis of
control hES and hES-DF teratomas shows mixed regions of
endodermal, mesodermal, and ectodermal differentiation
(Fig. 5b). Thus, the histological data suggest that lentiviral
transduction did not significantly affect the differentiation of
hES cells into the three germ layer cell types in vivo.
To date, the ability to manipulate stem cells for clinical
applications has been limited by techniques in basic molecular
and cellular biology, as well as our understanding of ES cell
biology. Protocols for gene transfer that had demonstrated
significant promise in mouse embryonic stem (mES) cells,
suchaselectroporationandlipofection,are lessefficientinhES
Control Electro+P Nucleo+PLentiV
Fluc Radiane / Control (fold)
Fluc Radiance
Lipo+P Plasmid
Fig. 2. Transfection and transduction of H9 and H1 hES cells in vitro with each of the transfection protocols. (a) H9 and H1
cells were seeded onto feeder-cell free Matrigel-coated six-well plates after transfection with plasmid alone (Plasmid),
lipofection (Lipo+P), electroporation (Electro+P), nucleofection (Nucleo+P), and transduction with lentivirus (LentiV). (b) The cells
were examined using bioluminescence imaging of Fluc activities after transfection. The quantitative measurements are shown
24 h after gene delivery.
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 6
cells [12]. In general, mES cells have demonstrated higher
levels of transfection efficiency, whereas hES cells are
notoriously difficult to transfect. This has led to optimizing
existing protocols, as well as the discovery of novel and
cutting-edge techniques that have increased transfection
efficiency into hES cells. It is important that if these protocols
method of gene transfer, the effect on hES cell growth and
should remain intact over prolonged periods of time. Using a
double-fusion reporter gene that allows for non-invasive "real-
time" imaging, we have examined the transfection and
transduction efficiency of various protocols on the H1 and
H9 hES cell lines in vitro and in vivo.
Few studies have performed side-by-side comparison of
the efficiency of the various transfection and transduction
methods available for gene transfer to hES cells. Nucleofec-
tion of hES cells has shown superior transfection efficiency
compared to lipofection and electroporation for H9.2 cells
[11], and in adipose-tissue-derived stem cells [21]. We have
examined the protocols from these studies, as well as
numerous other studies to identify the optimal experimental
conditions for electroporation, lipofection, nucleofection, and
lentiviral transduction using male H1 and female H9 cells,
and have compared their transfection or transduction efficien-
cy in vitro. The double-fusion construct (expressing eGFP and
the Fluc) allowed for rapid, reproducible, and real-time
monitoring of cells over an extended period of time.
Lentiviral transduction demonstrated the highest level of gene
transfer (22–25%) with the best viability following treatment
(>95%). The highest efficiency using a non-viral protocol was
demonstrated by nucleofection, though with a mildly de-
creased level of viability (70–75%). Electroporation and
lipofection demonstrated very low levels of transfection
efficiency, which is consistent with previous studies reported
[23]. Viability following treatment with electroporation was
poor (20–25%) despite attempts to optimize the specific
voltage and capacity settings. For each modality, expression
of the eGFP-Fluc double-fusion reporter gene did not appear
to affect cell growth and viability.
Fig. 3. Measurement of eGFP expression using flow cytometry analysis of H1 and H9 hES cell lines transfected with each
protocol. The bi-fusion pUb-eGFP-Fluc construct was transfected into H9 and H1 hES cells using lipofection (Lipo+P),
electroporation (Electro+P), nucleofection (Nucleo+P), and transduction with lentivirus (LentiV). eGFP expression was measured
using FACS analysis.
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 7
Previous studies have examined transient transfection of
various hES cell lines in vitro, and production of stable
human ES cell lines [16, 23, 25]. However, few studies have
generated stable hES cell lines and monitored their growth in
vivo using noninvasive bioluminescence imaging. In this
study, we have also generated stable cell lines with lentiviral
transduction and plasmid nucleofection protocols using the
double-fusion (DF) reporter gene construct and monitored
their growth and differentiation in vivo over several months.
Immunofluorescence and histological analysis demonstrates
that the stable hES cell lines consist of undifferentiated cells
that express numerous stem cell markers, including SSEA4
and Oct4. After several weeks, these undifferentiated cells
develop into subcutaneous teratomas. In addition, the DF
reporter gene did not affect hES cell viability, growth, and
differentiation, similar to previous report on mES cells [24].
Of note, the higher number of stable cell lines (~twofold
higher) generated by lentiviral transduction compared to
nucleofection is likely secondary to a higher gene-transfer
rate, in combination with a higher rate of viability.
