scholarly journals Fluorescence Tools Adapted for Real-Time Monitoring of the Behaviors ofStreptococcusSpecies

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
R. C. Shields ◽  
J. R. Kaspar ◽  
K. Lee ◽  
S. A. M. Underhill ◽  
R. A. Burne
Keyword(s):  
Streptococcus Mutans ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Major Influence ◽  
Natural State ◽  
Multicopy Plasmid ◽  
Oral Streptococci ◽  
Fluorescent Reporter ◽  
Biofilm Growth ◽  
Content Type

ABSTRACTTagging of bacteria with fluorescent proteins has become an essential component of modern microbiology. Fluorescent proteins can be used to monitor gene expression and biofilm growth and to visualize host-pathogen interactions. Here, we developed a collection of fluorescent protein reporter plasmids forStreptococcus mutansUA159 and other oral streptococci. Using superfolder green fluorescent protein (sfGFP) as a reporter for transcriptional activity, we were able to characterize four strong constitutive promoters inS. mutans. These promoter-sfgfpfusions worked both for single-copy chromosomal integration and on a multicopy plasmid, with the latter being segregationally stable in the absence of selective pressure under the conditions tested. We successfully labeledS. mutansUA159,Streptococcus gordoniiDL1, andStreptococcussp. strain A12 with sfGFP, DsRed-Express2 (red), and citrine (yellow). To test these plasmids under more challenging conditions, we performed mixed-species biofilm experiments and separated fluorescent populations using fluorescence-activated cell sorting (FACS). This allowed us to visualize two streptococci at a time and quantify the amounts of each species simultaneously. These fluorescent reporter plasmids add to the genetic toolbox available for the study of oral streptococci.IMPORTANCEOral streptococci are the most abundant bacteria in the mouth and have a major influence on oral health and disease. In this study, we designed and optimized the expression of fluorescent proteins inStreptococcus mutansand other oral streptococci. We monitored the levels of expression and noise (the variability in fluorescence across the population). We then created several fluorescent protein delivery systems (green, yellow, and red) for use in oral streptococci. The data show that we can monitor bacterial growth and interactionsin situ, differentiating between different bacteria growing in biofilms, the natural state of the organisms in the human mouth. These new tools will allow researchers to study these bacteria in novel ways to create more effective diagnostic and therapeutic tools for ubiquitous infectious diseases.

scholarly journals Oral streptococci growth on aging and non-aging esthetic restorations after radiotherapy

2010 ◽  
Vol 21 (4) ◽  
pp. 346-350 ◽  
Author(s):  
Adriana D. da Cruz ◽  
Karina Cogo ◽  
Cristiane de C. Bergamaschi ◽  
Frab N. Bóscolo ◽  
Francisco C. Groppo ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Bacterial Biofilm ◽  
Oral Streptococci ◽  
Restorative Material ◽  
Biofilm Growth ◽  
Aged Group ◽  
Dental Resins ◽  
Colony Forming Units ◽  
Therapeutic Doses ◽  
Therapeutic Irradiation

The aim of this study was to examine Streptococcus mutans biofilm growth on both aged and non-aged restorative dental resins, which were submitted to therapeutic irradiation. Sixty-four disks of an esthetic restorative material (Filtek Supreme) were divided into 2 groups: aged group (AG) and a non-aged group (NAG). Each group was subdivided into 4 subgroups: non-irradiated and irradiated with 10Gy, 35Gy, and 70Gy. The biofilms were produced by Streptococcus mutans UA159 growing on both AG and NAG surfaces. The colony-forming units per mL (CFU/mL) were evaluated by the ANOVA and the Tukey LSD tests (α=0.05). AG presented smaller amounts of CFU/mL than the NAG before irradiation and after 10Gy of irradiation (p<0.05). AG irradiated with 35 and 70Gy showed increased amount of bacterial biofilm when compared to non-irradiated and 10Gy-irradiated disks (p<0.05). The exposure to ionizing radiation at therapeutic doses promoted changes in bacterial adherence of aged dental restorative material.

scholarly journals Genetic Analysis of Mutacin B-Ny266, a Lantibiotic Active against Caries Pathogens

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Delphine Dufour ◽  
Abdelahhad Barbour ◽  
Yuki Chan ◽  
Marcus Cheng ◽  
Taimoor Rahman ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Dental Plaque ◽  
Oral Bacteria ◽  
Oral Streptococci ◽  
Chromosomal Locus ◽  
Content Type ◽  
Genes Encoding ◽  
Plaque Biofilm

ABSTRACT Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans. In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA′, were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA′ enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA′ expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA′. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA′ peptides were restricted to the most invasive serotypes of S. mutans. IMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

scholarly journals Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci andStreptococcus mutans

