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1.  Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems 
PLoS ONE  2014;9(4):e95128.
Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.
PMCID: PMC3990623  PMID: 24743311
2.  Identification of the translational start site of codon-optimized mCherry in Mycobacterium tuberculosis 
BMC Research Notes  2014;7:366.
Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined.
We determined the translational start site for functional (fluorescent) mCherry in mycobacteria. Several potential translational start codons were identified; introduction of downstream stop codons by mutagenesis was used to determine which start codon was utilized in the bacterial cells. Fluorescent protein was expressed from a construct which would allow translation of a protein of 226 amino acids or a protein of 235 amino acids. No fluorescence was seen when a construct which could give rise to a protein of 219 amino acids was used. Similar results were obtained in mycobacteria and in Escherichia coli. Western blotting confirmed that mCherry was expressed from the constructs encoding 235 or 226 amino acids, but not from the plasmid encoding 219 amino acids. N-terminal sequencing and mass determination confirmed that the mature protein was 226 amino acids and commenced with the amino acid sequence AIIKE.
We conclude that mCherry is expressed in M. tuberculosis as a smaller protein than expected lacking the GFP-derived N-terminal sequence designed to allow efficient fusions.
PMCID: PMC4091752  PMID: 24934902
Fluorescence; Gene expression; Mycobacteria; Reporter genes
3.  Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters 
PLoS ONE  2010;5(3):e9823.
Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the PmtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.
PMCID: PMC2843721  PMID: 20352111
4.  Distribution, Cleavage and Lipidation of Atg8 Fusion Proteins in Spodoptera litura Sl-HP Cells 
PLoS ONE  2014;9(5):e96059.
Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.
PMCID: PMC4008565  PMID: 24788719
5.  Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry 
World Journal of Stem Cells  2012;4(7):71-79.
AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.
METHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.
RESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival.
CONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.
PMCID: PMC3443714  PMID: 22993664
Human embryonic stem cells; Fluorescent marker; mCherry; Pluripotency; Cellular motility
6.  A novel multigene expression construct for modification of glycerol metabolism in Yarrowia lipolytica 
High supply of raw, residual glycerol from biodiesel production plants promote the search for novel biotechnological methods of its utilization. In this study we attempted modification of glycerol catabolism in a nonconventional yeast species Yarrowia lipolytica through genetic engineering approach.
To address this, we developed a novel genetic construct which allows transferring three heterologous genes, encoding glycerol dehydratase, its reactivator and a wide-spectrum alcohol oxidoreductase under the control of glycerol-induced promoter. The three genes, tandemly arrayed in an expression cassette with a marker gene ura3, regulatory and targeting sequences (G3P dh promoter and XPR-like terminator, 28S rDNA as a target locus), were transferred into Yarrowia lipolytica cells. The obtained recombinant strain NCYC3825 was characterized at the molecular level and with respect to its biotechnological potential. Our experiments indicated that the novel recombinant strain stably borne one copy of the expression cassette and efficiently expressed heterologous alcohol oxidoreductase, while glycerol dehydratase and its reactivator were expressed at lower level. Comparative shake flask cultivations in glucose- and glycerol-based media demonstrated higher biomass production by the recombinant strain when glycerol was the main carbon source. During bioreactor (5 L) fed-batch cultivation in glycerol-based medium, the recombinant strain was characterized by relatively high biomass and lipids accumulation (up to 42 gDCW L-1, and a peak value of 38%LIPIDS of DCW, respectively), and production of high titers of citric acid (59 g L-1) and 2-phenylethanol (up to 1 g L-1 in shake flask cultivation), which are industrially attractive bioproducts.
Due to heterogeneous nature of the observed alterations, we postulate that the main driving force of the modified phenotype was faster growth in glycerol-based media, triggered by modifications in the red-ox balance brought by the wide spectrum oxidoreductase. Our results demonstrate the potential multidirectional use of a novel Yarrowia lipolytica strain as a microbial cell factory.
