Display Settings:

Items per page

Results: 7

1.
Figure 7

Figure 7. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Equine infectious anemia virus (EIAV)-transduced cells differentiate into neurons in the olfactory bulbs (Obs). (a) EIAV-CMV-EGFP-transduced cells were distributed widely in the OB 1-month post-injection. Layers of the OB: PGL, periglomerular layer, EPL, external plexiform layer; MCL, mitral layer; GCL-S, superficial granule cell layer; GCL-D, deep granule cell layer; RMS, rostral migratory stream. Morphology of cell types in the distinct layers indicates that EIAV-transduced cells are highly suitable for morphometric assessment of differentiation in the OB. (b) The EGFP(+) population (green) in the OB includes some calretinin (CR, red)-expressing interneurons, a few tyrosine hydroxylase (TH, red)-expressing neurons in the PGL, but not calbindin (CB, red)-expressing neurons in the PGL. A portion of the EGFP cells expressed polysialylated neuronal cell adhesion molecule (PSA-NCAM) suggesting that they were undifferentiated cells. Arrows indicate double labeled cells for each marker. A significant number of EGFP(+) cells could not be labeled with any of the known markers. CMV, cytomegalovirus; EGFP, Enhanced Green Fluorescent Protein.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
2.
Figure 3

Figure 3. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Equine infectious anemia virus (EIAV) targets slowly dividing cells in the adult stem cell niche (SCN). To assess the proliferative capacity of putative neural stem cells targeted by EIAV, Bromodeoxyuridine (BrdU) was administered thrice per day, for 7 days, in 1-month post EIAV-injected mice. Mice were allowed to survive a 2-week ‘chase’ period after the last BrdU injection so as to dilute out BrdU from TAPs and migrating neuroblasts. (a) In the subventricular zone (SVZ), some EIAV-CMV-EGFP-transduced cells (arrows) retained BrdU (blue) and co-labeled with the mitotic (M)-phase marker phosphohistone-3 (PH3) (red). (b) Likewise, some EGFP(+)/BrdU(+) cells expressed the cell cycle marker Ki67 (red, arrows), indicating that EIAV-transduced cells in the SVZ included slowly dividing stem cells. (c and d) EGFP(+)/BrdU(+) cells included S100β(+) ependymal cells and glial fibrillary acidic protein (GFAP)(+) astrocytes in the SVZ (both markers are red in their respective panels). CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
3.
Figure 4

Figure 4. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Equine infectious anemia virus (EIAV) transduced stem cells form neurospheres in vitro. EIAV-CMV-EGFP-transduced brains were harvested 2 weeks post EIAV injection, and EGFP(+) cells were fluorescence-activated cell sorted and cultured for neurosphere formation. (a) Neurospheres maintained EGFP expression after 10 passages (Pass 10) indicating that the EIAV transgene had stably integrated into the genome of the transduced neural stem cells. Data in chart are number of neurospheres per 6 mm culture dish after 1, 5 and 10 passages (error bars, standard error of the mean). (b) Passaged neurospheres were plated on laminin/poly-L-lysine-coated glass slides and after 7 days in vitro (DIV) differentiated cells were readily visible surrounding the core of the neurospheres. (c) Cells derived from the neurospheres included TuJ1(+) neurons (red); NG2(+) oligodendrocytes (red); and glial fibrillary acidic protein (GFAP)(+) astrocytes (red). CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
4.
Figure 5

Figure 5. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

The life of migrating cells in the rostral migratory stream (RMS). (a) Organotypic slices harvested from EIAV-CMV-EGFP-injected newborn (P0) mice were time-lapse imaged (spanning 150 min) using a confocal microscope. Three dynamic migratory states in cells within the RMS were identified by assessment of EGFP cells in the RMS. Cells in the three states were colorized for diagrammatic purposes. (b) In the first state, rapidly migrating (RM) cells (green panel) rapidly translocate from one location to another, in a forward fashion toward the olfactory bulb (for example, cell indicated by arrowhead). Many RM cells entered a phase of non-directed, hyperactive motion, hallmarked by multi-process extensions and retractions (arborizing multiple processes, AMP cells, red panel). In this state, nuclear translocation is dramatically less rapid and covers less distance compared with the first state. AMP cells occasionally underwent mitosis (M cells, blue panel, asterisks indicate cell division). Bars = 50 μm for each panel. (c) Percentage of cells within each of the identified states in the RMS (n = 150 cells). (d) Length of processes(s) at each of the identified states (n = 25 cells per state). (e) Number of process branches in cells within each of the migratory states (n = 10 cell per state). (f) Average number of process extensions and process retractions during 150 min of observation (n = 30 cell per state). Data are average±s.e.m. CMV, cytomegalovirus; EGFP, Enhanced Green Fluorescent Protein.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
5.
Figure 2

