Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.     

Treatment of intracerebral haemorrhage in rats with intraventricular transplantation of human amniotic epithelial cells

We explored the effects on brain oedema and neurological functional recovery after transplantation of hAECs (human amniotic epithelial cells) into the lateral ventricle of rats with ICH (intracerebral haemorrhage). hAECs were isolated from human term placenta and seeded for primary culture. We delivered hAECs labelled with Hoechst33258 and transfected with EGFP (enhanced green fluorescent protein) gene using lentiviral vectors into ICH rat models. The behaviour of the animals and brain oedema were evaluated after 28 days, and brain sections were made for morphological and immunohistochemical analyses with fluorescence microscopy. Our results were as follows. Transplanted hAECs were observed along the lateral wall and survived for at least 4 weeks. Some of the cells were stained with human specific antibody to vimentin and nestin. Around the injury site, activated microglia stained with OX42 were reduced. The water content of ICH rats decreased in the treatment group. The behaviour test scores were improved in the treatment group compared with those in the control groups. In conclusion, hAECs cannot only survive in the lateral ventricle of ICH rats after transplantation, but also express vimentin and nestin. hAEC transplantation reduced brain oedema and improved the motor deficits of ICH rats.     

Identification of the rostral migratory stream in the canine and feline brain

In the adult rodent brain, neural progenitor cells migrate from the subventricular zone of the lateral ventricle towards the olfactory bulb in a track known as the rostral migratory stream (RMS). To facilitate the study of neural progenitor cells and stem cell therapy in large animal models of CNS disease, we now report the location and characteristics of the normal canine and feline RMS. The RMS was found in Nissl-stained sagittal sections of adult canine and feline brains as a prominent, dense, continuous cellular track beginning at the base of the anterior horn of the lateral ventricle, curving around the head of the caudate nucleus and continuing laterally and ventrally to the olfactory peduncle before entering the olfactory tract and bulb. To determine if cells in the RMS were proliferating, the thymidine analog 5-bromo-2-deoxyuridine (BrdU) was administered and detected by immunostaining. BrdU-immunoreactive cells were present throughout this track. The RMS was also immunoreactive for markers of proliferating cells, progenitor cells and immature neurons (Ki-67 and doublecortin), but not for NeuN, a marker of mature neurons. Luxol fast blue and CNPase staining indicated that myelin is closely apposed to the RMS along much of its length and may provide guidance cues for the migrating cells. Identification and characterization of the RMS in canine and feline brain will facilitate studies of neural progenitor cell biology and migration in large animal models of neurologic disease.     

Palmitic acid-modified poly-L-lysine for non-viral delivery of plasmid DNA to skin fibroblasts

Palmitic acid conjugates of poly-L-lysine (PLL-PA) were prepared, and their ability to deliver plasmid DNA into human skin fibroblasts was evaluated in vitro. The conjugates were capable of condensing a 4.7 kb plasmid DNA into 50-200 nm particles (mean +/- SD = 112 +/- 34 nm), which were slightly smaller than the particles formed by PLL (mean +/- SD = 126 +/- 51 nm). Both PLL and PLL-PA were readily taken up by the cells, but PLL-PA delivered the plasmid DNA into a higher proportion of cells. DNA delivery was found to be reduced by endocytosis inhibitor Brefeldin A, suggesting an active mechanism of particle uptake. Using enhanced green fluorescent protein (EGFP) as a reporter gene, PLL-PA was found to give the highest number of EGFP-positive cells among several carriers tested, including polyethyleneimine, Lipofectamine-2000, and an adenovirus. Although some carriers gave a higher percentage of EGFP-positive cells than PLL-PA, they were also associated with higher toxicities. We conclude that PLL-PA is a promising gene carrier for non-viral modification of human fibroblasts.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice

To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes.     