PMCID: PMC3827991  PMID: 24188724
Yarrowia lipolytica; Novel genetic construct; Glycerol metabolism; Heterologous expression; Biomass
7.  Expression of native and mutant extracellular lipases fromYarrowia lipolytica in Saccharomyces cerevisiae 
Microbial biotechnology  2012;5(5):634-641.
Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low‐cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S. cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y. lipolytica extracellular lipases in S. cerevisiae. The LIP2 gene of Y. lipolytica DSM3286 and its mutant Y. lipolytica U6 were isolated and cloned by expression vector in S. cerevisiae. New recombinant S. cerevisiae strains FDS100 containing the native LIP2 gene, and FDS101 containing the mutant LIP2 gene were produced 10 and 15 U ml −1 extracellular lipase respectively, on a production medium containing olive oil. New recombinant S. cerevisiae strains produce acceptable amount of extracellular lipase in comparison with Y. lipolytica wild‐type strains. These strains can utilize olive oil and lipids as low‐cost substrates to produce bioethanol, single cell protein and other biotechnologically valuable products. The recombinant S. cerevisiae strain with mutant LIP2 produced lipase with 1.5‐fold higher activity. The LIP2 gene of Y. lipolytica was expressed in S. cerevisiae as a heterologous protein without any modifications. Strong components of the Y. lipolytica expression/secretion system could be used for high‐level production of recombinant proteins in S. cerevisiae.
PMCID: PMC3815875  PMID: 22702371
8.  Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP 
BMC Microbiology  2010;10:248.
When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.
We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.
The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.
PMCID: PMC2957392  PMID: 20920169
9.  Visualization of HIV Protease Inhibition Using a Novel FRET Molecular Probe 
Biotechnology progress  2011;27(4):1107-1114.
The in vivo high-throughput screening of HIV protease inhibitors is a significant challenge due to the lack of reliable assays that allow for visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Förster resonance energy transfer (FRET) to visualize HIV-1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV-1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV-1 protease leads to AcGFP1’s separation from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV-1 protease inhibition can be determined by measuring the FRET signal’s change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV-1 protease inhibition. By co-transfecting HIV-1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV-1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is anticipated that this new probe will allow high-content screening of new anti-HIV drugs.
PMCID: PMC3163052  PMID: 21584951
molecular probe; FRET; HIV-1 protease inhibition; AcGFP1; mCherry; high-content screening
10.  Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging 
Journal of Virology  2014;88(1):82-98.
Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.
PMCID: PMC3911689  PMID: 24131714
11.  Biological Production of an Integrin αvβ3 Targeting Imaging Probe and Functional Verification 
BioMed Research International  2015;2015:681012.
The aim of the present study is to establish a bacterial clone capable of secreting an integrin αvβ3 targeting probe with bioluminescent and fluorescent activities, and to verify its specific targeting and optical activities using molecular imaging. A bacterial vector expressing a fusion of secretory Gaussia luciferase (sGluc), mCherry, and RGD (sGluc-mCherry-RGDX3; GCR), and a control vector expressing a fusion of secretory Gaussia luciferase and mCherry (sGluc-mCherry; GC) were constructed. The GCR and GC proteins were expressed in E. coli and secreted into the growth medium, which showed an approximately 10-fold higher luciferase activity than the bacterial lysate. Successful purification of GCR and GC was achieved using the 6X His-tag method. The GCR protein bound with higher affinity to U87MG cells than CHO cells in confocal microscopy and IVIS imaging, and also showed a high affinity for integrin αvβ3 expressing tumor xenografts in an in vivo animal model. An E. coli clone was established to secrete an integrin αvβ3 targeting imaging probe with bioluminescent and fluorescent activities. The probe was produced feasibly and at low cost, and has shown to be useful for the assessment of angiogenesis in vitro and in vivo.
PMCID: PMC4310313  PMID: 25654118
12.  Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core 
PLoS ONE  2012;7(6):e39976.
Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.
PMCID: PMC3386995  PMID: 22768188
13.  Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein 
PLoS ONE  2014;9(3):e92783.
Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.
