22nd Meeting of DSFCM
Applications of Green Fluorescent Protein in Biology and
Medicine
Joint symposium of the Danish Society for Flow Cytometry and the Danish Society for Biochemistry and
Molecular Biology
Auditorium 2, Rigshospitalet, Blegdamsvej
9,
Organizers/chairmen:
Sponsors:
The Danish Medical Society, BD (Becton Dickinson/Clontech), and RAMCON A/S (Beckman Coulter)
All are welcome!
Program:
Janet Jansson, Section for
Natural Sciences, Södertörns Högskola,
Use of GFP to monitor specific bacterial populations in
environmental samples
Raphael H. Valdivia,
Bacterial Genetics and flow cytometry:
Novel approaches to the study of bacterial pathogenesis
Ole Thastrup, BioImage
A/S
The usage of green fluorescent proteins in drug discovery
Pause - Sandwiches/refreshments
Lene Martini and Kristian Kirk
Jensen, Laboratory
of Molecular Pharmacology, Panum Institute,
Green fluorescent protein in studies of 7 transmembrane receptors
Bjarke Bak
Christensen, Claus Sternberg, Jens Bo Andersen, Janus
Haagensen, and Søren Molin, Department of Microbiology, Technical University of Denmark
Applications of GFP as an in situ marker of plasmid
transfer, and microbial activity in biofilms
Bo Normander, Department of Marine Ecology and
Microbiology, National Environmental Research Institute
GFP as a reporter of bacterial distribution, activity and
gene transfer in the plant environment
Pause - Coffee/tea/cakes
Solveig Krogh
Christiansen,
Department of Plant Biology and Biogeochemistry, Risø
National Laboratory
GFP expression in an obligate plant pathogenic fungus
Uffe Birk
Jensen and Lars Bolund, Institute of Human Genetics,
Use of GFP as a reporter gene in the study of the efficacy
of non-viral gene transfer to epidermal cells
Simultaneous use of three different fluorescent proteins as
transduction markers in gene therapy research
Salmonella-GFP in macrophage phagocytosis
Pause - Refreshments
17:00-
General assembly of the Danish Society for Flow cytometry
ABSTRACTS
Use of GFP to monitor specific bacterial populations in
environmental samples
Janet K. Jansson. Section for
Natural Sciences, Södertörns högskola,
The use of bacterial inoculants for different environmental
applications is becoming increasingly popular. For example, bacteria can be
used to prevent plant diseases (biocontrol) or to
degrade toxic pollutants (bioremediation). We are interested in monitoring of
bacterial inoculants in environmental samples in order to determine their
numbers, activity, distribution and mode of action. Bacteria of interest were
tagged with marker genes so that they could be specifically identified amidst
members of the natural mixed microbial community. Some strains were
chromosomally tagged with one or two copies of the gfp
gene, encoding GFP (green fluorescent protein). The gfp-tagged
strains were monitored by flow cytometry and by
different fluorescence microscopy techniques in soil and/or on plant surfaces.
In some cases, the cells were additionally tagged with the luc
or luxAB genes encoding luciferase
enzymes. Luciferase activity was used as an indicator
of cell metabolic activity, whereas GFP fluorescence was used for enumeration
of the total number of cells, regardless of their activity. Flow cytometry was found to be an excellent tool for enumeration
of gfp-tagged cells in soil and in plant homogenates, however the background level of fluorescent
particles was considerable. We incorporated the use of Nycodenz
density gradient centrifugation as a method to separate the bacterial cell
fraction from soil particles or plant homogenates before injection into the
flow cytometer. By incorporation of an internal
standard of microscopic beads, the number of fluorescent
cells in the samples were accurately enumerated. Usually, the number of
cells counted by flow cytometry was greater than that
enumerated by selective plate counting. Therefore, a proportion of the cells
counted by flow cytometry are non-culturable
or possibly even dead cells. We are currently investigating the reason for this
discrepancy.