Transfection and transduction protocols that can success-
fully transfer genes to the nucleus in a cell-cycle-indepen-
dent manner will be most effective in hES cells. Lipofection
(including Fugene and Exgen 500), transduction with many
pseudotyped retroviral constructs (e.g., murine leukemia
virus), and polymer- or polycation-based protocols require
cell proliferation (mitosis) and breakdown of the nuclear
envelope, or nuclear localization signal (NLS) directing
transport from the cytoplasm to the nucleus. The trafficking
of gene products to the nucleus has been challenging, with
mixed success using well-characterized NLS signals, such as
the SV40 NLS for example [26–28]. By contrast, lentivirus,
adenovirus, and AAV can infect both dividing and non-
dividing cells, have low cytogenicity, and can integrate their
genome into the host chromosome. The use of vectors
derived from retroviruses as gene-transfer vehicles in this
setting has had limited success because of silencing of
transgene expression. Two major mechanisms have been
identified for retrovirus silencing: trans-acting factors that
bind to the viral promoters in the long terminal repeats
(LTRs) and methylation of the integrated retroviral genome
and flanking host DNA sequences [29]. Similar to the
lentiviral system, electroporation is independent of cell cycle
and can directly transport DNA to the nucleus. Despite
efforts to optimize the specific voltage and capacity settings,
survival rates and levels of transfection efficiency in hES
Cell Viability (%)
Nucleo+P LentiV
Fig. 4. Viability of H9 and H1 hES cells in vitro following transfection with the various gene-transfer protocols. H9 and H1 hES
cells were examined at 24 h following transfection with lipofection (Lipo+P), electroporation (Electro+P), nucleofection (Nucleo+P)
(a) Fluorescence microscopy of representative H1 cells. Scale bar=100µm. (b) Viability of each transfected cell lines at 24 h as
measured using the trypan blue assay.
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 8
35 42
Control hES-DF
27 14 (days)56
Fig. 5. Optical bioluminescence imaging of stably expressed hES cells in vivo over time, with demonstration of proliferation
and teratoma formation. The pUb-eGFP-Fluc-SV40-Puro construct was stably expressed in H9 cells by transduction with
replication-incompetent lentivirus. Stable colonies (hES-DF) were selected by drug selection over 2 weeks and then injected
subcutaneously into right shoulder of nude mice. Control untransduced H9 hES cells were injected into left shoulder. (a) In vivo
bioluminescence imaging of the hES-DF cells on days 2, 7, 14, 35, 42, and 56. (b) Histology of the H9 hES cells stably
expressing eGFP-Fluc in nude mice were examined through 56 days. Teratoma formation was demonstrated by histology at
week 8 weeks after subcutaneous injection: (A) rosette consistent with neuroectodermal differentiation (ectoderm), (B) cartilage
formation (mesoderm), (C) respiratory epithelium with ciliated columnar, and (D) mucin-producing goblet cells (endoderm)
surrounded with mesenchymal cells (mesoderm). Scale bar=50µm.
F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells
Page 9
cells have been poor. Nucleofection, on the other hand, has
demonstrated higher levels of transfection efficiency with
significantly improved levels of cell viability over electro-
poration. This technique has demonstrated the ability to
ubiquitously transfect various cell lines [30, 31], including
CD34+cells and H1 cells [14]. In this study, the nucleo-
fector settings and buffer were selected based on previous
studies in our lab, as well as other published reports [27, 28].
The ability to adapt the nucleofection parameters and buffers
to various primary cell lines may further improve transfec-
tion efficiency. In addition, other studies using nucleofection
have demonstrated efficient expression of siRNA [32],
mRNA [22, 33], and various small and macromolecules [9].
In this study, both lentiviral transduction and plasmid
nucleofection protocols successfully generated stable hES
clones that stably expressed the DF reporter gene. Although
lentivirus generated higher numbers of stable clones,
nucleofection is a simple, reproducible, and efficient
technique for transient as well as stable transfection. The
double-fusion construct allowed for effective, non-invasive
tracking of proliferating hES cells in nude mice, with no
noticeable disruption of cell differentiation during prolonged
gene expression. The lack of significant transgene silencing
following in vivo transplantation has been previously
reported [24].
In summary, the ability to study the biology of hES cells
requires genetic manipulation to express various transgenes.
Nucleofection is an attractive transfection protocol with
minimal safety concerns. It is a simple and efficient system
and can be used to generate hES cell lines stably expressing
reporter genes. Although this protocol can generate stable
hES cell lines, the lentiviral system is more efficient and
could still play a role in various applications.
Acknowledgments. This work was supported by CIRM RS1-00322, NIH
HL089027 (JCW) as well as Shanxi Scientific and Technical Key Project
(2008K13-01), NIH HL091453, and Xijing Research Boosting Program
Open Access. ThisarticleisdistributedunderthetermsoftheCreativeCommons
Attribution Noncommercial License which permits any noncommercial use,
source are credited.