Figure 2. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Cellular targets of Equine infectious anemia virus (EIAV) in the subventricular zone (SVZ). (ae) At 1 week after EIAV-CMV-EGFP injections, the majority of transduced cells in the SVZ (positive for EGFP, green) were S100β(+) ependymal cells (red, a) and the glial fibrillary acidic protein (GFAP)(+) astrocytes (red, b). Few, if any, distaless 2 (DLX2)(+) transit amplifying progenitors (TAPs) (red, c), polysialylated neuronal cell adhesion molecule (PSA-NCAM)(+) migrating neuroblasts (red, d) or NeuN(+) olfactory neurons (red, e) expressed EGFP. EIAV-transduced cells in the rostral migratory stream (RMS) were largely GFAP(+) astrocytes (d). (a′/a″–e′/e″) 1 month (a′–b′) and 6 months (a″–b″) post EIAV-CMV-EGFP injection, ependymal and astrocytic cells maintained their expression of EGFP. In contrast to the 1-week survival time, a significant number of TAPs (c/c″), migrating neuroblasts (d/d″) and olfactory neurons (e/e″) were EGFP(+), suggesting that they were derived from either the transduced astrocytes or ependymal cells. (f) Percentage of distinct cell types in the SVZ described above that express EGFP was calculated using stereological estimates of cell density for double-labeled EGFP(+)/marker(+) cells in the SVZ. Data are average±s.e.m. Asterisks indicate significance, Student’s t-test, P<0.001. CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; RMS, rostral migratory stream; OB, olfactory bulb.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
6.
Figure 1

Figure 1. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Equine infectious anemia virus (EIAV)-mediated gene transfer to the ventricular zones of the adult brain. (a) EIAV vectors were constructed as described before.23,24 Diagram illustrates the three plasmids for generating the EIAV-CMV-EGFP vector. A line of 293 cells was triple transfected with EIAV constructs and 48 h later EIAV-CMV-EGFP or EIAV-pCA-EGFP viruses were harvested from the supernatant, concentrated by ultracentrifugation and resuspended in a carrier medium for in vivo and in vitro transduction. Consistent yields of 1010 infectious units per mililiter of carrier medium were obtained. (bd) EIAV-CMV-EGFP vectors were injected (5 μl per hemisphere; 5 × 107 total infectious units) into the anterior lateral ventricles of young adult mice (6 weeks old) using stereotaxic apparatus. Mice were then allowed to survive for 1 week (b), 1 month (c) or for 6 months (d). Sagittal views of the brains reveal a gradual increase in density of EGFP-positive cells (green) in the olfactory bulbs (OBs) with time. Note that the entire rostro-caudal and dorsal-ventral boundaries of the subventricular zone (SVZ) were transduced with the bolus injections. CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; pCA, chicken β-actin promoter; RMS, rostral migratory stream.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.
7.
Figure 6

Figure 6. From: Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Equine infectious anemia virus (EIAV)-transduced cells in the rostral migratory stream (RMS) are highly suitable for assessment of chemotactic cues using a novel assay. (a) To assess the responsiveness of EIAV-CMV-EGFP-transduced cells in the RMS to chemotactic cues, Cos7 cells were transfected with various constructs encoded secreted factors and a monomeric red fluorescent protein (mRFP) reporter construct. Organotypic slices were harvested from P7 animals as described in Figure 5, and aggregates of Cos7 cells were explanted on the slices proximal to, in the middle or distal to the subventricular zone (SVZ). (b) Migrating cells do not respond to mock-transfected (mRFP alone) Cos7 cells. We earlier identified the expression and function of a class of growth factors called the neuregulins (NRGs) and their receptor tyrosine kinase, ErbB4, in migration and differentiation in the RMS and olfactory bulb (OB).34,35 The majority of neuroblasts migrating in the RMS express the receptor ErbB4. A secreted isoform of NRG1 has a potent attractive effect on the migrating cells in the RMS. Another ErbB4 ligand, NRG2, has a different effect on migrating neuroblasts, wherein it disrupts their rapid migration and increases the number of AMP and M cells in the RMS. Another secreted factor, the semaphorin type 3a protein (Sema3a) has distinct effects on migrating cells in the sector of the RMS proximal to the SVZ versus its effect on cells in the sectors of the RMS distal to the SVZ (near the OB). Pie orientation charts indicate direction of 20 randomly selected EGFP(+) cells in the RMS relative to the appropriate explants (red). (ce) Percentage of cells within the identified migratory states in the RMS when exposed to different ligands (n =50 per experiment). Data are average±s.e.m. Asterisks indicate significance, Student’s t-test, P<0.01. AMP, arborizing multiple processes; CMV, cytomegalovirus; EGFP, Enhanced Green Fluorescent Protein; M, mitosis; RM, rapidly migrating.

BV Jacquet, et al. Gene Ther. ;16(8):1021-1033.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk