Detection Of Green Fluorescent Protein Research Articles

Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes.

Switchgrass (Panicum virgatum) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, ‘Summer’ and ‘Blackwell’, by ectopic overexpression of morphogenic genes, Baby boom (Bbm) and Wuschel2 (Wus2). Two auxotrophic Agrobacterium strains, LBA4404Thy- and EHA105Thy-, each harboring the same construct containing ZmBbm, ZmWus2, and a green fluorescence protein (GFP) gene, ZsGreen1, were used to infect immature leaf segments derived from in vitro grown seedlings. The Agrobacterium strains also contain a transformation helper plasmid that carry additional copies of Agrobacterium virulence genes. GFP-expressing calli were identified and selected for regeneration. The highest transformation efficiency of 6% was obtained for the tetraploid cultivar Summer when LBA4404Thy- was used for infection, which is twice of that for the octoploid cultivar Blackwell. LBA4404Thy- consistently outperformed EHA105Thy- on transformation frequency across the two cultivars. Fifteen randomly selected putative transgenic plants of Summer and Blackwell, representing independent callus events, were confirmed as transgenic by the presence of the transgene, ZmAls, and the absence of AtuFtsZ, a chromosomal gene specific to the Agrobacterium strain LBA4404 using polymerase chain reaction. Transgene integration and expression was further confirmed by the detection of GFP in roots, and the resistance to herbicide injury to leaves of selected putative transgenic plants. The ZmBbm and ZmWus2 genes were successfully removed from 40 to 33.3% of the transgenic plants of Summer and Blackwell, respectively, via the Cre-Lox recombination system upon heat treatment of GFP-expressing embryogenic calli. Our successful transformation of recalcitrant upland switchgrass provides a method for gene function analysis and germplasm enhancement via biotechnology.

Intramuscular injection of mesenchymal stem cells activates anabolic and catabolic systems in mouse skeletal muscle

Skeletal muscle mass is critical for good quality of life. Mesenchymal stem cells (MSCs) are multipotent stem cells distributed across various tissues. They are characterized by the capacity to secrete growth factors and differentiate into skeletal muscle cells. These capabilities suggest that MSCs might be beneficial for muscle growth. Nevertheless, little is known regarding the effects on muscle protein anabolic and catabolic systems of intramuscular injection of MSCs into skeletal muscle. Therefore, in the present study, we measured changes in mechanistic target of rapamycin complex 1 (mTORC1) signaling, the ubiquitin–proteasome system, and autophagy-lysosome system-related factors after a single intramuscular injection of MSCs with green fluorescence protein (GFP) into mouse muscles. The intramuscularly-injected MSCs were retained in the gastrocnemius muscle for 7 days after the injection, indicated by detection of GFP and expression of platelet-derived growth factor receptor-alpha. The injection of MSCs increased the expression of satellite cell-related genes, activated mTORC1 signaling and muscle protein synthesis, and increased protein ubiquitination and autophagosome formation (indicated by the expression of microtubule-associated protein 1 light chain 3-II). These results suggest that the intramuscular injection of MSCs activated muscle anabolic and catabolic systems and accelerated muscle protein turnover.

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta-Brief Report.

Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.

Small Nppa and Myl2 Promoters Are Sufficient to Maintain Chamber‐specific Expression on an AAV9 Platform

