Levels Of Enhanced Green Fluorescent Protein Research Articles

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling five cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Background The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results The addition of a PolyR group to the C-terminal of EGFP carrying C-terminal 6×His-tag showed similar fluorescence expression levels compare to EGFP without addition of PolyR as well. Moreover, yeast with plasmid insertion showed decrease S. cerevisiae growth curve but still preserving the fluorescence EGFP. Conclusions The expression of the EGFP modified protein in S. cerevisiae is not affected by the addition of arginine.

Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting.

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.

N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes.

Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines, such as those containing a modified nucleic acid, N1-methylpseudouridine-5′-triphosphate (m1ψ). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression from IVT mRNA in vitro, primary cultured fibroblast-like synoviocytes (FLS) and MH7A human synovial fibroblast cells were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1ψ incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. Gene expression levels were measured using reverse transcription polymerase chain reaction. In both MH7A cells and FLS, cells transfected with EGFP mRNA containing m1ψ generated higher levels of EGFP than those transfected with unmodified EGFP or control mRNAs. The multiplex assay of the FLS culture supernatant and reverse transcription polymerase chain reaction for FLS revealed that both concentration and expression of IL-6, TNF-α, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1ψ. Overall, m1ψ incorporation enhanced protein expression and decreased the expression of cytokines. These findings may contribute to arthritis research.

A Model System to Explore the Detection Limits of Antibody-Based Immuno-SPECT Imaging of Exclusively Intranuclear Epitopes.

Imaging of intranuclear epitopes using antibodies tagged to cell-penetrating peptides has great potential given its versatility, specificity, and sensitivity. However, this process is technically challenging because of the location of the target. Previous research has demonstrated a variety of intranuclear epitopes that can be targeted with antibody-based radioimmunoconjugates. Here, we developed a controlled-expression model of nucleus-localized green fluorescent protein (GFP) to interrogate the technical limitations of intranuclear SPECT using radioimmunoconjugates, notably the lower target-abundance detection threshold. Methods: We stably transfected the lung adenocarcinoma cell line H1299 with an enhanced GFP (EGFP)-tagged histone 2B (H2B) and generated 4 cell lines expressing increasing levels of GFP. EGFP levels were quantified using Western blot, flow cytometry, and enzyme-linked immunosorbent assay. An anti-GFP antibody (GFP-G1) was modified using dibenzocyclooctyne-N3-based strain-promoted azide-alkyne cycloaddition with the cell-penetrating peptide TAT (GRKKRRQRRRPPQGYG), which also includes a nuclear localization sequence, and the metal ion chelator N3-Bn-diethylenetriamine pentaacetate (DTPA) to allow radiolabeling with 111In. Cell uptake of 111In-GFP-G1-TAT was evaluated across 5 cell clones expressing different levels of H2B-EGFP invitro. Tumor uptake in xenograft-bearing mice was quantified to determine the smallest amount of target epitope that could be detected using 111In-GFP-G1-TAT. Results: We generated 4 H1299 cell clones expressing different levels of H2B-EGFP (0-1 million copies per cell, including wild-type H1299 cells). GFP-G1 monoclonal antibody was produced and purified in house, and selective binding to H2B-EGFP was confirmed. The affinity (dissociation constant) of GFP-G1 was determined as 9.1 ± 3.0 nM. GFP-G1 was conjugated to TAT and DTPA. 111In-GFP-G1-TAT uptake in H2B-EGFP-expressing cell clones correlated linearly with H2B-EGFP expression (P < 0.001). In vivo xenograft studies demonstrated that 111In-GFP-G1-TAT uptake in tumor tissue correlated linearly with expression of H2B-EGFP (P = 0.004) and suggested a lower target-abundance detection threshold of approximately 240,000 copies per cell. Conclusion: Here, we present a proof-of-concept demonstration that antibody-based imaging of intranuclear targets is capable both of detecting the presence of an epitope of interest with a copy number above 240,000 copies per cell and of determining differences in expression level above this threshold.

Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding α-1,3-galactosyltransferase (GGTA1), which synthesizes the α-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection.

Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors

Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

Direct Evaluation of the Effect of Gene Dosage on Secretion of Protein from Yeast Pichia pastoris by Expressing EGFP

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines

Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1F2A-Cre-F2A-EGFP). While the ‘skipping’ was highly efficient between Bapx1 and Cre, the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.