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Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from
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ation in vitro. Nat Biotechnol 18:399–404
3. Unger E, Hersh E, Vannan M, McCreery T (2001) Gene delivery using
ultrasound contrast agents. Echocardiography 18:355–361
4. Nakashima M, Tachibana K, Iohara K, Ito M, Ishikawa M, Akamine A
(2003) Induction of reparative dentin formation by ultrasound-mediated
gene delivery of growth/differentiation factor 11. Hum Gene Ther 10
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(1999) Mice cloned from embryonic stem cells. Proc Natl Acad Sci U S
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9. Mohr J, de Pablo J, Palecek S (2006) Electroporation of human
embryonic stem cells: Small and macromolecule loading and DNA
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10. Sukhorukov V, Reuss R, Zimmermann D, Held C, Muller KJ, Kiesel
M, Gessner P, Steinbach A, Schenk WA, Bamberg E, Zimmermann U
(2005) Surviving high-intensity field pulses: strategies for improving
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Nucleofection of human embryonic stem cells. Stem Cells Dev 14:378–
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Abbayes H, Mercier B, Abgrall JF, Ferec C (1997) Cationic phospho-
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lines and CD34+ cells. Blood Cells Mol Dis 23:69–87
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Kaufman D, Verfaillie C (2004) Efficient transfection of embryonic and
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15. von Levetzow G, Spanholtz J, Beckmann J, Fischer J, Kogler G,
Wernet P, Punzel M, Giebel B (2006) Nucleofection, an efficient
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16. Ma Y, Ramezani A, Lewis R, Hawley R, Thomson J (2003) High-level
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Reubinoff B (2003) Stable genetic modification of human embryonic
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Singh P (2004) Highly efficient mRNA-based gene transfer in feeder-
free cultured H9 human embryonic stem cells. Cloning Stem Cells
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Hennink WE, Crommelin DJ (2005) An NLS peptide covalently linked
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Coupling of nuclear localization signals to plasmid DNA and specific
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F. Cao et al.: Gene-Transfer Efficiency in Human Embryonic Stem Cells


text:
Scott Dixons Car Wipes Out Two In Pit Crew [VIDEO]
I watched this video a few times and it does not look like it was on purpose.
I watched this video a few times and it does not look like it was on purpose.
Know the law and know your rights.
Well the Chargers had it, lost it, Cardinals pick it up then loose it, Chargers get it again finally and make a touchdown!
This is a short film about one girl that left her phone somewhere for one day!
This is some funny stuff right here!
There is always misconceptions about the way things are named and are in this world. For example German Chocolate cake was named after a guy not Germany!
Check this video out and see for yourself!
On this day in 1966 an amazing guitar player was born! Dimebag Darrel Abbott!
This is an interesting outlook for chicks about 'The Girls' and why not!
Ball State University, located in Indiana, sports famous alum like late night T.V.'s David Letterman.
The most famous man on campus today, though, may be incoming freshman Markus Burden.
These crazy kids should do this for a show!
They were in the practice studio when the played 'Train Kept A Rollin' for the first time on this day in 1968.
This movie looks so good! Based of the book by Orson Scott Card!


text:
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For those of you who have already been captivated and taken into Neil Finn's mysterious sexy world of contrasts, I am simply preaching to the choir. Read morePublished 14 months ago by Janet Zemel
While I LOVE Crowded House and Neil Finn, this is just a little bit disappointing overall. There are some awesome moments, but there are also some skip-able ones. Read morePublished on February 18, 2013 by Don Parker
My husband and I had our first date at a Crowded House concert. This live album brought us back to a great evening ! Well recorded and a joy to listen to.Published on January 8, 2013 by Patricia H
Yet another CH release that is amazing as ever. I had to write just to say, I'm an AMERICAN who started listening in 1986 and I never stopped. Read morePublished on August 5, 2012 by common folk songs
Their last album with the original lineup. This two cd set was recorded in Sidney harbor and is a live greatest hits mix. It is lovingly recorded. Read morePublished on August 29, 2010 by P. robb
Crowded house are so much more than "don't dream it's over". They are quite simply one of the best,if not THE best band of the 80's. Read morePublished on March 4, 2010 by Amazon Customer
I know quite a number of music fans who avoid "live" albums in favor of studio recordings. They perceive that the live albums sometimes do not add much over the studio recordings,... Read morePublished on March 18, 2009 by P. Opus
Ok true confessions first--I'm a huge Neil Finn fan and also had this when it was released 10 years ago as part of the Crowded House Fanclub live releases. Read morePublished on July 14, 2007 by Wayne Klein
I have been a Crowded House / Finn Brothers fan for a long time, but this live CD is amazing. Neil Finn is such a talented and gracious musician and performer. Read morePublished on May 6, 2007 by Amy A. Camisa
|From this Discussion
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text:
displaying 61-90 out of 191
Women vs Men : The garage
Another creation for the unending fight between the sexes!!
Different POV
Cause... you know... he is a man... and they are women... like... not a man...
Looking at the whole picture is important when you are talking about women.
read the joke
Two women that are dog owners are arguing about which dog is smarter...
-Stop taking pictures of me bitch! -Nan please, that's a ...read the joke
Bed nagging
That's also an option to divide the bed!
Be polite.
Yeah, right...
The power of carats
Commercial for Natan Jewerly.
Reasons girls like winter
Great pie chart! And sooo true!


text:
Unions and the Fallacy Behind Them
Written November 18, 2011 Generations of people have grown up with the misconceptions that unions are the God's of the people, by the people for the people. Like the current state of affairs with our government nothing could be further from the truth. Union heads are strong supporters of the current administration with huge contributions… Read More »


text:
Milltown, County Antrim
Milltown is a small settlement in County Antrim, Northern Ireland. It is within the townland of Derriaghy,[1] about one mile to the north of Lisburn. Once a rural village, it is now part of the Greater Belfast conurbation. However, it is separated from the surrounding urban area by a narrow stretch of countryside. In the 2001 Census it had a population of 1,361 people.