Bacterial genetics and flow cytometry:
Novel approaches to the study of bacterial pathogenesis
Raphael H. Valdivia. Department of Molecular and Cell Biology, 634
Barker Hall,
Salmonella typhimurium survives and
replicates in the intracellular environment of a variety of mammalian cells by
activating the transcription of bacterial factors that block the destruction of
the organism by lysosomal enzymes, protect the
bacterium from antimicrobial peptides, and provide the nutrient-scavenging
capabilities to survive in an intracellular vacuole. In order to further our
understanding of the molecular and genetic basis of the interaction between S. typhimurium and its host cell, we have developed a flow
sorting-based gene selection strategy to identify S. typhimurium
bearing gfp gene fusions that are preferentially
expressed in the intracellular environment of host cells. In this manner,
bacteria were separated by fluorescence-activated cell sorting on the basis of
stimulus-dependent fluorescence induction. This selection technology, termed
differential fluorescence induction (DFI) permitted the isolation of eight
genes expressed in response to low pH and fourteen genes preferentially
expressed in the macrophage environment. These macrophage-inducible genes (mig) were found to code for cell envelope proteins,
cell-surface maintenance enzymes, stress response proteins, transcriptional
activators, and a component of a type III secretion system required for
virulence. All mig analyzed were found to be
expressed in the intracellular environment of a variety of non-macrophage
cells, and in the splenocytes and hepatocytes
of infected animals. DFI selections were also used to identify regulatory loci
for mig. Intracellular expression of migs was found to be dependent on at least four different
genetic regulatory systems including PhoP/PhoQ, OmpR/EnvZ, PmrA/PmrB and SsrA/SsrB. Other applications of flow sorting in the study
of host-pathogen interaction will be discussed.
The usage of Green Fluorescent Proteins in Drug Discovery
Ole Thastrup. BioImage A/S, Moerkhoej Bygade 28, DK-2860
Green Fluorescent Proteins (GFPs)
have opened for a wealth of new opportunities in drug discovery. GFPs thus allow development of new assay systems that in
physiologically correct scenarios are capable of screening compound libraries
against previously intractable drug targets.
The pharmaceutical industry is under a considerable
pressure to utilize the constantly growing resource of potential drug targets
coming out of genomics. Essential to a successful exploitation is the creation
of novel ways to analyse for the functional
characteristics of these targets and a fast development of robust drug
screening systems.
In BioImage A/S, a recent
spin-out of Novo Nordisk, we have developed a broad
proprietary discovery platform, which allows us to screen against various
families of drug targets involved in intracellular signalling.
Important components of our screening systems are GFPs
engineered to monitor signalling events that are
involved in the redistribution of specific signalling
components. GFPs have proven indispensable in the characterisation of these important signalling
phenomena and in the identification of compounds that are capable of modulating
them.
GFPs from various marine organisms, and
derivatives thereof, have already revolutionised the
way functional cell biology is conducted, it is most likely that these genetic
tools also will form the basis for a paradigm shift in the drug discovery
industry.
Green Fluorescent Protein Studies of 7 Transmembrane
Receptors
Lene Martini and Kristian Kirk Jensen
Lab. for Molecular Pharmacology, Dept. Pharmacology, The Panum Institute, Build 18.6,
Email: martini@molpharm.dk
and kirk@molpharm.dk
The superfamily of 7 transmembrane receptors comprises the largest group of
membrane proteins with numerous important physiological functions, and is
therefore a key target in drug development.
Signalling through the receptor commences by
the binding of a ligand to its extracellular
domains. Following conformational changes it activates specific members of the
signal transducing guanine nucleotide-binding
regulatory proteins, the G proteins, on the cytoplasmic
side of the cell membrane. In turn these modulate the activity of
down-stream-effectors, thereby changing the physiology of the cell. In order to
turn off this signalling-cascade, a cytosolic protein termed arrestin
is recruited to the membrane, where it has a dual role both terminating the G
protein signalling and mediating receptor internalisation, thereby removing it from any external
stimuli.