Ocular Delivery of Compacted DNA-Nanoparticles Does Not Elicit Toxicity in the Mouse Retina

Subretinal delivery of polyethylene glycol-substituted lysine peptide (CK30PEG)-compacted DNA nanoparticles results in efficient gene expression in retinal cells. This work evaluates the ocular safety of compacted DNA nanoparticles. CK30PEG-compacted nanoparticles containing an EGFP expression plasmid were subretinally injected in adult mice (1 µl at 0.3, 1.0 and 3.0 µg/µl). Retinas were examined for signs of inflammation at 1, 2, 4 and 7 days post-injection. Neither infiltration of polymorphonuclear neutrophils or lymphocytes was detected in retinas. In addition, elevation of macrophage marker F4/80 or myeloid marker myeloperoxidase was not detected in the injected eyes. The chemokine KC mRNA increased 3–4 fold in eyes injected with either nanoparticles or saline at 1 day post-injection, but returned to control levels at 2 days post-injection. No elevation of KC protein was observed in these mice. The monocyte chemotactic protein-1, increased 3–4 fold at 1 day post-injection for both nanoparticle and saline injected eyes, but also returned to control levels at 2 days. No elevations of tumor necrosis factor alpha mRNA or protein were detected. These investigations show no signs of local inflammatory responses associated with subretinal injection of compacted DNA nanoparticles, indicating that the retina may be a suitable target for clinical nanoparticle-based interventions.     

Efficient adeno-associated virus-mediated gene expression in human placenta-derived mesenchymal cells

Mesenchymal cells from various sources are pluripotent and are attractive sources for cell transplantation. In this study, we analyzed recombinant adeno-associated virus (rAAV)-mediated gene expression in human placenta-derived mesenchymal cells (hPDMCs), which reside in placental villi. After transduction of AV-CAG-EGFP, a rAAV expressing enhanced green fluorescence protein (EGFP), hPDMCs showed much higher level of EGFP expression than human umbilical vein endothelial cells or rat aortic smooth muscle cells. The number of EGFP-positive hPDMCs infected by AV-CAG-EGFP alone did not increase significantly by coinfection of adenovirus, which enhanced expression level of the rAAV vector. Moreover, flow cytometric analysis showed discrete positive fraction of EGFP-expressing hPDMCs, which is about 15-20% of the cells infected with AV-CAG-EGFP. Therefore, some cell population in hPDMCs might be highly susceptible to rAAV-mediated gene transduction. In addition, stable EGFP expressions were observed in about 1% of hPDMCs infected with AV-CAG-EGFP at 4 weeks post-infection. Collectively, hPDMCs have characters favorable for rAAV-mediated gene expression.     

Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r-EGFP transgene reporter

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r (c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.     

Properties of doublecortin expressing neurons in the adult mouse dentate gyrus

The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses.     

Cell Proliferation in the Adult Rat Rostral Migratory Stream Following Exposure to Gamma Irradiation

Summary One of the few areas of the adult CNS, that are known to be competent for neuronal proliferation, is the subventricular zone (SVZ) lining the brain lateral ventricles. Cells proliferating in the SVZ migrate along a defined pathway, the rostral migratory stream (RMS), where their proliferation continues until reaching the olfactory bulb.1. In relation to the fact that brain is, in general, regarded as a radioresistant organ composed from non dividing cells, the aim of the present study was to investigate effect of ionizing radiation on proliferating cell numbers in the RMS of adult rats.2. Male Wistar rats were investigated 25 and 80 days after whole body gamma irradiation with the dose of 3 Gy. Dividing cells were labeled by bromodeoxyuridine (BrdU). BrdU-positive cells were counted by Disector program. The mean number of BrdU⁺ cells in the whole RMS and in its individual parts (vertical arm, elbow, and horizontal arm) was evaluated.3. Temporary increase in proliferating cell number (by 30%) was seen in the whole RMS at the 25th day after irradiation.4. The most expressive increase occurred in the vertical arm (by 60%) and elbow (about 37%). The values reduced till the 80th day after exposure.Our results show that ionizing irradiation significantly influences the extent of cell proliferation and migration in the adult rat RMS.     

Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain

Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90?% on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.     

Mechanism of migration of olfactory bulb interneurons

Neuroblasts in the subventricular zone of the walls of the lateral ventricle in the brain of young and adult rodents migrate into the olfactory bulb where they differentiate into local interneurons. These cells move closely associated with each other, forming chains without radial glial or axonal guidance. The migrating neuroblasts express PSA-NCAM on their surface and PSA residues are crucial for cell-cell interaction during chain migration. This migration occurs throughout the lateral wall of the lateral ventricle, where the precursors form an extensive network of chains. Cells remain organized as chains until they reach the olfactory bulb, where they disperse organized as chains until they reach the olfactory bulb, where they disperse radially as individual cells. Chain migration defines a novel form of neuronal precursor translocation which is based on homotypic interactions between cells.     