Dual transgene expression by foamy virus vectors carrying an endogenous bidirectional promoter

Several gene therapy applications require the transfer and simultaneous expression of multiple genes in the same cell. In this study, we analyzed the potential for coordinated expression of an endogenous bidirectional promoter located on chromosome X, which controls the expression of the heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) and alpha-galactosidase (GLA) genes. The promoter was cloned in both transcriptional orientations in a foamy virus (FV) vector backbone, whereas the enhanced green fluorescent protein (EGFP) and low-affinity nerve growth factor receptor (DeltaLNGFR) reporter genes were cloned in the 5'-3' and 3'-5' transcriptional orientations, respectively. In all the cell lines tested, both vectors showed high levels of transgene coexpression that reached 76% of total positive cells (range from 76 to 18%). Comparison of EGFP and DeltaNGFR levels revealed that the side of the promoter that drives the expression of the HNRNPH2 gene in the genome was stronger and in accordance to its in situ activity. When tested with CD34(+) cells, transgene coexpression reached 35.3% of all positive cells in progenitor assays and 16.8% of all positive cells after transplantation in NOD/severe combined immunodeficient mice. In summary, we show that the endogenous promoter used in this study holds bidirectional activity in the context of FV vectors and can be used in gene therapy applications requiring synchronized expression of two genes.

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

BackgroundDetection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells.Methodology/Principal findingsMurine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow.Conclusions/SignificanceThe use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections.

Transcriptional control of Flt3 ligand targeted by fluorouracil-induced Egr-1 promoter in hematopoietic damage

BackgroundIonizing radiation (IR) activate the early growth response-1 (Egr-1) promoter by production of radical oxygen intermediates (ROIs). Egr-EF, an expression vector pCIneo containing Egr-1 promoter cloned upstream of the cDNA for Flt3 ligand, was used to treat hematopoietic damage. 5-fluorouracil, a commonly used chemotherapeutic agent, cause tumor cell death by producing DNA damage and generating ROIs. We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EF in a chemoinducible gene therapy strategy. The goal of this study was to explore the effect of Flt3 Ligand gene transcription regulated by fluorouracil-induced Egr-1 promoter on hematopoietic recovery.MethodsHuman Flt3 Ligand (FL) cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EF). The vector was transfected into the HFCL human bone marrow stromal cell line, and these cells were exposed to 5-FU, a chemotherapeutic drug. Expression of FL by HFCL/EF cells after 5-FU treatment was determined with ELISA, western blot and RT-PCR assays. In addition, the effect of FL from HFCL/EF cell culture supernatants on growth of CD34+ cells from cord blood was also studied. HFCL/EF cells were injected into CB-17 combined immunodeficient (SCID) mice with B16 melanoma. 5-FU was given three days after injection of the HFCL/EF cells. In the recipient mice, white blood cell levels in peripheral blood and expression of EGFP and FL in human stromal cells were measured. Tumor volumes in tumor-bearing mice were also measured.Results5-FU treatment increased EGFP levels and secreted FL levels in HFCL/EF cells. Supernatants from HFCL/EF cell cultures treated with 5-FU increased CD34+ cell growth significantly. HFCL/EF exhibited an increase in the number of white blood cells after chemotherapy.ConclusionThe data presented here support the use of transcriptional control mediated by chemoinducible gene therapy to reduce hematopoietic injury associated with 5-FU.

Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle1

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells

Human pituitary tumor-transforming gene 1 (PTTG1) encodes a securin protein critically important in regulating chromosome separation. Murine PTTG (mPTTG) is 66% homologous to human PTTG1 and PTTG-null (PTTG-/-) mice exhibit pancreatic beta-cell hypoplasia and abnormal nuclear morphology with resultant diabetes. As we show that ductal beta-cell neogenesis is intact in PTTG-/- mice, we explored mechanism for defective beta-cell replication. We tested whether mPTTG exhibits securin properties in mouse insulin-secreting insulinoma MIN6 cells, using a live-cell system to monitor mitosis in cells transfected with an enhanced green fluorescent protein (EGFP)-tagged mPTTG conjugate (mPTTG-EGFP). To fulfill the criteria for securin properties, the protein should undergo degradation immediately before the metaphase-to-anaphase transition when expression levels are low, and should inhibit metaphase-to-anaphase transition when expression levels are high. EGFP itself did not undergo degradation throughout mitosis and high levels of EGFP per se did not affect normal mitosis progression (n=25). However, mPTTG-EGFP was degraded 2 min before the metaphase-to-anaphase transition when expression levels were low (n=19), and high mPTTG-EGFP levels blocked metaphase-to-anaphase transition in 13 cells. mPTTG-EGFP inhibited MIN6 cell proliferation and caused apoptosis. Immunocoprecipitation demonstrated binding of mPTTG-EGFP and separase. These results show that mPTTG exhibits properties consistent with a murine securin in insulin-secreting mouse cells and mPTTG overexpression inhibits cell proliferation, suggesting that defective beta-cell proliferation observed in PTTG-/- mice is likely due to abnormal cell-cycle progression.

In Vivo Selection of Murine Long-Term Repopulating Cells Transduced with a Foamy Virus Vector That Expresses MGMTP140K.