Milltown is a local service centre with facilities including retail units, the former Derriaghy Primary School, Christ Church, Church of Ireland and hall, Derriaghy Gospel Hall and a Community Centre. There is a railway halt in Derriaghy, to the east.
Milltown owes much of its history to the succession of mills that were established by the early 19th century, and which continued to develop over the next century. Christ Church, a local landmark, lies to the eastern end of the settlement and is situated on an earlier site that dates back to at least the medieval period. Throughout the 19th century the village of Milltown was fairly modest in size, but the settlement expanded significantly in the later 20th century.
2001 Census[edit]
Milltown is classified as a village by the NI Statistics and Research Agency (NISRA) (i.e. with population between 1,000 and 2,250. On Census day (29 April 2001) there were 1,361 people living in Milltown. Of these:
- 28.0% were aged under 16 years and 12.9% were aged 60 and over
- 48.3% of the population were male and 51.7% were female
- 28.4% were from a Catholic background and 67.7% were from a Protestant background
- 4.1% of people aged 16–74 were unemployed
For more details see: NI Neighbourhood Information Service
- ^ Ordnance Survey Ireland: Online map viewer (choose "historic" to see townland boundaries)
See also[edit]
Coordinates: 54°32′32″N 6°2′30″W / 54.54222°N 6.04167°W


text:
The Top 10 In Action Sports
01This is What It Looks Like To Break The World Record For The Fastest Time on a Bike
02This All-Action Edit Encapsulates Every Single Reason We Should All Love Motocross
03A Glacier The Size Of Manhattan Has Disappeared, And These Guys Got It On Film
04Cork 720s and Flipping Madness: The World's Best Slopestyle Stars Just Opened the MTB Season in Style
05Welcome to Qasr Al-Farid: The Rock With A Door That Will Rock Your World
06Already Got MTB Withdrawal Symptoms? Here Are The Crazy Downhill and Enduro Highlights From Crankworx New Zealand
0724 Reasons Why Snowboarding Was Better In The Early Noughties
08Win a Holiday for 2 in Chamonix with Three #holidayspam
0918 Things That Will Give Anyone Who Did Duke Of Edinburgh A Serious Flashback
1016 Things That Will Give 90s Skater Kids A Serious Flashback
Obstacle Series: Protest Parents In Law
Protest helps everyone to ride more. We protest against all the obstacles that stand between you and your board and help you to get there. Being fresh powder, a perfect pointbreak or a wake session at a glassy lake.
The 'Protest Obstacle Series' are short movies about riders and their obstacles, made by creative minds within and outside the industry. They are made to inspire you. Watch them and get the hell out of here. See you on the slopes or in the water.
Protest Parents In Law is produced by Kaaps
Actors in order of appearance:
Jette Edens
Oli Adams
Addie van de Walle
Bram Kwekkeboom
Screenplay: Eelco de Winter
Director and D.O.P.: Frank Borghstijn
Assistant director: Sjoerd Schipper
Camera assistant, light and grip: Wouter Boes
Editing: Frank Borghstijn
Sound design: Ronnie van der Veer
Thanks to Millers, The Hague
For more info about Protest Boardwear check: http://www.protest.eu


text:
Welcome to the League of Women Voters of Illinois.
Today is
The League of Women Voters of Illinois encourages the informed and active participation of citizens in government and works to influence public policy through education and advocacy. Any citizen, 18 or older, male or female, may join. The LWV is nonpartisan and neither supports nor opposes candidates for elected office but does act on issues after member study and consensus. Read more.
Did You Know?
Featured Resources
February 2015 E-News
2015 Issues Briefing
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Patricia V. Connor Resch
unknown - April 17, 2013
Patricia V. Resch, 78 of Syracuse, passed away peacefully Wednesday at St. Joseph's Hospital. A lifetime Syracuse resident she was a graduate of Most Holy Rosary High School, and received a BA in Education from LeMoyne College. Patty was a substitute teacher in the Liverpool School District and taught religious education. She was a Health Ade for the elderly and a volunteer for the Right to Life Committee for many years. She enjoyed spending time with her grandchildren and was a life long Elvis Fan.
She was predeaced by her husband John E. Resch, her sister Margaret Casey-Pointkowski and one brother Joseph Connor.
Patricia is survived by her three daughters, Mary Pat (James) Leonard of Fairmount, Teresa (Nick) Lore of Liverpool, Ellen Resch of Liverpool, two sons, John and James Resch of Syracuse, five grandchildren and four great-grandchildren. Also, a brother Martin (Janet) Connor, and a sister Mary Ellen Lozo, a sister in law Rita Connor and several nieces and nephews.
A Mass of Christian Burial will be at Most Holy Rosary Church, 111 Roberts Avenue, Syracuse, NY, 13207.
Burial will follow in Woodlawn Cemetery.
There are no calling hours.