Cell-specific expression of enhanced green fluorescence protein under the control of neuropeptide gene promoters in the brain of the silkworm,Bombyx mori,using Bombyx mori nucleopolyhedrovirus-derived vectors

Using the larvae of the silkworm, Bombyx mori, we examined the baculovirus expression vector system for the expression of the enhanced green fluorescence protein (EGFP) gene under the control of several gene promoters in vivo. To investigate the gene-delivery efficiency of the baculovirus into various larval tissues, we constructed two recombinant baculoviruses carrying the EGFP gene downstream of the Drosophila melanogaster hsp70 gene promoter from B. mori nucleopolyhedrovirus (BmNPV) and Autographa californica nucleopolyhedrovirus (AcNPV). After injection of these recombinant baculoviruses into newly ecdysed 5th instar larvae, hsp70::EGFP-BmNPV, but not hsp70::EGFP-AcNPV, caused intense expression of EGFP not only in various non-neural tissues, but also in the neural organs including the brain 5 days postinfection. To investigate the cell-specific expression in the brain, we constructed recombinant C4/B3::EGFP-BmNPV and PTTH::EGFP-BmNPV which carry the EGFP gene under the control of bombyxin B3 and prothoracicotropic hormone (PTTH) gene promoters, respectively. Injection of these recombinant baculoviruses caused specific expression of EGFP with a high gene-expression efficiency in the neurosecretory cells of the brain depending on the neurohormone gene promoters. Present results indicate that this in vivo gene-expression system mediated by the baculovirus can serve as an efficient system permitting gene delivery into neural tissues in insects.     

Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells

Baculovirus is a promising gene delivery vector and can be titrated by constitutive EGFP expression in HeLa cells, which, however, might interfere with target transgene expression and impart cytotoxicity. Here we constructed Bac-ME accommodating egfp under the inducible metallothionein promoter and Bac-MECB harboring an additional BMP-2 gene. Bac-ME effectively transduced HeLa cells with minimal leaky expression, but expressed EGFP robustly upon induction with ZnSO(4), hence allowing for virus titration by transducing HeLa cells with serially diluted virus, subsequent ZnSO(4) induction and flow cytometry analysis of EGFP-positive cells. The titration protocol enabled the generation of discernable titration curves, determination of transducing titers, and discrimination of the transducing abilities of different virus batches. After titration, cell transduction with pre-determined Bac-ME dose revealed consistent transduction efficiency dependence on the dose, regardless of virus batch and cell type. Bac-MECB was similarly titrated by inducible EGFP expression and used to transduce de-differentiated articular chondrocytes without EGFP induction. BMP-2 expression was proportional to the Bac-MECB dose and promoted cartilage-specific matrix synthesis, implicating the potential of Bac-MECB in restoring chondrocyte differentiation. These data confirmed that regulatable EGFP expression enabled rapid, reliable baculovirus titration without interference with subsequent applications.     

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.     

Developmental incompatibility of human parthenogenetic embryonic stem cells in mouse blastocysts

Human parthenogenetic embryonic stem (pES) cells can be clinically used in the future to avoid immunological rejection. However, the developmental potential of human pES cells remains to be elucidated. In this study, we generated a human pES-enhanced green fluorescent protein (EGFP) cell line (chHES-32-EGFP), which shows pluripotency thus far and maintains stable and robust EGFP expression in the undifferentiated and differentiated states in vivo and in vitro. Using this pES-EGFP cell line, we found that when human pES-EGFP cells were injected into mice blastocysts, EGFP-positive cells progressively decreased with the development of blastocysts in vitro. Only 4 out of 23 embryos (17.4%) contained EGFP-positive cells and all of these embryos exhibited abnormal morphology or delayed development when the chimera blastocysts were implanted into the pseudopregnant recipient mouse uterus. These results raise serious questions regarding the feasibility of the generation of interspecific chimeras between mouse blastocysts and human pES cells.