PMCID: PMC3963934  PMID: 24664106
14.  Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane▿  
Journal of Bacteriology  2008;191(1):161-168.
The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.
PMCID: PMC2612452  PMID: 18978053
15.  Imaging of mRNA–Protein Interactions in Live Cells Using Novel mCherry Trimolecular Fluorescence Complementation Systems 
PLoS ONE  2013;8(11):e80851.
Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA–protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA–protein interactions such as that between influenza viral protein NS1 and the 5’ UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA–protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA–protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.
PMCID: PMC3829953  PMID: 24260494
16.  Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines 
Molecular Vision  2011;17:1090-1102.
With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly for non-ocular tissues. A human retinal pigment epithelial cell line (ARPE19) and a mouse cone photoreceptor cell line, 661W, have been well characterized and are used for many retinal metabolism and biologic pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self-complementary (sc) adeno-associated virus (AAV) vectors in these biologically relevant ocular cell lines using high-throughput fluorescence-activated cell sorting (FACS) analysis.
ARPE19 and 661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at multiplicity of infections (MOIs) ranging from 100 to 10,000. Three days post infection fluorescent images verified mCherry expression. Following microscopy, FACS analysis was performed to quantify the number of positive cells and the mean intensity of mCherry fluorescence, the product of which is reported as transduction efficiency for each vector. The scAAV vectors containing cone-specific (sc-mCARpro-green fluorescent protein [GFP]), rod-specific (sc-MOPS500-eGFP), retinal pigment epithelium (RPE)-specific (sc-VMD2-GFP), or ubiquitous (sc-smCBA-GFP) promoters were used to infect both cell lines at an MOI of 10,000. Three days post infection, cells were immunostained with an antibody raised against GFP and imaged. Finally, based on our in vitro results, we tested a prediction of transduction efficiency in vivo.
Expression from unmodified scAAV1, scAAV2, scAAV5, and scAAV8 vectors was detectable by FACS in both ARPE19 and 661W cells, with scAAV1 and scAAV2 being the most efficient in both cell lines. scAAV5 showed moderate efficiency in both ARPE19 and 661W cells. scAAV8 was moderately efficient in 661W cells and was by comparison less so in ARPE19 cells; however, transduction was still apparent. scAAV9 performed poorly in both cell types. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with the increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases, efficiency improved; in others, it was unchanged or marginally reduced. Retinal-specific promoters were also active in both cell lines, with the 661W cells showing a pattern consistent with the in vivo activity of the respective promoters tested. The prediction based on in vitro data that AAV2 sextuple Y-F mutants would show higher transduction efficiency in RPE relative to AAV2 triple Y-F capsid mutants was validated by evaluating the transduction characteristics of the two mutant vectors in mouse retina.
Our results suggest that this rapid and quantifiable cell-based assay using two biologically relevant ocular cell lines will prove useful in screening and optimizing AAV vectors for application in retina-targeted gene therapies.
PMCID: PMC3087449  PMID: 21552473
17.  Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm 
Genesis (New York, N.y. : 2000)  2011;49(3):124-133.
Live imaging of genetically-encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells is being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene iss based on, and recapitulates reporter expression of a previously described Afp::GFP strain. However we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes, as well as in pancreas, digestive tract and brain of postnatal mice.
PMCID: PMC3081534  PMID: 21442721
mouse embryo; visceral endoderm; endoderm morphogenesis; yolk sac; liver; intestine; alpha-fetoprotein; live imaging; confocal; fluorescent protein; mCherry
18.  A HA2-Fusion Tag Limits the Endosomal Release of its Protein Cargo Despite Causing Endosomal Lysis † 
Biochimica et biophysica acta  2011;1810(8):752-758.
Protein transduction domains (PTDs) can be fused to a protein to render it cell-permeable. The delivery efficiencies of PTDs are, however, often poor because PTD-protein conjugates cannot escape from endosomes. A potential solution this problem consists in adding HA2 analogs to the PTD-protein construct as these peptides can cause endosomal lysis upon acidification of the endosomal lumen. To date, however, the utility of HA2-based PTDs has not been clearly established.
