Retroviral vectors have been invaluable tools for studies of development in vertebrates. Their use has been somewhat constrained, however, by the low viral titers typically obtained with replication-incompetent vectors, particularly of the avian type. We have addressed this problem in several ways. We optimized the transient production of avian replication-incompetent viruses in a series of cell lines. One of the optimal cell lines was the mammalian line 293T, which was surprising in light of previous reports that avian viral replication was not supported by mammalian cells. We also greatly increased the efficiency of viral infection. Pseudotyping with the vesicular stomatitus virus G (VSV-G) protein led to an over 350-fold increase in the efficiency of infection in ovo relative to infection with virus particles bearing an avian retroviral envelope protein. To further increase the utility of the system, we developed new Rous sarcoma virus (RSV)-based replication-incompetent vectors, designed to express a histochemical marker gene, human placental alkaline phosphatase, as well as an additional gene. These modified retroviral vectors and the VSV-G pseudotyping technique constitute significant improvements that allow for expanded use of avian replication-incompetent viral vectors in ovo.
We previously reported that retrovirally marked clones in the mature chick diencephalon were widely dispersed in the mediolateral, dorsoventral and rostrocaudal planes. The current study was undertaken to define the migration routes that led to the dispersion. Embryos were infected between stages 10 and 14 with a retroviral stock encoding alkaline phosphatase and a library of molecular tags. Embryos were harvested 2.5-5.5 days later and the brains were fixed and serially sectioned. Sibling relationships were determined following PCR amplification and sequencing of the molecular tag. On embryonic day 4, all clones were organized in radial columns spanning the neuroepithelium, which was composed primarily of a ventricular zone at this age. No tangential migration was seen in the ventricular zone. On embryonic day 5, most clones remained radial with many cells located in the ventricular zone; however, a few clones had cells migrating perpendicular to the radial column, in either a rostrocaudal or dorsoventral direction. The tangential migration began just beyond the basal limit of the ventricular zone. On embryonic days 6 and 7, many clones had cells migrating perpendicular to the radial column, which spanned from the ventricular to the pial surface. The migrating cells appeared to be aligned along axes that were perpendicular to the radial column. Using a combination of DiI tracing, immunohistochemistry and electron microscopy, we have determined that axonal tracts are present and are aligned with the migrating cells, suggesting that they support the non-radial cell migration. These data indicate that migration along pathways independent of radial glia occur outside of the ventricular zone in more than 50% of the clones in the chick diencephalon.
CASE DESCRIPTION A 1-year-old castrated male German Shepherd Dog was examined because of an acute onset of lethargy, tachypnea, and inappetence. CLINICAL FINDINGS On initial physical examination, the dog was tachypneic with muffled heart sounds on thoracic auscultation and a palpable abdominal fluid wave. Transthoracic echocardiography revealed focal intramural pericardial effusion and cardiac tamponade. TREATMENT AND OUTCOME The patient underwent emergency therapeutic pericardiocentesis, followed by right lateral intercostal thoracotomy and subtotal pericardiectomy. A 3 × 5-cm mass located between the parietal and visceral layers of the pericardium was resected. The histologic diagnosis was necrotic adipose tissue with granulomatous inflammation and fibroplasia. The patient also underwent exploratory laparotomy and umbilical herniorrhaphy during the same anesthetic episode and recovered from surgery without apparent complications. There were no further clinical signs of cardiac disease. CLINICAL RELEVANCE The patient described in the present report underwent successful subtotal pericardiectomy for treatment of a benign focal lesion causing recurrent pericardial effusion and cardiac tamponade. Prompt diagnosis and intervention may have contributed to the positive outcome in this case.
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