Contributions may be made in Patricia's name to NYS Right To Life Committee, 41 State Street, Albany, NY 12207.


text:
"Ok story, terrible narrator"
Her delivery, tone, accents, voices, everything. Just not that great.
It was a good Sandra Brown book, with some good story development and twists, but the narration made it really, really hard to get through.
Among the best. The story is gripping and the narration is amazing. The best.
EVERYTHING! He's perfect. He lent the perfect tone to this amazing story.
Both! What an amazing story. I have such a great appreciation (even more than ever) for WWII vets and POWs. And I learned so much. What an incredible book and story.
You're missing out if you don't listen to this book.
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This article is part of the Seeking Alpha PRO subscriber-only research library, which covers more than 3,800 companies. Click here for more on subscribing to Seeking Alpha PRO.
Organovo: Modest Expectations For The Liver Tox Assay
Jan. 6, 2014 12:17 PM ET | About: Organovo Holdings, Inc. (ONVO) This article is now exclusive for PRO subscribers.


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Blog Archives
Lastest sneek-peak from Cabal US
Original Source : cabal.com
New Mercenaries
New items
Ring of Luck +3
Critical Ring +3
Killian Ring +3
Prideus' Bracelet
Battle Mode 3 screenshots of 6 battle styles
Force Shielder after activating "Hammer Crusher" – as modeled by yours truly!
Warrior after activating "Axe Destroyer"!
Wizard after activating "Elemental Dominator"!
Blader after activating "Knucklet Striker"!
Force Archer after activating "Launcher Punisher"!
Force Blader after activating "Sword Splitter"!
Battle Mode 3 utilizes the new Skill Chaining system to unleash three levels of deadly Fatal Attacks, granting revolutionary Synergy Effects unique to each Battle Style.
Embark on an epic quest for power and glory. Conquer never before seen enemies residing in previously undiscovered locations. Stop at nothing to become one of the most powerful champions the world of Nevareth has ever seen.
Your journey will not be easy. Only the strongest and bravest of warriors will survive. If you are one of them, you will hold the power of Minesta's Legacy in the palm of your hand.
New DX
Some new images of the Next Update – EP8
Appearance Change
Original Weapon
Trying to Change the Appearance
Result Weapon
You can restore back to the Original appearance
Epaulet with Slot
Adding Slot to Epaulet by using Conversion Formula Card
RoL3 / CR3
Killian Ring
Material for crafting Killian Ring
Infinity Chaos
Enty Item
1st Stage – 1st Boss
2nd Stage – 2nd Boss
3rd Stage – 3rd Boss
4th Stage – 4th Boss
5th Stage – 5th Boss
6th Stage – 6th Boss
7th Stage – 7th Boss
8th Stage – 8th Boss
9th Stage – 9th Boss
10th Stage – 10th Boss
11th Stage – 11th Boss
12th Stage (Final Stage) – 12th Boss (Final Boss)
Cleared Infinity Chaos


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My daughter turned one year old the other day! It's been an incredible experience to be a daddy and I am so thankful for the gift that she is to my wife and me. But as many parents know, you learn a lot about yourself and the important things in life as you try to […]
My One Year Old and Ebola: It's all about Preparation and Prevention
In Colossians 1:27, Paul wrote about the riches of the mystery "which is Christ in you, the hope of glory." This week the US staff of Food for the Hungry (FH) is having an awards day. Several members of our team will be honored for their contributions to our FH community and work. The […]
The Seen and Unseen World
Paul wrote in Ephesians 6: Put on the whole armor of God, that you may be able to stand against the schemes of the devil. For we do not wrestle against flesh and blood, but against the rulers, against the authorities, against the cosmic powers over this present darkness, against the spiritual forces of evil in the heavenly places. (ESV) This is a profound […]
Mommy, When Can We Go Home?
There is a longing inside each one of us to go home. Whether it is a child away at camp or visiting grandma, or an adult on vacation or away on business, or a young soldier off to serve at a distant post, our hearts long to return to home. While we all feel that […]
Fleeing to Find a Safe Place to Call "Home"
I have many choices living in America – but millions who suffer from religious persecution around the world don't… Have you ever felt like you had the perfect plan and you wake up the next morning and the plan had changed drastically? In the U.S. we are taught, "You can do anything you put your […]
Filling the Food Gap in Ethiopia
Bugna district The late onset rainy season is about to cease. From a distance, all mountains and valleys look like they are covered with well-woven green and yellow carpets. It is breathtaking scenery. I am riding in a 4-wheel drive Toyota Land Cruiser from Lalibela to Ayina, a town in the Bugna district, in Amhara […]
Building a Foundation For Life
The Word of God In the Bible, the term 'foundation' refers to the foundation of a building during construction and the foundation of reading, believing and practicing God's Word. God wanted us to know that His word is the key for our development, starting when we are children. Why do you call me, 'Lord, Lord,' […]
A Thousand Pictures Tell A Single Word
I love photography! Out of any qualifying hobbies out there, being a shutterbug is definitely at the top of my list. It's so intriguing to me that there's a device that can literally capture a "slice" of time, even down to 1/1000 of a second. Any picture that's ever been taken, since the invention of […]
Witch doctors and the Gospel
When you believe something for most of your life, it can be very hard to eventually realize it isn't true. How do you reject the lifestyle you knew for so long, and suddenly start believing in something different? Believing the Lies Fidelina is from a Dominican Republic community where witchcraft is very common. She was […]
Let me tell a story of grace. My father grew up on a 600-acre family estate in Virginia. The land was granted by the English Earl of Dunmore in the mid-1700s. "Back Creek" is situated in the Blue Ridge Mountains, some of the most beautiful country in the world. My first ancestor there built a […]
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Econo Lodge
2331 South Cedar
Ottawa, KS 66067
Phone: (785) 242-3400
Fax: (785) 241-2193
Kansas City Zoo
6700 Zoo Drive
Kansas City, MO 64132
+1 816 513 5700
Su to Sa from 09:30 AM to 04:00 PMThis zoo is a must-see for children and adults interested in getting back to nature. This sprawling park, which attracts 700,000 visitors annually, is divided into sections named for areas of the world, from Africa and Australia to Farmland USA. Visitors can walk the 202-acre park or ride trams from one place to another. There is also a guided train tour that allows guests to see park highlights without the fatigue.