ObjectiveTo generate an AAV9 vector with small promoters that selectively express transgenes in a chamber‐specific manner.IntroductionThe adeno‐associated virus serotype 9 (AAV9) vector exhibits high transduction efficiency and cardiotropism which has allowed for the elucidation of tissue‐specific functions of genes implicated in cardiac diseases. However, cardiac diseases such as atrial fibrillation, require a more selective strategy to allow for the study of gene function in a chamber‐specific manner. AAV9 vectors carrying chamber‐specific promoters, such as Nppa and SLN for atrial‐specific expression, have been published; however, these vectors contain 632 and 1000 base pairs (bp), respectively, limiting the carrying capacity for transgenes.HypothesisLess than 500 bp of the Nppa and Myl2 promoters is sufficient to confer potent AAV9‐mediated atrial and ventricular cardiomyocyte specific expression, respectively.MethodsFragments of the Nppa promoter, consisting of about 600 and 400 bp, and of the Myl2 promoter, consisting of about 700 and 300 bp, were inserted into a Green Fluorescent Protein (GFP) expressing vector. These vectors were electroporated into primary neonatal rat atrial and ventricular cardiomyocytes (NRAMS and NRVMs). Because pathological hypertrophy is known to upregulate expression of Nppa in ventricular cardiomyocytes, NRAMs and NRVMs were treated with 50 uM of phenylephrine (PE), a chemical inducer of hypertrophy, for 48 hours, to assess the faithfulness of our promoters to maintain chamber‐specific GFP expression. Promoter activity was analyzed by immunoblot (IB) for GFP and fluorescent microscopy. The promoters which expressed GFP robustly and retained chamber‐specificity with PE treatment were selected for testing in mice in an AAV9 vector. The mouse model used was a two‐color fluorescent Cre Recombinase (Cre) reporter, consisting of membrane targeted Tomato (mT) and GFP (mG). In this model, mT fluorescence is exclusively expressed; upon introduction of Cre, mT is excised, allowing mG to express. We generated AAV9‐Cre driven by the Nppa or Myl2 promoters, injected mice via tail‐vein with 1×1011 viral particles, performed TAC for 4 weeks, and determined chamber‐specific expression of Cre by IB detection of GFP from atrial and ventricular tissue.ResultsIn NRAMs and NRVMs, IB and fluorescent microscopy confirmed that promoters as small as 400 bp and 300 bp for Nppa and Myl2, respectively, elicited robust atrial and ventricular specific GFP expression, respectively. Interestingly, 600 bp of the Nppa promoter expressed GFP in NRVMs with PE treatment, while 400 bp did not. In an AAV9‐Cre vector, 400 and 300 bp of the Nppa and Myl2 promoter, respectively, were sufficient to mediate chamber‐specific excision of mT, as determined by IB for GFP.ConclusionSmall promoter regions of Nppa and Myl2, consisting of about 400 and 300 bp, respectively, can be utilized in an AAV9 vector to selectively express transgenes in atrial and ventricular cardiomyocytes, respectively. Importantly, the smaller size of the Nppa and Myl2 promoters leaves room for larger transgenes, alleviating the limitations imposed upon the small carrying capacity of AAV9 vectors.Support or Funding InformationAmerican Heart AssociationNational Institute of Health

6 Invitro validation of gene edited phenotypes using CRISPR-dCas9 transcriptional activators

Despite the extensive advantages of gene-edited large animals for agriculture and biomedical purposes, they represent a large monetary and time investment due to high husbandry costs, long gestation lengths, and few offspring; that is, 9 months for one calf and almost 4 months for pigs. Even with known DNA sequences before somatic cell nuclear transfer (SCNT), inserted transgenes are often not expressed as expected. Therefore, there is a need to phenotypically validate the gene modifications invitro before investing time and resources in the creation of a gene-edited large animal; however, many gene targets are tissue specific and not expressed in SCNT donor cells. In this work, we show that CRISPR-dCas9 transcriptional activators (TAs) can be used to validate functional transgene insertion in nonexpressing SCNT donor cells, in our case fetal fibroblasts. To demonstrate this concept, we first generated a DNA knockin of the H2B-GFP sequence into the porcine LGR5 locus. CRISPR/Cas9 nuclease was used to create a double-stranded break in the genomic DNA downstream of the LGR5 promoter. A homology-directed repair template plasmid containing H2B-GFP flanked by 1000bp homology arms flanking the cut site was co-transfected with the Cas9 and gRNA, and cells were seeded at low density for colony outgrowth. Colonies were genotyped by PCR and sequencing to verify successful targeted transgene integration. To test whether TAs allow for invitro validation of transgene expression, 5×105 wildtype or gene-edited fibroblasts were nucleofected (Lonza) with 500ng total of four gRNA plasmids (Addgene #43860) designed to target the 1-kb region upstream of the LGR5 transcriptional start site in combination with 500ng VP64-dCas9 (Addgene #47107). Detection of green fluorescent protein (GFP) was analysed by fluorescent microscopy followed by flow cytometry; at least 30 000 events were recorded for each treatment (Cytoflex). Our results show that GFP was detected in on average 28.6% of the gene-edited cells transfected with LGR5 TAs but not detected in gene-edited cells that were not transfected with LGR5 TAs (0%) or in wild-type cells transfected with the LGR5 TAs (0%). The experiment was repeated three times. Next, to prove that our invitro validation replicates the invivo phenotype, the gene-edited colonies heterozygous for the insertion were used for SCNT to generate piglets. Epidermal cells, which contain a population of LGR5+ stem cells, were isolated from the skin and sorted for GFP expression. The RT-qPCR results from GFP+ or GFP− cells showed the presence of LGR5 transcripts in the GFP+ cells but not GFP− cells. In conclusion, TAs were necessary and sufficient to detect LGR5-promoter driven H2B-GFP expression in gene-edited fibroblasts invitro, which faithfully recapitulates the invivo phenotype of the gene-edited animal. Further preliminary data from our laboratory suggest that our novel method can be used to detect successful gene knockouts in addition to transgene knockins and can be used to validate phenotypic outcomes of DNA modifications before the generation of gene-edited animals.