For many years the molecular structure and function of 7 transmembrane receptors has been elucidated mainly through
mutagenesis studies. Their interaction with accessory proteins has in addition
to mutagenesis been detected through the use of appropriately derived
antibodies. Now the revolutionary discovery of GFP has made it possible to
study the kinetics of the receptor and its associated proteins in real time in
living cells by fluorescence microscopy. In addition, the synthesis of spectral
variants of GFP, such as the cyan (CFP) and yellow (YFP) mutants,
has made it possible to study the interactions and the kinetics of multiple labelled proteins simultaneously.
In our study, numerous chimeric
proteins were synthesised comprising the neurokinin receptor, NK1, fused to CFP at various sites,
and the following radioligand binding and functional
assays assessed their performance. Also chimeric
proteins consisting of beta-arrestin fused to YFP, were made. Combinations of one type of labelled receptor and one type of labelled
beta-arrestin were then transiently co-expressed in
COS7 cells and observed by epifluorescence microscopy
prior to and after treatment with the ligand,
Substance P. Thereby, the relative spatial and temporal distribution of the two
proteins may be taken as another measure of receptor performance.
Applications of GFP as an in situ marker of plasmid
transfer, and microbial activity in biofilms
Bjarke Bak
Christensen, Claus Sternberg, Jens Bo Andersen, Janus
Haagensen, and Søren Molin. Department of Microbiology,
During the last 5-10 years the development of in situ
markers for analysis of microbial communities, has evolved tremendously. In
particular, the use of fluorescent in situ rRNA
hybridization (FISH) for identification and localization of specific species
combined with Gfp as an in situ reporter for
gene expression has proven to be extremely successful. At the Microbial ecology
group (DTU) we have used this combination of biomarkers in studies of microbial
interactions in biofilms.
Different types of microbial interaction have been studied
like: i) metabolic interactions, ii) communication
mediated by Acyl-Homoserine-Lactone (AHL)
signal molecules, and iii) plasmid transfer. For example, in a Benzyl alcohol
degrading biofilm, combining quantitative
determination of donor, recipient and transconjugants
with in situ monitoring of single cells through zygotic interaction of Gfp fluorescence provided hitherto unknown details about
spread of the TOL-plasmid in the biofilm. In the same
biofilm, we also studied metabolic interactions
between the two dominant species (P. putida RI
and Acinetobacter C6). By inserting, into
P. putida RI, a cassette carrying the growth
phase regulated ribosomal RNA promoter rrnB P1
fused to an unstable variant of the gfp gene
it was possible to monitor the metabolic activity of P. putida
RI at different locations in the biofilm. The
activity of P. putida RI was shown to be
higher near micro-colonies of Acinetobacter
C6, which was further shown to be caused by leakage of the metabolic
intermediate, benzoate, from Acinetobacter C6
into the surrounding where it is degraded by P. putida
RI. This metabolic cross talk turned out to have a significant impact on the structural
organization of the two species in the biofilm.
Recently a number of new monitor strains for in situ
detection of various AHL signal molecules has been developed. These are based
on a cassette carrying the luxR gene and the luxI promoter (or homologues to lux)
fused to gfp. Thus, in the presence of high
concentrations of AHL the monitor strain becomes green fluorescent. Preliminary
studies have shown that the production of signal molecules in large
micro-colonies in biofilms may have a significant
impact on development of other micro-colonies in the regions near the large
micro-colonies.
GFP as a reporter of bacterial distribution, activity and
gene transfer in the plant environment
Bo Normander. Department of Marine Ecology and
Microbiology, National Environmental Research Institute, DK-4000
The green fluorescent protein (GFP) has proved to be a
powerful tool in in situ studies of
bacteria introduced into the terrestrial environment. By confocal
laser scanning microscopy (CLSM), the spatial localisation
of the gfp-tagged bio-control strain, Pseudomonas
fluorescens DR54-BN14 in the barley rhizosphere was studied. Within short distances (<30 µm)
on the root surface, DR54-BN14 varied in size from small coccoid
cells to large dividing rod-shaped cells. Also, it was found that DR54-BN14 was
closely associated with the indigenous bacteria and commonly situated near or
in the crevices between the epithelial root cells. A micro-colony assay,
involving the enumeration of single cells and cells forming micro-colonies,
showed a high viability of DR54-BN14 on roots. However, the activity of
DR54-BN14 on roots, as measured by image-analysis of single cells, was low and
comparable to the activity of starved cells. Finally, by using GFP it has been
possible to detect hot-spots for conjugal gene transfer on plant leaves.