Earn valuable points toward free nights.


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Dave Granlund cartoon about the midterm election results.
FALL RIVER — Democratic incumbents Carole Fiola and Michael Rodrigues cruised to re-election victories Tuesday in the 6th Bristol District for state House and 1st Bristol & Plymouth District for state Senate, respectively.
1. The election features races for U.S. Senate, U.S. House, governor, lieutenant governor, secretary of state, treasurer, auditor, Governor's Council, state Legislature and several county positions. Local contested...
In victory speech, candidate promises to build economy on 'our terms'
Stephen Kerrigan won the Democratic primary for lieutenant governor on Tuesday night, defeating Cambridge City Councilor Leland Cheung and nonprofit executive Mike Lake to capture a place on the party�s ticket.
BOSTON ��With the primary behind him, Republican Charlie Baker wasted no time setting his sights on the general election in November.
Taunton has lost its seat on Bristol County's Board of Commissioners. Taunton's Dan Dermody waited for election results Tuesday night at the Italian Social Club, as he ran for a position on the three-member board. He conceded shortly before 11...
Attorney general candidate Maura Healey scored a slam dunk on Tuesday night when she locked up her spot on November's ballot.
Although The Herald News has not endorsed candidates in partisan primaries in recent years, we felt that our readers would be well served this year by our vetting of the statewide candidates. Our endorsements are not intended to tell our readers how...
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Shelia you will be missed
Submitted by Kat Harrell (not verified) on Mon, 06/18/2012 - 7:08pm.
Shelia you will be missed greatly by many dear. RIP and i will always love you. You were a very warm and loving woman and you were the best friend any one could ask for. Ms. Dot will greatly miss you. Love you always


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Been "Gifted" by a Pet?
Been "Gifted" by a Pet?
I still remember being at a childhood friend's house in the country when her cat brought in a dead mouse and placed it at her little brother's feet. Because I must have been about six or seven at the time, I remember screaming and jumping on the couch, and recall the whole thing to be a tad traumatic. Now, flash forward many years later as I got a call tonight from a high school friend to tell me (as PetSugar) that her indoor/outdoor cat brought in a Koi from her pond and left it on her back stoop . . . she was a bit freaked out, shocked at the cat's ability and sudden hunting desire, but still handling herself rather well. I tried to offer moral support as she went through her options to (a) call her boyfriend; (b) close her eyes and remove it herself; or (c) ignore it and hope kitty decides to become doglike and digs a hole outside. We decided on the first option and she just called back to tell me that the situation has been taken care of. Now, North is likely too greedy to present anything to me, but I want to hear what your (insert pet here) brought in!
- Find out what Taylor Swift said about her doppelgänger — Us Weekly
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- Land a copy of Madonna's latest album — popbytes


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A Song of Ice and Fire/Game of Thrones Spoiler-Filled Discussion
View Single Post
June 4 2012, 11:47 PM
Re: A Song of Ice and Fire/Game of Thrones Spoiler-Filled Discussion
CaptainCanada wrote:
Shurik wrote:
Here, he's a fucking moron and doesn't merit an ounce of sympathy.
Ultimately, he's a fucking moron in both versions. In the books, he married some girl because he slept with her, completely ignoring the fact that he swore an oath and Frey men fought for him - and forsaking your oaths is not a very honorable thing to do IMO. Just dumb stuff a teenager might do.
In the book he was despondent when his brothers were seemingly killed and slept with a girl. Having already done that, he felt he had no other choice but to marry her, since he's taken her virginity.
In the show it's Robb quite consciously choosing to break his oath for no reason at all other than he feels like it.
I agree with your views on it. In the books Robb is at this time what 16 years old? Being King of the North with his Father dead... He leads an attack on the Westerling's keep and takes an arrow during it. Jeyne is the oldest daughter of the noble household and she nurses him and tends to his injury. While he's there he receives word that Bran and Rickon are dead. Jeyne comforts him, they sleep together and he marries her the very next door because of feeling he had dishonored her.