Recombinant foamy virus vectors transduce noncycling and cycling cells, are stable episomally, and integrate into the host genome during cell division. Due to the cytoplasmic stability of this vector, a substantial lag period between transduction and cell division required for provirus integration is possible. Therefore, in transplantation studies that use minimally stimulated hematopoietic stem and progenitor cells (HSC), integration of the foamy virus vector in HSC may occur once HSC divide post-transplantation. We used a foamy virus vector, MD9-P140K-EGFP, that co-expresses a mutant form of O6-methylguanine DNA methyltransferase (MGMTP140K) and the enhanced green fluorescent protein (EGFP) to test the hypothesis that HSC could be transduced with a foamy virus vector and selected in vivo by alkylator-based chemotherapy. We also compared foamy virus transduction and selection to our previously optimized strategy using an oncoretrovirus vector to express MGMTP140K (SF1-P140K-IRES-EGFP). Lineage-depleted bone marrow (BM) from C57BL/6 mice was transduced for 10-16 hours with the foamy virus vector or transduced following a 2-day prestimulation with the oncoretrovirus vector. Data presented are from three primary transplant experiments analyzed over 6 months and one secondary transplantation experiment analyzed for 6 months. The bulk transduction efficiency using the foamy virus vector ranged from 12–25% and the CFU transduction efficiency was 55–57%. Transductions with the oncoretrovirus vectors resulted in similar bulk and CFU transduction efficiencies (55–60%). Similar numbers of progenitor colonies (oncoretrovirus vs. foamy virus) were observed. MGMT activity in pooled progenitor colonies was ~10-fold higher in EGFP+ versus EGFP− colonies. Although similar numbers of CFU were transduced using the two vector systems, significantly different levels of in vivo selection were obtained in primary recipient mice. Consistent with previous studies, selection of oncoretroviral vector-transduced cells resulted in high and sustained levels of EGFP+ cells in the PB and BM in primary and secondary recipient mice (80–99% EGFP+ with 2–3 cycles of 6BG/BCNU). For primary transplants using cells transduced with the foamy virus vector, EGFP expression in the PB peaked at 3 months post-treatment (26.2+/−4.0%) which represented a 4–6 fold increase compared to vehicle-treated mice. However, by 6 months EGFP expression dropped by 3-fold (9+/−1%). Western analysis of MGMT protein levels found in the BM at 6 months post-transplantation also showed a 3-fold decline in expression. In secondary reconstitution experiments, however, flow cytometry and Western analysis of MGMT expression indicated that EGFP expression in foamy virus-transduced HSC no longer correlated with MGMT expression. In fact MGMT expression levels following drug treatment were similar to those found in secondary recipient mice transplanted with oncoretroviral vector-transduced cells, suggesting that stem cells expressing MGMT were selected over time. These data demonstrate that although foamy virus transduction is not as efficient as the more commonly studied oncoretrovirus transduction strategy, a simple overnight protocol can be used to transduce minimally stimulated HSC with a foamy virus vector. These cells can be selected in vivo, can reconstitute mice for up to one year, and can maintain high levels of MGMT expression.

Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase

Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase

Sequence‐specific gene silencing in murine muscle induced by electroporation‐mediated transfer of short interfering RNA

Post-genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence-specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation-mediated transfer of short interfering RNA (siRNA). siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde-3-phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or beta-galactosidase (beta-gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT-PCR, respectively. When Luc-specific siRNA was co-delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 micro g of siRNA almost completely blocked the reporter gene expression from 10 micro g of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of beta-gal plasmid and GFP-specific siRNA. beta-Gal-positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA. The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post-genomic era.

Successive increases in human cyclin A1 promoter activity during spermatogenesis in transgenic mice

The human cyclin A1 gene is highly expressed in pachytene spermatocytes and is essential for spermatogenesis. To analyze mechanisms of cyclin A1 gene expression in vivo, we cloned a 1.3 kb fragment of the promoter upstream of the cDNA of enhanced green fluorescent protein (EGFP). Four lines of transgenic mice were generated that carried the transgene. Cyclin A1 promoter activity in the organs of the transgenic mice was analyzed using fluorescence microscopy and flow cytometry. Expression of EGFP was seen in male germ cells of all four murine lines. Spermatogonia at the basal membrane expressed low levels of EGFP, but bright green fluorescence was present in spermatocytes entering meiosis. Interestingly, a further sharp increase in EGFP expression was found in spermatocytes approximately at the stage of the first meiotic division. EGFP levels stayed high thereafter and EGFP was present in mature spermatozoa. A portion of c-kit expressing cells in the testis also expressed EGFP indicating cyclin A1 promoter activity in a subpopulation of spermatogonia. These data suggest that cyclin A1 is active not only in pachytene spermatocytes but also in earlier phases of spermatogenesis.