2019 ◽  
Vol 85 (10) ◽  
Author(s):  
Lulu Chen ◽  
Brinta Chakraborty ◽  
Jing Zou ◽  
Robert A. Burne ◽  
Lin Zeng
Keyword(s):  
Streptococcus Mutans ◽  
Arginine Deiminase ◽  
Clinical Isolates ◽  
Amino Sugars ◽  
Oral Streptococci ◽  
Streptococcus Gordonii ◽  
Content Type ◽  
Oral Biofilms ◽  
Passage Number ◽  
Low Passage

ABSTRACTN-Acetylglucosamine (GlcNAc) and glucosamine (GlcN) enhance the competitiveness of the laboratory strain DL1 ofStreptococcus gordoniiagainst the caries pathogenStreptococcus mutans. Here, we examine how amino sugars affect the interaction of five low-passage-number clinical isolates of abundant commensal streptococci withS. mutansby utilizing a dual-species biofilm model. Compared to that for glucose, growth on GlcN or GlcNAc significantly reduced the viability ofS. mutansin cocultures with most commensals, shifting the proportions of species. Consistent with these results, production of H2O2was increased in most commensals when growing on amino sugars, and inhibition ofS. mutansbyStreptococcus cristatus,Streptococcus oralis, orS. gordoniiwas enhanced by amino sugars on agar plates. All commensals exceptS. oralishad higher arginine deiminase activities when grown on GlcN and, in some cases, GlcNAc. Inex vivobiofilms formed using pooled cell-containing saliva (CCS), the proportions ofS. mutanswere drastically diminished when GlcNAc was the primary carbohydrate. Increased production of H2O2could account in large part for the inhibitory effects of CCS biofilms. Surprisingly, amino sugars appeared to improve mutacin production byS. mutanson agar plates, suggesting that the commensals have mechanisms to actively subvert antagonism byS. mutansin cocultures. Collectively, these findings demonstrate that amino sugars can enhance the beneficial properties of low-passage-number commensal oral streptococci and highlight their potential for moderating the cariogenicity of oral biofilms.IMPORTANCEDental caries is driven by dysbiosis of oral biofilms in which dominance by acid-producing and acid-tolerant bacteria results in loss of tooth mineral. Our previous work demonstrated the beneficial effects of amino sugars GlcNAc and GlcN in promoting the antagonistic properties of a health-associated oral bacterium,Streptococcus gordonii, in competition with the major caries pathogenStreptococcus mutans. Here, we investigated 5 low-passage-number clinical isolates of the most common streptococcal species to establish how amino sugars may influence the ecology and virulence of oral biofilms. Using multiplein vitromodels, including a human saliva-derived microcosm biofilm, experiments showed significant enhancement by at least one amino sugar in the ability of most of these bacteria to suppress the caries pathogen. Therefore, our findings demonstrated the mechanism of action by which amino sugars may affect human oral biofilms to promote health.

scholarly journals Strain-Dependent Recognition of a Unique Degradation Motif by ClpXP in Streptococcus mutans

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
Author(s):  
Biswanath Jana ◽  
Liang Tao ◽  
Indranil Biswas
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Streptococcus Mutans ◽  
Protein Turnover ◽  
Fluorescent Protein ◽  
Biological Process ◽  
Polyacrylamide Gel Electrophoresis ◽  
Adaptor Protein ◽  
Protein Homeostasis ◽  
Content Type

ABSTRACT Regulated proteolysis in bacteria is an important biological process that maintains protein homeostasis. ClpXP, an intracellular proteolytic complex, is the primary protease that is responsible for protein turnover. While the substrates for ClpXP were identified in Escherichia coli, the substrates for vast majority of bacteria are currently unknown. In this study, we identified a unique substrate for ClpXP-mediated degradation in Streptococcus mutans, a dental pathogen. We also found that a small motif composed of 3 amino acids is sufficient for ClpXP-mediated degradation. Identification of this motif will clearly help us to understand the pathogenesis of this organism and other related pathogens. Streptococcus mutans, a dental pathogen, has a remarkable ability to cope with environmental stresses. Under stress conditions, cytoplasmic proteases play a major role in controlling the stability of regulatory proteins and preventing accumulation of damaged and misfolded proteins. ClpXP, a well-conserved cytoplasmic proteolytic system, is crucial in maintaining cellular homeostasis in bacteria. ClpX is primarily responsible for recognition of substrates and subsequent translocation of unfolded substrates into the ClpP proteolytic compartment for degradation. In Escherichia coli, ClpX recognizes distinct motifs present at the C-terminal end of target proteins. However, recognition sequences for ClpXP in other bacteria, including S. mutans, are not known. In this study, using two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) analysis, we have identified several putative substrates for S. mutans ClpXP. SsbA, which encodes a small DNA binding protein, is one such substrate that is degraded by ClpXP. By sequential deletions, we found that the last 3 C-terminal amino acids, LPF, are sufficient for ClpXP-mediated degradation. Addition of LPF at the C-terminal end of green fluorescent protein (GFP) rendered the protein completely degradable by ClpXP. Alterations of this tripeptide motif impeded ClpXP-mediated degradation. However, recognition of LPF by ClpXP is highly specific to some S. mutans strains (UA159, UA130, and N3209) since not all S. mutans strains recognize the motif. We speculate that an adaptor protein is involved in either substrate recognition or substrate degradation by ClpXP. Nevertheless, this is the first report of a unique recognition sequence for ClpXP in streptococci. IMPORTANCE Regulated proteolysis in bacteria is an important biological process that maintains protein homeostasis. ClpXP, an intracellular proteolytic complex, is the primary protease that is responsible for protein turnover. While the substrates for ClpXP were identified in Escherichia coli, the substrates for vast majority of bacteria are currently unknown. In this study, we identified a unique substrate for ClpXP-mediated degradation in Streptococcus mutans, a dental pathogen. We also found that a small motif composed of 3 amino acids is sufficient for ClpXP-mediated degradation. Identification of this motif will clearly help us to understand the pathogenesis of this organism and other related pathogens.