GFP expression in an obligate plant pathogenic fungus
Solveig Krogh
Christiansen. Department of
Plant Biology and Biogeochemistry, Risø National
Laboratory, DK-4000
The obligate parasitic fungus, Blumeria graminis f.sp. hordei (Bgh), causing the barley powdery mildew disease
reduces grain yield in all temperate climate zones. Huge amounts of fungicides
are used to control the disease. The inhibition of specific processes rather
than whole organisms will have less impact on the environment and our research
aims at providing alternative strategies for management of the disease. GFP
reporter gene technology provides a unique tool for studying the infection
process of obligate parasites because it is based on a non-destructive assay. Using
differential screening techniques we have identified a number of stage specific
Bgh genes that are expressed during the
infection process. To analyse the function of novel
genes it is essential to know exactly in which cells and when in the infection
process the genes are expressed. This can be accomplished in transgenic Bgh where a translational fusion has been made
between the gene of interest and the GFP gene. Conidia from transgenic Bgh colonies will be transferred to new leaves and
the cellular expression profile will be recorded following the infection
process with a fluorescence microscope coupled to a camera. Among the most
interesting are genes expressed in the fungal feeding organ, the haustorium, inside the barley epidermal cells. The in
vivo expression pattern of these genes can only be determined using a Confocal Lazer Scanning
Microscope in combination with the GFP reporter gene. Fungal gene products that
are necessary for infection permit specific control measures to be developed in
the form of targeted pesticides.
Transfection mediated cell cycle arrest in
human epidermal keratinocytes
Uffe Birk Jensen and Lars Bolund.
We have previously shown that one problem with non-viral
gene transfer into human primary epidermal keratinocytes
is cell cycle arrest of the productively transfected
cells.
This was independent on vector construct and the p53 levels
were unaffected by the procedures.
We are currently investigating the role of INK4 family and
CIP/Kip family members in the observed phenomenon and using DNA labelled with dyes before transfection
we are analysing the role of DNA entry into the cells
as the precipitating factor.
Simultaneous use of three different
fluorescent proteins as transduction markers in gene therapy research.
Abstract
By incorporating the gene encoding GFP in the vector used
for transduction successfully transduced cells can be
identified and sorted on the flow cytometer due to
their green fluorescence. For many purposes different markers that can be used
simultaneously are required. We have outlined a project requiring three
different markers and are trying to find a combination of three different
fluorescent proteins for use at the FACS Vantage flowcytometer.
GFP and the yellow fluoreescent protein YFP are both
effectively excited by the 488 nm laser, and even
though their emission spectra are slightly overlapping it is possible to distinguish
between the two by using appropriate filters and electronic compensation. So
far we have not found a suitable third marker, but our candidates are the blue
fluorescent protein, BFP and the newly available red fluorescent protein, DsRed. BFP is excited by the UV-laser and emits at a
wavelength easily distinguished from the green and yellow fluorescence. DsRed has excitation optimum at 558 nm, but the excitation
by the 488 nm laser should be sufficient for detection on the FACS. The
emission of red light can easily be distinguished from the green and yellow
light.
Salmonella-GFP in macrophage phagocytosis
The GFP-gene inserted in the plasmids or chromosome of the
bacteria has been used as reporter-gene system to study many problems related
to clinical, food and environmental microbiology. However, various aspect of
immunity against micro-organisms can also gain from this technology. The
macrophage uptake of bacteria can be easily visualized and quantitated
using GFP-labelled bacteria. This presentation
concerns our attempt to measure the functional activity of chicken macrophages
exposed to Salmonella-GFP. Two different video-imaging systems were
applied for epifluorescense microscopy visualization
of phagocytosis, an electronically intensified CCD
system (