Simple and Robust Intravital Microscopy Procedures in Hybrid TIE2GFP-BALB/c Transgenic Mice.

The endeavor of deciphering intricate phenomena within the field of molecular medicine dictates the necessity to investigate tumor/disease microenvironment real-time on cellular level. We, hereby, design simple and robust intravital microscopy strategies, which can be used to elucidate cellular or molecular interactions in a fluorescent mouse model. We crossbred transgenic TIE2GFP mice with nude BALB/c mice, allowing the breeding of immunocompetent and immunodeficient mouse models expressing green fluorescent protein (GFP) in vascular endothelium. Then, we surgically exposed various tissues of interest to perform intravital microscopy. By utilizing simple tissue preparation procedures and confocal or two-photon microscopy, we produced high-resolution static snapshots, dynamic sequences, and 3D reconstructions of orthotopically grown mammary tumor, skin inflammation, brain, and muscle. The homogenous detection of GFP expressed by endothelial cells and a combination of fluorescence agents enabled landmarking of tumor microenvironment and precise molecular tagging. Simple intravital microscopy procedures on TIE2GFP mice allowed a real-time multi-color visualization of tissue microenvironment, underlining that robust microscopy strategies are relatively simple and can be readily available for many tissues of interest.

CD40L inhibits cell growth of THP-1 cells by suppressing the PI3K/Akt pathway.

IntroductionAcute myeloid leukemia (AML), the hematological malignant tumor with high mortality, is still difficult to treat. CD40L is a type II transmembrane protein, which has been reported to have the potential to inhibit growth of some cancer cells.Materials and methodsIn order to determine the role of CD40L on AML-M5 cell line THP-1, we overexpressed CD40L in the cells using a lentiviral vector system (pHBLV-CMVIE-Zs Green-T2A-puro vector); overexpression was confirmed by the detection of green fluorescent protein and CD40L protein expression.ResultsCellular apoptosis, proliferation, and cycle assays showed that CD40L could promote the apoptosis of, suppress the proliferation of, and stimulate the arrest of the G1/S phase of THP-1 cells. Finally, the protein expression of P53, Bax/Bcl-2, cyclinD1, PCNA, PTEN, and p-Akt illustrated that CD40L may partly influence cell growth of THP-1 cells through those genes, which was confirmed by immunohistochemistry and a PI3K/Akt activator.ConclusionTaken together, CD40L could inhibit cell growth of THP-1 cells through the PI3K/Akt pathway, indicating that the overexpression of CD40L may be a potential target to treat the AML-M5 disease.

High-Resolution Imaging of GFP-Tagged Cardiac Ryanodine Receptor in Intact Heart and Brain

The cardiac ryanodine receptor (RyR2) is most abundantly expressed in the heart and brain. RyR2 is located in the sarco(endo)plasmic reticulum (ER/SR) membrane and functions as an intracellular calcium release channel, important for a number of fundamental processes, such as muscle contraction, secretion, neurotransmitter release, learning and memory. Proper function of RyR2 critically depends on its subcellular distribution. In ventricular myocyte of the heart, RyR2 is organized in highly-ordered arrays of clusters, which is thought to be important for synchronous, stable calcium release during excitation-contraction coupling. However, little is known about RyR2 distribution in other cardiac cells. In the brain, RyR2 is thought to play a central role in learning and memory, but the cellular/subcellular distribution of RyR2 in the brain remains largely undefined. Recently, we have generated a knock-in mouse model that expresses a green fluorescence protein (GFP)-tagged RyR2. This GFP-RyR2 mouse model allows us to directly and specifically monitor the cellular/subcellular distribution and expression of RyR2 in various cells and tissues. To improve the detection of GFP, we have also developed novel GFP-specific probes based on anti-GFP single domain antibodies (i.e. nanobodies). High-resolution confocal imaging of intact GFP-RyR2 heart sections and brain slices revealed highly ordered arrays of GFP-RyR2 in various regions of the heart, but disperse distribution of GFP-RyR2 in hippocampus and other regions of the brain. Using Alexa-Fluor 647 (AF647)-conjugated GFP-specific probes, we will perform super-resolution imaging to further define the subcellular distribution of GFP-RyR2 in the heart and brain (Supported by NSERC, CFI, CIHR, and LCIA).

Anticancer activity of stoppin based on a novel peptide delivery system.

Stoppin (L1) is a newly identified anticancer peptide, which is a potent p53‑MDM2/MDMX inhibitor. Due to its limitation in cell delivery efficiency, a new peptide delivery system was developed based on a nucleic acid‑polypeptide‑liposome complex and its stability and effectiveness in vitro was investigated. The nucleic acid‑stoppin‑liposome complex was prepared and characterization of the complex was conducted. The stability of the complex was evaluated by enzyme digestion. Following transfection of the A549 cells with the complex, detection of green fluorescent protein (GFP) and luciferase activity was conducted to evaluate transfection efficiency. In addition, the anticancer activity of the complex was determined by 3‑(4,5‑dimethyl‑thiazolyl‑2)‑2,5 diphenyltetrazolium bromide assay and apoptosis was detected by flow cytometry. The results indicated that the particle size of the complex was 102±10 nm and the encapsulation rate was ~100% when the ratio of liposome, L1 and plasmid was: 4 µl:1 µg:2 µg. The enzyme digestion experiment demonstrated that the complex was resistant to pancreatic and DNA enzyme degradation, indicating that the complex had biological stability. Cell transfection demonstrated that it had a mutual promotion effect on delivery, which could be confirmed by GFP fluorescence and luciferase assay. The cell‑killing efficiency of this novel delivery system was three times higher than with stoppin alone at a low concentration. In conclusion, this novel stoppin peptide delivery system was stable. The nucleic acid‑peptide‑liposome complex can protect the internal component from the degradation of enzymes, promote entry of the peptide into the cells and enhance the anti‑tumor activity of stoppin. Therefore, it is a promising approach for peptide delivery, which can be characterized and visualized using plasmids with GFP or luciferase.

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events.

Excitation-scanning hyperspectral imaging microscope.

Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300 ms/wavelength band with excitation scanning versus 3 s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications.

Direct detection of green fluorescent protein in histological preparations of recombinant vaccinia virus in mice (1050.9)

Green fluorescent protein (GFP)‐expressing recombinant viruses have proven to be invaluable tools for the in‐vivo monitoring of viral kinetics and disease progression in infectious disease models. Histopathological evaluation of the ex‐vivo tissues by way of immunohistochemical (IHC) staining for viral antigens further assists in the characterization of disease pathology, but can be insensitive, nonspecific, and time consuming. In this study, we determine if the direct detection of GFP signal provides a suitable alternative to IHC staining in GFP‐expressing tissues. Fixation and histological processing protocols were developed in a GFP‐expressing 293T cells and then optimized and validated in a GFP‐recombinant rat model. The optimized protocol was then applied to GFP‐recombinant cowpox virus (VACV‐TK‐GFP) infected mouse lungs in addition to lungs infected with a wild‐type virus strain (VACV‐WR). While animals infected with both virus strains manifested lesions reactive to anti‐Vaccinia IHC, GFP fluorescence was only detected in lesions from animals infected with the recombinant virus strain. The method was further validated through IHC staining of both virus strains with anti‐GFP antibodies, which were only reactive in tissues infected with VACV‐TK‐GFP. Digital alignment of sections showed a strong correlation between GFP fluorescence and IHC staining for GFP and viral proteins suggesting direct‐detection of fluorescent proteins is a promising alternative to traditional methods.

A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13–20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.

In Vivo Targeting of Activated Leukocytes by a β2-Integrin Binding Peptide

In immunopathological conditions, clinical diagnosis is commonly made on the basis of patient symptoms, measurement of blood leukocyte levels or proinflammatory biomarkers, non-specific radiological findings and, regarding infection, microbiological analysis. Nevertheless, frequently the exact spatial location of inflammation or even infection cannot be reliably identified, despite the use of up-to-date clinical, laboratory and imaging techniques. For this reason, new tools are warranted for use in advanced diagnosis and therapy targeting in patients. The peptide CPCFLLGCC (LLG), known to bind activated β2-integrins in vitro, was fused with green fluorescent protein (GFP) to test the ability of LLG fusions to target and bind activated leukocytes in vivo. A murine skin scratch inflammation model was chosen for the convenient non-surgical detection of GFP. The murine skin lesion inflammation model demonstrated in vivo targeting of LLG-GFP to sites of inflammation. Targeting by LLG-GFP fusion construct depends on the ability of the LLG-moiety to bind activated leukocytes. Control construct unable to bind β2-integrins appeared biologically inert. The data support the possibility of using this fluorescently labeled peptide as a tool for both the detection of and targeting to inflammatory sites characterized by robust leukocyte activation.

Regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata

Protocols for regeneration and Agrobacterium-mediated transformation of the apomictic species Eulaliopsis binata were developed. Initially, seeds of four genotypes of E. binata were incubated on a callus induction Murashige and Skoog (MS) basal medium supplemented with three concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). It was found that 36.2% of explants developed highly friable callus on medium containing 3.0mgl(-1) 2,4-D. Based on frequency of callus induction, the genotype Neixiang was selected for regeneration and transformation. Callus incubated on MS basal medium supplemented with 0.2mgl(-1) α-naphthalene acetic acid and 6.0mgl(-1) 6-furfuryl-aminopurine developed shoots. Subsequently, Agrobacterium tumefaciens strain EHA105-harboring a plasmid pCAMBIA1381 carrying a hygromycin phosphotransferase (hpt) resistance gene and a synthetic green fluorescent protein (GFP) gene, both driven by the cauliflower mosaic virus 35S promoter-was used for transformation system. Putative transgenic callus was obtained following two cycles of hygromycin selection. Expression of the transgene(s) in putative transgenic callus was analyzed using the GFP detection. Molecular identification of putative transformed shoots was performed by polymerase chain reaction and Southern blot analysis to confirm presence and integration of the hpt gene.

Photooxidation-Guided Ultrastructural Identification and Analysis of Cells in Neuronal Tissue Labeled with Green Fluorescent Protein

The ultrastructural characterization of neuronal compartments in intact tissue labeled with green fluorescent protein (GFP) remains a frequently encountered challenge, despite work establishing photooxidation of GFP in cultured cells. However, most applications require the detection of GFP or GFP fusion proteins expressed in intact tissue. Here, we report that illumination of GFP variants in oxygen-enriched environment reliably generated electron-dense 3,3′-diaminobenzidine (DAB) precipitates in slices from rat brain. The method is applicable to GFP variants tagged to presynaptic proteins as well as to soluble GFP in various brain regions. Serial section scanning electron microscopy was used to examine genetically labeled presynaptic terminals at high resolution and to generate three-dimensional representations of the synapses. Thus, we introduce a generally applicable correlative approach for the identification of presynaptic terminals genetically labeled with green fluorescent proteins in tissue slices and their ultrastructural characterization.

331 OPTIMIZATION OF BRANCHED 25kDa POLYETHYLENIMINE FOR EFFICIENT GENE DELIVERY IN BOVINE FETAL FIBROBLASTS

Cost-effective, highly efficient, and noncytotoxic transfection of bovine fetal fibroblasts (BFF) has proven difficult to achieve by regular chemical and physical methods. The aims of this study were to evaluate transient transfection efficiency and toxicity of commercially available branched 25 kDa polyethylenimine (25 kDa PEI, Sigma-Aldrich, St. Louis, MO, USA) and to optimize the transfection conditions leading to high percentages of PEI-transfected fibroblasts with minimum cytotoxic effects. Bovine fetal fibroblast (BFF) cells were seeded a day before transfection in 24-well plates at a density of 3 × 104 cells per well in DMEM with antibiotics and 10% SFB. When 70 to 90% confluence was reached, cells were washed with PBS and incubated in DMEM without antibiotics or SFB. For the transfection-mix preparation, increasing amounts of plasmidic DNA (pZsGreen1; 2 to 6 µg) were added to 50 µL of DMEM without antibiotics or SFB, incubated for 5 min at room temperature, and complexed with 0.5 to 4 µg of PEI (from 1 mg mL–1 solution) in 50 µL of PBS for 10 min. This transfection mix was added to the cell cultures and, 2 h later, 500 µL of DMEM with antibiotics and 10% SFB was added to each well. Detection of green fluorescent protein (GFP) expression by flow cytometry (reported as percentage of green fluorescent cells) was performed 48 h after transfection. Results were analysed by ANOVA and Tukey test and expressed as mean ± SEM (P &lt; 0.05). We found no significant difference between the percentage of GFP-positive cells transfected with 1 or 2 µg of 25 kDa PEI at 2 µg of DNA/well (15.2 ± 1.3 v. 16.9 ± 0.9%, respectively; P &gt; 0.05), whereas cells transfected with 1 or 2 µg of low-molecular-weight PEI (2 kDa) showed extremely low transfection efficiencies. Increasing the DNA load up to 6 µg significantly enhanced cell transfection (3.5- and 6-fold comparing 2 µg v. 4 µg and 6 µg of DNA, respectively; P &lt; 0.05) at 1 and 2 µg of 25 kDa PEI/well. In order to evaluate the cytotoxic effect of PEI, cell viability was determined using the MTT assay in 96-well plates (cells/well), with each condition scaled down to replicate the effect of 2 kDa or 25 kDa PEI in a 24-well plate. The MTT results (expressed as % of the control) indicated that PEI became cytotoxic at concentrations equivalent to 2 and 4 µg/well (54.7 ± 3.4 and 18.5 ± 5.7, respectively), whereas 1 µg/well produced a slight detrimental effect on cell viability (90.0 ± 2.6). No evidence of cytotoxicity was observed when the BFF were incubated with 0.5 µg/well of 25 kDa PEI and 1 or 2 µg/well of 2 kDa PEI. To study if a combination of low- and high-molecular-weight PEI could improve transfection efficiency and reduce toxicity, we tested a mixture (1 : 1) of 2 kDa and 25 kDa PEI. Even though the 1 : 1 mixture was less cytotoxic, the efficiency of gene delivery was not improved. We conclude that, under our experimental conditions, the highest percentage of GFP-expressing cells with good viability was obtained when 1 µg of 25 kDa PEI was added per well. Therefore, branched 25 kDa PEI transfection represents an efficient, simple, and cost-effective alternative for gene delivery in bovine fibroblast cells in culture.