He attacked her home, captured it, she nurses him from injury he sustained while attacking her home... He then takes her virginity in her own home (not that she was resisting). But the circumstances are more understandable than what the tv show did.
They give him a foreign woman who comes across as being around the bend 'once or twice'.
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Speak up—or forever fall behind
In the study, 68 unacquainted students were divided into 17 same-gender groups. The groups had 45 minutes to design a mock nonprofit environmental organization or for-profit Web site. The winning team would receive a $400 prize. Researchers videotaped the groups, and independent observers judged the effectiveness of the four-person teams. What the researchers found was surprising. Observers who watched the on-screen activity said the team members with more talkative, dominant personalities were more intelligent, dependable, and disciplined. Those who were less outspoken were called conventional or uncreative.
But were these outspoken leaders just more competent? No. In the second round of the experiment, the teams were asked to solve computational problems from old standardized tests. When asked to pick the most competent leaders of the teams, observers picked the most dominant, outspoken team members—even when those team members didn't answer problems correctly. These dominant personalities just answered more questions out loud than the other team members.
Researchers say their findings are important life for two reasons. Managers may want to look beyond outspokenness and look into objective productivity when judging employees. And employees should consider speaking up to improve their managers' perception of their value to the team.
The study was published in the February 2009 Journal of Personality and Social Psychology.
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Pol on defense as Michigan veterinary board discusses negligence charges
Controversial reality TV veterinarian calls his approach 'common sense.'
Photo gallery: The top 10 veterinary schools in America, according to U.S. News
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Everyone's favorite receptionist is at it again. Would you handle this situation differently?
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Parasitology expert Michael Dryden, DVM, MS, PhD, reveals prime hideouts for fleas—and gives tips to clear them out of clients' homes for good.
Veterinarians: Your clients are going to Google with these cat questions
You might be surprised by what your clients are researching. Plus, get an educational client handout.


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Ronald (Ron) G. Jones, 76, passed away March 30, 2015 in Orangeville, Utah.
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PRICE-Our beloved wife, mother, grandmother, sister and friend, Faye Florine Clark Mager, age 70, passed away March 26, 2015 in Grand Junction, Colo. She was born July 31, 1944 in Poteau, Okla. to Harmon Clark and Hazel Crumpton. ...
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Date: 1920 ca.
Description: Hawk's Tavern, front door entrance.
Description: View of the main building of the Moor (Mud) Baths.
Description: Carl, Robert, and Ray Breecher at work on their modified parasol Heath airplane. After months of hard work, the plane was completely destroyed during a tes...
Date: 1960 ca.
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Description: Nashotah Mission Blue House. Built in 1842, it was the mission's first building.
Date: 1911 ca.
Description: Colored bird's-eye map of Oconomowoc and the Lakes Region, Waukesha County. Looking North from Government Hill. Location key below image identifies 24 Hote...
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Date: 1873
Description: Plat map of the township of Eagle (T5N, R17E) at a scale of 2 inches to one mile.
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CE Support Services JV
B-406542.2: Sep 28, 2012
Additional Materials:
- Full Report:
Ralph O. White
(202) 512-8278
(202) 512-4800
(202) 512-8278
Office of Public Affairs
(202) 512-4800
CE Support Services JV, of Kathleen, Georgia, protests the award of a contract to Solution One Industries, of Killeen, Texas, under request for proposals (RFP) No. FA8501-11-R-0017, issued by the Department of the Air Force for base-wide civil engineering support at Robins Air Force Base (AFB), Georgia. CE challenges the agency's evaluation of its past performance and the selection decision.
We deny the protest.