We investigate the biophysical and cellular properties of the glutamate-rich HA2 analog E5 fused to the model protein TAT-mCherry.
E5-TAT-mCherry causes the release of fluorescent dextrans trapped with the protein inside endosomes. Yet, E5-TAT-mCherry itself is not released in the cytosol of cells, indicating that the protein remained trapped inside endosomes even after endosomal lysis takes place. Cytosolic delivery of the protein could be achieved, however, by insertion of a disulfide bond between E5 and its cargo.
These results show that E5 causes the retention of its fused protein inside endosomes even after lysis takes place.
These data establish that HA2 analogs might not be useful PTDs unless cleavable linkers are engineered between PTD and protein cargo.
PMCID: PMC3128663  PMID: 21664431
protein delivery; HA2 peptide; endosomal escape; protein transduction domain; lysis
19.  Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose 
Analytical biochemistry  2008;386(1):1-8.
Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.
PMCID: PMC2654400  PMID: 18834849
Cyclodextrin; Sepharose; Prenylation; Affinity chromatography; FPLC
20.  An acidic oligopeptide displayed on AAV2 improves axial muscle tropism after systemic delivery 
The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones.
In this study six-L aspartic acids (D6) were inserted into the AAV2 capsid protein sequence between amino acid residues 587 and 588. 129SVE mice were injected with double-stranded wild-type- (WT-) or D6-AAV2 mCherry expression vectors (3.24 x 1010 vg per animal) via the superficial temporal vein within 24 hours of birth.
Fluorescence microscopy and quantitative polymerase chain reaction confirmed higher levels of mCherry expression in the paraspinal and gluteus muscles in the D6-AAV2 injected mice. The results revealed that although D6-AAV2 was less efficient in the transduction of immortalized cells stronger mCherry signals were detected over the spine and pelvis by live imaging in the D6-AAV2-injected mice than were detected in the WT-AAV2-injected mice. In addition, D6-AAV2 lost the liver tropism observed for WT-AAV2.
An acidic oligopeptide displayed on AAV2 improves axial muscle tropism and decreases liver tropism after systemic delivery. This modification should be useful in creating AAV vectors that are suitable for gene therapy for diseases involving the proximal muscles.
PMCID: PMC3416570  PMID: 22709483
Adeno-associated virus; Tropism; L-aspartic acid; Acidic oligopeptide
21.  Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis 
BMC Research Notes  2010;3:303.
Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium Bacillus subtilis has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.
While observing the recruitment of the transcription machinery into the forespore of sporulating Bacillus subtilis, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.
Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.
PMCID: PMC2994887  PMID: 21073756
22.  Genetic approach to track neural cell fate decisions using human embryonic stem cells 
Protein & Cell  2014;5(1):69-79.
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry+ NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
PMCID: PMC3938840  PMID: 24474203
Nestin; knock-in; human embryonic stem cells; neural progenitor cells
23.  Genetic approach to track neural cell fate decisions using human embryonic stem cells 
Protein & Cell  2014;5(1):69-79.
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry+ NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
PMCID: PMC3938840  PMID: 24474203
Nestin; knock-in; human embryonic stem cells; neural progenitor cells
24.  Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes 
Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase.
For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.
Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system.
Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains.
Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.
PMCID: PMC3544689  PMID: 22876969
Yarrowia lipolytica; Biphasic sytem; Cytochrome P450; Steroid; Whole-cell bioconversion
25.  Sertoli Cell Behaviors in Developing Testis Cords and Postnatal Seminiferous Tubules of the Mouse1 
Biology of Reproduction  2010;84(2):342-350.
Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.
Sertoli cells are visualized using novel fluorescent protein reporters in transgenic mice to study their morphology and behavior in embryonic and postnatal testes.
PMCID: PMC3071268  PMID: 20944081
developmental biology; early development; fluorescent proteins; Gata4; germ cells; live imaging; seminiferous tubules; Sertoli cells; testis; testis cords

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