Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm.

BACKGROUND: Development of spectrally distinct green fluorescent protein (GFP) variants has allowed for simultaneous flow cytometric detection of two different colored mutants expressed in a single cell. However, the dual-laser methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm exhibits considerable spectral overlap. The purpose of this work was to evaluate detection of enhanced cyan fluorescent protein (ECFP) in combination with the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent proteins by flow cytometry. METHODS: Cells transfected with expression constructs for EGFP, EYFP, or ECFP were analyzed by flow cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filter configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (ECFP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filters. RESULTS: All three fluorescent proteins when expressed individually or in combination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. CONCLUSIONS: This method demonstrates the detection of three fluorescent proteins expressed simultaneously in living cells using single laser excitation and is applicable for use on flow cytometers equipped with a tunable argon ion laser.     

TdTomato and EGFP identification in histological sections: insight and alternatives

Abstract

Tandem dimer Tomato (tdTomato) provides a useful alternative to enhanced green fluorescent protein (eGFP) for performing simultaneous detection of fluorescent protein in histological sections together with fluorescence immunohistochemistry (IHC). eGFP has many properties that make it useful for cell labeling; however, during simultaneous fluorescence IHC, the usefulness of eGFP may be limited. This limitation results from a fixation step required to identify eGFP in histological tissue sections that can mask antibody epitopes and adversely affect staining intensity. An alternative fluorescent protein, tdTomato, may assist concurrent detection of fluorescent protein within tissue sections and fluorescence IHC, because detection of tdTomato does not require tissue fixation. Tissue sections were obtained from various organs of mice ubiquitously expressing eGFP or tdTomato that were either unfixed or fixed with 4% paraformaldehyde. These tissues later were combined with fluorescence IHC. Both eGFP and tdTomato displayed robust signals in fixed frozen sections. Only tdTomato fluorescence, however, was detected in unfixed frozen sections. Simultaneous detection of fluorescence IHC and fluorescent protein in histological sections was observed only in unfixed frozen tdTomato tissue. For this reason, tdTomato is a useful substitute for eGFP for cell labeling when simultaneous fluorescence IHC is required.     

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated.     

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/μg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.     

基于FMDV IRES的双顺反子载体的构建及体外表达分析

利用RT-PCR扩增出口蹄疫病毒小核糖体进入位点（IRES）序列,并定向克隆进pcDNA3.1(+)载体,构建成双顺反子真核表达载体。为了验证该载体是否能够转录出双顺反子mRNA,在IRES起始密码（ATG）下游正确插入增强型的绿色荧光蛋白基因（egfp）,把重组质粒转染BHK-21细胞,培养20～48 h,在紫外显微镜下观察,能够看到典型的绿色荧光,表明载体能够体能够利用FMDV的IRES能够介导非帽依赖性表达外源基因。并通过流式细胞仪,与同样是CMV启动转录egfp的pGFPN1质粒在细胞中的表达的水平进行了比较。该载体的成功构建为体外表达双基因、双顺反子逆转录载体构建以及相关应用奠定基础,并有作为基因疫苗和标记定位基因治疗载体的潜力。     

Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.     

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.     

Two human MARs effectively increase transgene expression in transfected CHO cells

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.     

Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background

To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S or < tm1(ACTB-cre,-EGFP)Ics> and Gt(ROSA) 26S or < tm2(CAG-flpo, EYFP)Ics>, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

TwinGFP, a marker for cell cycle analysis in transiently transfected cells

Green fluorescent protein (GFP) is widely used as a marker to identify transfected cells either by fluorescence microscopy or flow cytometry. However, cell cycle analysis with propidium iodide typically employs ethanol for cell permeabilization. During this treatment, soluble GFPs generally leak out of cells, probably due to their small size. We have now significantly improved cellular retention by creating an in-frame fusion of two GFP DNA sequences, thereby generating a double-sized GFP (TwinGFP, 57 kDa). Permeabilized HeLa cells transfected with pTwinGFP showed a strong green fluorescent signal localized throughout the cells that could easily be detected by fluorescence microscopy and flow cytometry, in contrast to cells transfected with a standard single GFP construct. The experiment indicates that protein size constitutes the major determinant of the loss of fluorescence in permeabilized cells. As a proof of principle, pTwinGFP was cotransfected with the p53 tumor suppressor gene into HeLa cells, and cells transiently expressing p53 could be identified and phenotypically characterized by flow cytometry.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

Apoptosis of liver-derived cells induced by parvovirus B19 nonstructural protein

Parvovirus B19 has been implicated in some cases of acute fulminant non-A, non-B, non-C, non-G liver failure. Our laboratory previously demonstrated that B19 infection of hepatocytes induces apoptosis and that the B19 viral nonstructural protein, NS1, may play a critical role. To study the involvement of NS1 in apoptosis of liver cells, we generated a fusion protein of NS1 with enhanced green fluorescent protein (eGFP) in a system allowing for inducible gene expression. Transfection of the liver-derived cell line HepG2 with the eGFP/NS1 vector allowed expression of the fusion protein, which was visualized by fluorescence microscopy and demonstrated by immunoblotting. The fusion protein localized to discrete domains in the nucleus. Transfection of HepG2 cells with the eGFP/NS1 vector led to apoptosis of 35% of transfected cells, a sevenfold increase over cells transfected with the parent eGFP expression vector. Mutation of the eGFP/NS1 vector to eliminate the nucleoside triphosphate-binding site of NS1 significantly decreased apoptosis, as did treatment of transfected cells with inhibitors of caspase 3 or 9. Neutralization of tumor necrosis factor alpha or Fas ligand had no effect on apoptosis. These results demonstrate that NS1 is sufficient to induce apoptosis in liver-derived cells and that it does so through the initiation of an intrinsic caspase pathway.     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.     

Construction and co-expression of grass carp reovirus VP6 protein and enhanced green fluorescence protein in the insect cells

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein) genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5 days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro. Undergraduate training student from College of Life Sciences, Wuhan University.     

Easy In Vitro Synthesis of Optimised Functioning Reporter mRNA from Common eGFP Plasmid

The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p?<?0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5′ element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.     

Hematoporphyrin/DAPI staining: simplified simultaneous one-step staining of DNA and cell protein and trial application in automated cytological screening by flow cytometry

We describe a procedure for simplified, simultaneous one-step staining in 10 min for DNA and cell and tissue proteins using a newly developed staining solution containing 0.03% hematoporphyrin (HP) with 0.001% DAPI [or with Hoeschst 33342 (HO)]. These HP/DAPI or HP/HO solutions were especially developed to facilitate a trial of automated cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of DNA and that of protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of DNA and protein. On flow cytometry, a single UV beam (370 nm) from the argon laser was used for excitation of both dyes. Measurement of DNA was done using a 470-nm bandpass filter and of protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of protein and DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and cancer cells easier.