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Dimensions: 17in X 17-3/8in X 1/2in H. Stylish. Sturdy. Flexible. Waterproof. Stackable. What more could you want in a storage bin? Use Life Bins indoors or out to store shoes, laundry, toys, magazines and more. They even double as ice buckets. Lids... more
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Genuine OEM Whirlpool Affresh Cooktop Cleaner 10oz. / W10355051
Affresh Cooktop Cleaner removes cuts through the toughest baked-on residue. This non-abrasive cleaner and pads are proven to remove even the toughest baked-on residue more
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Plastic Cleaner Kit 8CK Red Camois (12 PACK) Brillianize Kit 8CK
Brillianize Plastic Cleaning Kit Now Packaged With Ourbest Micro Fiber Polishing Cloth ! Super Soft Super AbsorbentAnti-Static Non-ToxicAlcohol & Ammonia Free Features: 8 oz. Brillianize Cleaning fl Uid Pump Spray Bottle With Super Fine Atomizer Large... more
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Mrs. Meyer's Mrs. Meyer's Lemon Verbena Toilet Bowl Cleaner35% Off Sitewide - CODE: PRESIDENTS
Who wants to clean the toilet? Mrs. Meyer's Clean Day Lemon Verbena Toilet Bowl Cleaner makes this job more bearable. Mrs. Meyer's toilet bowl cleaner cleans and deodorizes your entire bowl without using chlorine or solvents. Completely biodegradable.... more
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Gedy 2209-67 Kyoto Leather (Orange) Argenta Square Waste Bin
Gedy 2209-67 Kyoto Leather (Orange) Argenta Square Waste Bin. Bathroom Accessories > Waste Baskets. Gedy Square Waste Bin, Argenta Collection, Dimensions: 9.84" x 6.50" x 8.46", Kyoto Leather covering., Pop-up lid with foot pedal.. more
$109.00 $76.30go to store
Tube Cleaning Wand with Cleaning Pads
From AG Industries comes the Tube Cleaning Wand, ideal for easily maintaining a clean and healthy 22mm hose environment using a pre-moisten pad. The wand is in 3 sections, packaged with a 7 day supply of cleaning pads. Additional pads can be purchased... more
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Dragonfly Organix Odor Eliminator
The Dragonfly Organix Odor Eliminator destroys odors naturally. The special odor-cleaning action absorbs the odor on contact, leaving a refreshing fra... more
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Steel Eagle Professional Light Weight 24" Surface Cleaner w/ Aluminum Deck - ASE0017T
Steel Eagle Professional Light Weight 24" Surface Cleaner w/ Aluminum Deck - ASE0017T - 24-Inch Aluminum Surface Cleaner 1/8" spun aluminum deck resists corrosion & breakage Cuts cleaning time up to 66% over regular spray nozzles Heavy Duty Talon 4... more
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FUSE Combo - Lens Cleaner 4 oz bottle and Micro-Fiber Cloth / Bag
FUSE Combo - Lens Cleaner 4 oz bottle and Micro-Fiber Cloth / Bag more
$9.95 $4.95
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Sparkle Pipe Cleaners - Pack of 100
1/4 (6mm) diameter. 12 long assorted colors. more
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Rubbermaid Commercial Products Recycling Bins BRUTE 50 gal. Blue Rollout Recycling Trash Container with Lid FG9W2773BLUEFree Shipping on Over 500K Items!
Shop for Cleaning at The Home Depot. The Rubbermaid BRUTE 50 gal. Blue Rollout Container with Lid can serve as central collection containers or shipping containers, reducing the amount of handling. Heavy-duty, 8 in. wheels afford easy mobility, even... more
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ELIMINATOR Carpet Spot & Stain Remover, 20 Ounce
ELIMINATOR Carpet Spot & Stain Remover, 20 Ounce CRPT STN RMVR,20OZ - 10620 more
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Darlee Cast Aluminum Patio Trash Receptacle - Antique Bronze
Darlee Cast Aluminum Patio Trash Receptacle - Antique Bronze. AC80-28-TRSH-AB. Trash Cans & Containers. Bringing the quality and elegance of their patio furniture designs to outdoor living accessories with this cast aluminum trash receptacle.... more
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Shop Sweep Indoor/Outdoor Shop Vac - Improvements
19"-wide vac nozzle. Hard rubber wheels adjust to two heights: Low: 7/8"; High: 1-1/2". Safety switch. Built-in kickstand. 15"-long stubby cord. This dry shop sweeper devours dirt and debris indoors and out! A broom would take forever, but this... more
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Rev-A-Shelf RV-15KD-C S RV Series Double 27 Quart Waste Containers with Full Ext Metallic Silver
The popular RV series has all the functional necessities one would expect from a waste container system. A simple heavy duty wire frame with included door mounting brackets and bottom mount slides has everything needed to create a waste specific space... more
$99.75 $62.28
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Safco 38"H Trash Receptacle with Multiple Lid Options
38''H Waste Receptacle from the Trifecta collection is made of steel with a black base and stylish metal tops. The flexible design accommodates any workplace setting.The stainless steel lids feature laser cut inscriptions to specify use for waste or... more
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Lil Wayne claiming to be, well, Lil Wayne is a considerably more modest boast than Meek Mill declaring himself the second coming of 2Pac but, personally, I couldn't be happier to know that Tunechi's Back in fighting form. Here, the Mike Will & Marquel "Marz" Middlebrooks-produced instrumental from Mill's Tupac Back sets the stage for some characteristically brash-and-braggadocios bars from the Young Money heavyweight. Craving more? Wayne's Sorry 4 the Wait mixtape is set to hit the 'net this Wednesday (the 13th), paving the way for the August 29, arrival of Tha Carter IV.
Written by richard on 07/11/11
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Young Money
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"Hollywood Divorce" (2006)
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Envision Essential Oil
Envision™ stimulates creativity and resourcefulness, encouraging renewed faith in the future and the ability to maintain the emotional fortitude necessary to achieve goals and dreams. Sometimes in life, for a variety of reasons, people suppress their internal drive. Envision helps awaken and renew that drive to overcome fear and begin experiencing new, more rewarding dimensions.
Spruce (Picea mariana), geranium (Pelargonium graveolens), orange (Citrus aurantium), lavender (Lavandula angustifolia), sage (Salvia officinalis), and rose (Rosa damascena).
Aromatic | Topical | Dietary
Available Items
5 ml - $21.71
• Item # 3337
Use For:
Emotional Release
Renew Focus