Replication-incompetent Retrovirus Research Articles

A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.

ABSTRACTAn important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

Pseudotyped retroviruses for infecting axolotl.

The ability to introduce DNA elements into host cells and analyze the effects has revolutionized modern biology. Here we describe a protocol to generate Moloney murine leukemia virus (MMLV)-based, replication-incompetent pseudotyped retrovirus capable of infecting axolotls and incorporating genetic information into their genome. When pseudotyped with vesicular stomatitis virus (VSV)-G glycoprotein, the retroviruses can infect a broad range of proliferative axolotl cell types. However, if the retrovirus is pseudotyped with an avian sarcoma leukosis virus (ASLV)-A envelope protein, only axolotl cells experimentally manipulated to express the cognate tumor virus A (TVA) receptor can be targeted by infections. These strategies enable robust transgene expression over many cell divisions, cell lineage tracing, and cell subtype targeting for gene expression.

Pseudotyped retroviruses for infecting axolotl in vivo and in vitro

Axolotls are poised to become the premiere model system for studying vertebrate appendage regeneration. However, very few molecular tools exist for studying crucial cell lineage relationships over regeneration or for robust and sustained misexpression of genetic elements to test their function. Furthermore, targeting specific cell types will be necessary to understand how regeneration of the diverse tissues within the limb is accomplished. We report that pseudotyped, replication-incompetent retroviruses can be used in axolotls to permanently express markers or genetic elements for functional study. These viruses, when modified by changing their coat protein, can infect axolotl cells only when they have been experimentally manipulated to express the receptor for that coat protein, thus allowing for the possibility of targeting specific cell types. Using viral vectors, we have found that progenitor populations for many different cell types within the blastema are present at all stages of limb regeneration, although their relative proportions change with time.

The Third Transmembrane Segment of Orai1 Protein Modulates Ca2+ Release-activated Ca2+ (CRAC) Channel Gating and Permeation Properties

Orai1, the pore subunit of Ca(2+) release-activated Ca(2+) channels, has four transmembrane segments (TMs). The first segment, TMI, lines the pore and plays an important role in channel activation and ion permeation. TMIII, on the other hand, does not line the pore but still regulates channel gating and permeation properties. To understand the role of TMIII, we have mutated and characterized several residues in this domain. Mutation of Trp-176 to Cys (W176C) and Gly-183 to Ala (G183A) had dramatic effects. Unlike wild-type channels, which exhibit little outward current and are activated by STIM1, W176C mutant channels exhibited a large outward current at positive potentials and were constitutively active in the absence of STIM1. G183A mutant channels also exhibited substantial outward currents but were active only in the presence of 2-aminoethoxydiphenyl borate (2-APB), irrespective of STIM1. With W176C mutant channels inward, monovalent currents were blocked by Ca(2+) with a high affinity similar to the wild type, but the Ca(2+)-dependent blocking of outward currents differed in the two cases. Although a 50% block of the WT outward current required 250 μm Ca(2+), more than 6 mm was necessary to have the same effect on W176C mutant channels. In the presence of extracellular Ca(2+), W176C and G183A outward currents developed slowly in a voltage-dependent manner, whereas they developed almost instantaneously in the absence of Ca(2+). These changes in permeation and gating properties mimic the changes induced by mutations of Glu-190 in TMIII and Asp-110/Asp-112 in the TMI/TMII loop. On the basis of these data, we propose that TMIII maintains negatively charged residues at or near the selectivity filter in a conformation that facilitates Ca(2+) inward currents and prevents outward currents of monovalent cations. In addition, to controlling selectivity, TMIII may also stabilize channel gating in a closed state in the absence of STIM1 in a Trp-176-dependent manner.

Postnatal subventricular zone progenitors give rise not only to granular and periglomerular interneurons but also to interneurons in the external plexiform layer of the rat olfactory bulb

Interneurons in the granule cell layer (GCL) and glomerular layer (GL) of the olfactory bulb (OB) are generated from progenitors in the subventricular zone (SVZ) of the lateral ventricle. However, little is known about the origin of interneurons in the external plexiform layer (EPL) of the OB. On the basis of the concept of corticogenesis, I hypothesized that interneurons in the EPL of the rodent OB also originate in the SVZ. In the present study, replication-incompetent retroviruses encoding a marker gene, human placental alkaline phosphatase (AP), were injected into the lateral ventricles of postnatal day 4 Wistar rats to label dividing cells in the SVZ. Two days after injection, some of the AP-labeled cells had migrated into the OB. Five weeks after injection, AP/NeuN double-labeled cells were found not only in the GCL and GL but also in the EPL of the OB. In the EPL, most AP-labeled cells were calcium-binding protein parvalbumin (PV)-immunoreactive (+) interneurons. A subset of these cells was made up of calcium-binding protein calretinin (CR)(+) interneurons. According to their structural features, AP-labeled cells in the EPL were Van Gehuchten cells, multipolar cells, and superficial short-axon cells. Thus, postnatal SVZ progenitors give rise not only to granular and periglomerular interneurons but also to interneurons in the EPL of the OB. Furthermore, these results suggest that SVZ progenitors give rise to virtually all subpopulations of interneurons in the OB.

TrkB/BDNF signaling regulates photoreceptor progenitor cell fate decisions

Neurotrophins, via activation of Trk receptor tyrosine kinases, serve as mitogens, survival factors and regulators of arborization during retinal development. Brain-derived neurotrophic factor (BDNF) and TrkB regulate neuronal arborization and survival in late retinal development. However, TrkB is expressed during early retinal development where its functions are unclear. To assess TrkB/BDNF actions in the early chick retina, replication-incompetent retroviruses were utilized to over-express a dominant negative truncated form of TrkB (trunc TrkB), or BDNF and effects were assessed at E15. Clones expressing trunc TrkB were smaller than controls, and proliferation and apoptosis assays suggest that decreased clone size correlated with increased cell death when BDNF/TrkB signaling was impaired. Analysis of clonal composition revealed that trunc TrkB over-expression decreased photoreceptor numbers (41%) and increased cell numbers in the middle third of the inner nuclear layer (INL) (23%). Conversely, BDNF over-expression increased photoreceptor numbers (25%) and decreased INL numbers (17%). Photoreceptors over-expressing trunc TrkB demonstrated no increase in apoptosis nor abnormalities in lamination suggesting that TrkB activation is not required for photoreceptor cell survival or migration. These studies suggest that TrkB signaling regulates commitment to and/or differentiation of photoreceptor cells from retinal progenitor cells, identifying a novel role for TrkB/BDNF in regulating cell fate decisions.

Glutamate regulates retinal progenitors cells proliferation during development

The precise coordination of cell cycle exit and cell fate specification is essential for generating the correct proportion of retinal cell types during development. The decision to exit the cell cycle is regulated by intrinsic and extrinsic cues. There is growing evidence that neurotransmitters can regulate cell proliferation and cell fate specification during the early stages of CNS development prior to the formation of synaptic connections. We found that the excitatory neurotransmitter glutamate regulates retinal progenitor cell proliferation during embryonic development of the mouse. AMPA/kainate and N-methyl-d-aspartate receptors are expressed in embryonic retinal progenitor cells. Addition of exogenous glutamate leads to a dose-dependent decrease in cell proliferation without inducing cell death or activating the p53 pathway. Activation of AMPA/kainate receptors induced retinal progenitor cells to prematurely exit the cell cycle. Using a replication-incompetent retrovirus to follow the clonal expansion of individual retinal progenitor cells, it was observed that blockade of AMPA/kainate receptors increased the proportion of large clones, showing that modulation of endogenous glutamatergic activity can have long-term consequences on retinal cell proliferation. Real time reverse transcriptase-polymerase chain reaction and immunoblot analyses demonstrated that glutamate does not alter the levels of the mRNA and proteins that regulate the G1/S-phase transition. Instead, the activity of the Cdk2 kinase is reduced in the presence of glutamate. These data indicate that glutamate regulates retinal progenitor cell proliferation by post-translational modulation of cyclin/Cdk2 kinase activity.

Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells

Fate maps based on quail-chick grafting of avian cephalic neural crest precursors and paraxial mesoderm cells have identified the majority of derivatives from each population but have not unequivocally resolved the precise locations of and population dynamics at the interface between them. The relation between these two mesenchymal tissues is especially critical for the development of skeletal muscles, because crest cells play an essential role in their differentiation and subsequent spatial organization. It is not known whether myogenic mesoderm and skeletogenic neural crest cells establish permanent relations while en route to their final destinations, or later at the sites where musculoskeletal morphogenesis is completed. We applied beta-galactosidase-encoding, replication-incompetent retroviruses to paraxial mesoderm, to crest progenitors, or at the interface between mesodermal and overlying neural crest as both were en route to branchial or periocular regions in chick embryos. With respect to skeletal structures, the results identify the avian neural crest:mesoderm boundary at the junction of the supraorbital and calvarial regions of the frontal bone, lateral to the hypophyseal foramen, and rostral to laryngeal cartilages. Therefore, in the chick embryo, most of the frontal and the entire parietal bone are of mesodermal, not neural crest, origin. Within paraxial mesoderm, the progenitors of each lineage display different behaviors. Chondrogenic cells are relatively stationary and intramembranous osteogenic cells move only in transverse planes around the brain. Angioblasts migrate invasively in all directions. Extraocular muscle precursors form tightly aggregated masses that en masse cross the crest:mesoderm interface to enter periocular territories, while branchial myogenic lineages shift ventrally coincidental with the movements of corresponding neural crest cells. En route to the branchial arches, myogenic mesoderm cells do not maintain constant, nearest-neighbor relations with adjacent, overlying neural crest cells. Thus, progenitors of individual muscles do not establish stable, permanent relations with their connective tissues until both populations reach the sites of their morphogenesis within branchial arches or orbital regions.

Postnatal cellular contributions of the hippocampus subventricular zone to the dentate gyrus, corpus callosum, fimbria, and cerebral cortex

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.

A comparison of cell transplantation and retroviral gene transfection as tools to study lineage and differentiation in the rat spinal cord

Establishing the cell lineage relationships of cells during development allows insight into when and where developmental decisions are made. In the developing spinal cord, the origin and fate of radial glial has yet to be determined. One way in which to address this question is to transplant enriched populations of radial glia into the ventricular zone (VZ) region of host embryos to examine the lineage and differentiation pattern of these cells. An indirect selection procedure using immunomagnetic beads (Dynabeads; Dynal Biotech) was used here to isolate spinal cord radial glia. This negative immunoselection procedure resulted in a high yield of radial glia. A fluorescent cytoplasmic dye (Cell Tracker Green CMFDA) was used to label radial glia before transplantation. The role of radial glia as progenitor cells can also be examined using a green fluorescent protein (GFP)-expressing retroviral vector. The retroviral vector allows dividing cells in the VZ region of the spinal cord to be tracked by labelling them with GFP. Both techniques were utilised here to successfully label and examine embryonic spinal cord radial glia in vivo after a microinjection of either fluorescently labelled radial glia or replication-incompetent GFP-expressing retrovirus in utero.

Topotecan Combination Chemotherapy in Two New Rodent Models of Retinoblastoma

Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein- or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats. The second model uses a replication-incompetent retrovirus (LIA-E(E1A)) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-E(E1A) is injected into the eyes of newborn p53-/- mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide. Vincristine had the lowest LC50 in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.

Rb regulates proliferation and rod photoreceptor development in the mouse retina.

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1(lox) allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.

Gene Therapy with Virus Vectors for specific Disease of the Nervous System: Recombinant Retrovirus Vectors for Treatment of Malignant Brain Tumors

Publisher Summary The susceptibility of malignant brain tumors of glial origin (gliomas) to virus vector-mediated gene therapy has been investigated only since the early 1990s. The initially favored and best-explored gene therapy approach included insertion of a gene into tumor cells that renders these and their clonal progeny differentially sensitive to drug treatment. The gene/vector system most widely utilized until now is the herpes simplex virus thymidine kinase (HSV-tk) gene transferred by a replication-incompetent retrovirus (RV) vector that is released in situ by fibroblast-derived retrovirus vector-producing cells (RV-VPC). The early work on RV vectors initiated current developments of highly sophisticated third- and fourth-generation virus vectors for human gene therapy. Although, there is an impressive variety of different gene therapy strategies for experimental treatment of brain tumors, their therapeutic success is still very limited and highly dependent on the qualities and limitations of the vector systems available. RV has been one of the earliest vectors used for gene transfer in the brain and employed as a tool for targeted and selective destruction of tumor cells. These vectors seem to be well tolerated and exceptionally biologically safe, although apparently not able to significantly improve survival of the patients in combined treatment protocols.

Pancreatic lineage analysis using a retroviral vector in embryonic mice demonstrates a common progenitor for endocrine and exocrine cells.

The origin of pancreatic endocrine cells is unknown. Some studies have suggested that there is a common pancreatic progenitor which gives rise to both endocrine and exocrine cells, while others have suggested separate endocrine and exocrine lineages. Previous conclusions have been based on indirect data, such as the co-expression of molecular markers. We directly assessed the relationship between endocrine and exocrine cells during development using a lineage tracer. A replication-incompetent retrovirus was used to introduce the reporter gene alkaline phosphatase into single cells in explants of mouse embryonic pancreas. After a week in culture, the subsequent fate of the infected cells could then be determined. The results show that a common pancreatic progenitor cell exists, which gives rise to both endocrine and exocrine cells.

In vivo analysis of retroviral enhancer mutations in hematopoietic cells: SP1/EGR1 and ETS/GATA motifs contribute to long terminal repeat specificity.

The objective of this work was to identify, in the context of chromosomally integrated DNA, the contribution of defined transcription factor binding motifs to the function of a complex retrovirus enhancer in hematopoietic cells in vivo. Repopulating murine hematopoietic cells were transduced with equal gene dosages of replication-incompetent retrovirus vectors encoding enhanced green fluorescent protein. Enhancer sequences were derived from mouse spleen focus-forming virus. Destruction of GC-rich sites representing overlapping targets for SP1 or EGR1 uniformly attenuated gene expression (approximately 25 to 70% of wild-type levels) in all hematopoietic lineages, as shown by multicolor flow cytometry of peripheral blood and bone marrow cells at various time points posttransplantation. In contrast, a point mutation within a dual ETS/GATA motif that abolished transactivation by ETS factors but not by GATA-1 slightly increased activity in erythroid cells and significantly attenuated enhancer function in T lymphocytes. This study shows that controlled gene transfer in transplantable hematopoietic cells allows a functional analysis of distinct cis elements within a complex retrovirus enhancer, as required for the characterization and engineering of various cellular and viral regulatory sequences in basic research and gene therapy.

In vitro and in vivo gene delivery using a lentiviral vector

The delivery of antiarthritic genes to the synovial lining of joints is an effective strategy for the treatment of experimental models of rheumatoid arthritis (RA). Moreover, in two clinical studies it has proved possible to transfer the human interleukin-1 receptor antagonist (hIL-1Ra) cDNA to human rheumatoid joints. These protocols, however, utilized an ex vivo approach to gene delivery. While useful for establishing proof of concept, ex vivo methods do not lend themselves well to widespread clinical application. For this reason, we are devoting increasing attention to developing clinically acceptable in vivo methods of gene delivery to synovium. Because chronic conditions such as RA will probably require extended periods of intra-articular gene expression, integrating vectors are more attractive. In preclinical experiments, two such vectors, adeno-associated virus and high-titer Moloney-based retrovirus, have shown promise for in vivo gene delivery to synovium. Lentiviral vectors also possess favourable properties in this regard, but there are no published data on their suitability for in vivo gene delivery to joints. Here we report preliminary data from the use of lentiviruses to deliver genes to articular tissues. The recent generation of packaging systems able to produce high titers of replication-incompetent HIV-based retroviruses, and the pseudotyping of lentiviral vectors with the vesicular stomatitis virus G-protein (VSV-G) which increase the target cell range and allow concentration by centrifugation have facilitated these studies. In culture we have found that human synoviocytes, and both human and rat chondrocytes were efficiently transduced by high titer (>109 pfu/ml) of VSV-G pseudotyped HIV-1-based lentiviral vectors containing the β-galactosidase gene (lacZ). Similar patterns of expression were observed using rabbit synovial fibroblast line, HIG-82, murine 3T3 cells, and primary cultures of rat skin cells. Direct, intra-articular gene delivery was performed by injecting similar lentiviral preparations into the knees of Wistar rats. Histological analyses of the knee joints revealed the expression of lacZ in the synovial membrane for at least one week following injection. No lacZ staining was observed following the injection of empty viruses. Based on the ability to successfully deliver and express the lacZ marker gene in synoviocytes in culture and in the synovial lining in vivo, a recombinant vector containing hIL-1Ra has been constructed and is being evaluated.

In vivo transfer of bacterial marker genes results in differing levels of gene expression and tumor progression in immunocompetent and immunodeficient mice.

To optimize gene delivery for the treatment of malignant mesothelioma, expression of the beta-galactosidase marker gene was examined in a murine model of intraperitoneal malignant mesothelioma. The beta-galactosidase gene was delivered to the peritoneal cavity of tumor-bearing mice by various plasmid-liposome complexes or by replication-incompetent retrovirus, used alone or complexed to liposomes. In tumor samples from immunodeficient nude mice, moderate levels of gene expression were achieved by liposome-complexed plasmids. Retroviral gene delivery was more effective, and was increased nearly 10-fold by complexing the retrovirus to liposomes. In contrast, in tumor samples from immunocompetent CBA mice treated with the same vectors, no marker gene expression was detected. In immunodeficient mice, tumor growth was not affected by beta-galactosidase gene transfer. However, immunocompetent mice showed a significant decrease in tumor size and increase in survival time after beta-galactosidase delivery. Induction of cytotoxic T cells capable of lysing beta-Gal-transfected tumor cells suggests that tumor cells transduced with the bacterial beta-galactosidase gene may be eliminated in immunocompetent hosts. Our findings also indicate that plasmid-liposome complexes, which achieve a low level of gene expression, and retrovirus-liposome complexes, which result in nearly 100 times higher levels of gene expression in tumor cells in vivo, are similarly effective in inducing an antitumor immune response.

A subpopulation of apoptosis-prone cardiac neural crest cells targets to the venous pole: multiple functions in heart development?

A well-described population of cardiac neural crest (NC) cells migrates toward the arterial pole of the embryonic heart and differentiates into various cell types, including smooth muscle cells of the pharyngeal arch arteries (but not the coronary arteries), cardiac ganglionic cells, and mesenchymal cells of the aortopulmonary septum. Using a replication-incompetent retrovirus containing the reporter gene LacZ, administered to the migratory neural crest of chicken embryos, we demonstrated another population of cardiac neural crest cells that employs the venous pole as entrance to the heart. On the basis of our present data we cannot exclude the possibility that precursors of these cells might not only originate from the dorsal part of the posterior rhombencephalon, but also from the ventral part. These NC cells migrate to locations surrounding the prospective conduction system as well as to the atrioventricular (AV) cushions. Concerning the prospective conduction system, the tagged neural crest cells can be found in regions where the atrioventricular node area, the retroaortic root bundle, the bundle of His, the left and right bundle branches, and the right atrioventricular ring bundle are positioned. The last area connects the posteriorly located AV node area with the retroaortic root bundle, which receives its neural crest cells through the arterial pole in concert with the cells giving rise to the aortopulmonary septum. The NC cells most probably do not form the conduction system proper, as they enter an apoptotic pathway as determined by concomitant TUNEL detection. It is possible that the NC cells in the heart become anoikic and, as a consequence, fail to differentiate further and merely die. However, because of the perfect timing of the arrival of crest cells, their apoptosis, and a change in electrophysiological behavior of the heart, we postulate that neural crest cells play a role in the last phase of differentiation of the cardiac conduction system. Alternatively, the separation of the central conduction system from the surrounding working myocardium is mediated by apoptotic neural crest cells. As for the presence of NC cells in both the outflow tract and the AV cushions, followed by apoptosis, a function is assigned in the muscularization of both areas, resulting in proper septation of the outflow tract and of the AV region. Failure of normal neural crest development may not only play a role in cardiac outflow tract anomalies but also in inflow tract abnormalities, such as atrioventricular septal defects.

Neural crest cell contribution to the developing circulatory system: implications for vascular morphology?

In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.

Moloney murine leukemia virus-derived retroviral vectors decay intracellularly with a half-life in the range of 5.5 to 7.5 hours.

Replication-incompetent recombinant retroviruses are currently used for gene delivery. The limited efficiency of gene transfer using these vectors hampers implementation of gene therapy. Successful integration of Moloney murine leukemia virus (MMuLV)-derived retroviral vectors into the host cell DNA requires cell division. The time difference between virus entry and cell division is variable and prolonged in slowly dividing cells. Therefore, the rate of intracellular decay of internalized vectors between the time of entry into the target cell and cell division may limit the probability of successful integration following viral entry. We present two methods that measure the intracellular stability of MMuLV-derived retroviral vectors in NIH 3T3 cells. The first is based on a temporary interruption of cell cycle progression by using cell detachment. This method provides an estimate, but not a direct measurement, of the half-life. The results show that the MMuLV intracellular half-life is on the order of but shorter than the total cell cycle time. The second method allows the direct measurement of the intracellular half-life by using two cell cycle-specific labels: 5-bromodeoxyuridine, a thymidine analog that labels cells in S-phase; and the viral vector that labels cells in mitosis. By varying the time between the administration of the two labels, the intracellular half-life is measured to be in the range of 5.5 to 7.5 h. Such a short intracellular half-life may restrict the efficiency of gene transfer by retroviral vectors, particularly in slowly dividing target cells.