scholarly journals Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms

2016 ◽  
Vol 82 (12) ◽  
pp. 3671-3682 ◽  
Author(s):  
Lin Zeng ◽  
Tanaz Farivar ◽  
Robert A. Burne
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Sugar Metabolism ◽  
Amino Sugar ◽  
Amino Sugars ◽  
Mrna Levels ◽  
Oral Streptococci ◽  
Streptococcus Gordonii ◽  
Carbohydrate Source ◽  
Content Type

ABSTRACTBiochemical and genetic aspects of the metabolism of the amino sugarsN-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogenStreptococcus mutanswere explored. MultipleS. mutanswild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms betweenS. mutansandStreptococcus gordoniishowed thatS. gordoniiwas particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced inS. mutansthan inS. gordonii. Exposure to H2O2, which is produced byS. gordoniiand antagonizes the growth ofS. mutans, led to reduced mRNA levels ofnagAandnagBinS. mutans. When the gene for the transcriptional regulatory NagR was deleted inS. gordonii, the strain produced constitutively high levels ofnagA(GlcNAc-6-P deacetylase),nagB(GlcN-6-P deaminase), andglmS(GlcN-6-P synthase) mRNA. Similar to NagR ofS. mutans(NagRSm), theS. gordoniiNagR protein (NagRSg) could bind to consensus binding sites (dre) in thenagA,nagB, andglmSpromoter regions ofS. gordonii. Notably, NagRSgbinding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm. This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries.IMPORTANCEAmino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much greater capacity to utilize amino sugars than the dental pathogenStreptococcus mutansand that the ability of the model commensalStreptococcus gordoniito compete againstS. mutansis substantively enhanced by the presence of amino sugars commonly found in the oral cavity. The mechanisms underlying the greater capacity and competitive enhancements of the commensal are shown to depend on how the genes for the catabolic enzymes are regulated, the role of the allosteric modulators affecting such regulation, and the ability of amino sugars to enhance certain activities of the commensal that are antagonistic toS. mutans.

scholarly journals Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+Regeneration by Lactate Dehydrogenase

2015 ◽  
Vol 197 (23) ◽  
pp. 3645-3657 ◽  
Author(s):  
J. L. Baker ◽  
A. M. Derr ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Oxidative Stress ◽  
Lactate Dehydrogenase ◽  
Stress Response ◽  
Streptococcus Mutans ◽  
Parent Strain ◽  
Nadh Oxidase ◽  
Oral Streptococci ◽  
Pathogenic Potential ◽  
Oral Pathogen ◽  
Content Type

ABSTRACTPrevious studies of the oral pathogenStreptococcus mutanshave determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded bynox) is thought to be critical for the regeneration of NAD+, for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD+/NADH ratio in anoxdeletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded byldh) activity andldhtranscription in the Δnoxstrain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis ofS. mutanscultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion ofnoxor the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnoxstrain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevatedmanLtranscription, mediated in part, like elevatedldhtranscription, by Rex. While the Δnoxstrain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnoxstrain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions.IMPORTANCEStreptococcus mutansis an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that theS. mutanswater-forming NADH oxidase is critical for both carbon metabolism and the prevention of oxidative stress. The results of this study show that upregulation of lactate dehydrogenase, mediated through the redox sensor Rex, overcompensates for the loss ofnox. Additionally,noxdeletion led to the upregulation of mannose and glucose transport, also mediated through Rex. Importantly, the loss ofnoxrenderedS. mutansdefective in its ability to compete directly with two species of commensal streptococci, suggesting a role fornoxin the pathogenic potential of this organism.

scholarly journals CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Rouzbeh R. Taghizadeh ◽  
James L. Sherley
Keyword(s):  
Stem Cells ◽  
Time Scales ◽  
Cell Biology ◽  
Adult Stem Cells ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Fluorescent Reporter ◽  
Green Fluorescent

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.

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