Generation of striatal projection neurons extends into the neonatal period in the rat brain

Substantial advances have been made in the last decade on our understanding of the basic physiology underlying neurogenesis in the postnatal mammalian brain. The bulk of the work in this area has been based on analysis of the adult brain. Relatively less is known about the capacity for neurogenesis in specific structures within the neonatal brain. Here we report that the production of medium spiny striatal projection neurons extends into the early neonatal period under normal physiological conditions in the rat brain. Birth-dating of newborn cells with bromodeoxyuridine at postnatal days 0, 2 and 5 showed a peak production close to birth, which sharply declined at the later time-points. Additionally, there was a low-level but stable contribution of neurons with interneuron identity over the same time-period. Importantly, retroviral labelling of new striatal projection neurons with green fluorescent protein showed long-term survival and terminal differentiation with characteristic morphology, including highly elaborated spiny dendrites, and appropriate axonal targeting of the globus pallidus and midbrain. This latent period of striatal neurogenesis in the early neonatal brain represents an interesting target for regenerative approaches aimed at restoring striatal circuitry in perinatal pathologies, such as hypoxic and ischaemic damage associated with cerebral palsy.

Pharmacokinetics and risk evaluation of DNA vaccine against Schistosoma japonicum

DNA plasmid immunization is a novel approach of preventive and therapeutic vaccine. More than 100 DNA vaccines have been on preclinical or clinical phase trials, and four kinds of DNA vaccines for livestock have been approved by USDA, CFIA, and APVMA. Schistosomiasis is a worldwide parasitic disease, and vaccine immunization is supposed to be a promising approach to control the health crisis. On the basis of former preclinical studies, we further focused on the pharmacokinetics and risk evaluation of DNA vaccine in vivo. In the present study, enhanced green fluorescent protein (EGFP) report gene was fused with Schistosoma japonicum 23kDa transmembrane protein antigen gene (Sj23) and constructed into DNA vaccine pVIVO2-Sj23.EGFP. After intramuscularly injecting 100μg of purified DNA vaccine plasmid to immunizate BALB/c mice, we studied the tissue distribution of DNA plasmid and expressed Sj23.EGFP antigen, the persistence time of elicited antibodies, and the risk of DNA vaccine transferred into intestinal microorganisms. The results showed that DNA vaccine plasmid could be distributed into all tissues of the body after injection; however, only few organs including the injected muscle were detected DNA vaccine at postimmunization until the 100days by PCR technology; the detection of green fluorescence protein displayed that DNA vaccine could be expressed in almost every tissue and organs; the ELISA assay indicated the immune antibody against Sj23 could persist over 70days; and the DNA vaccine transferring intestinal flora results was negative. The results indicated that the DNA vaccine has systemic protection and long-lasting effectivity and is safe to intestinal flora.

Antiretroviral agents effectively block HIV replication after cell-to-cell transfer.

Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4(+) T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo.